Safety Evaluation of Certain Contaminants in Food

WHO FOOD ADDITIVES SERIES: 82
Prepared by the ninety-first meeting of the

Joint FAO/WHO Expert Committee
on Food Additives (JECFA)
Safety evaluation
of certain
contaminants
in food
WHO FOOD ADDITIVES SERIES: 82
Prepared by the ninety-first meeting of the
Joint FAO/WHO Expert Committee
on Food Additives (JECFA)
Safety evaluation
of certain
contaminants
in food
The summaries and evaluations contained in this book are, in most cases, based on unpublished proprietary data
submitted for the purpose of the JECFA assessment. A registration authority should not grant a registration on the basis
of an evaluation unless it has first received authorization for such use from the owner who submitted the data for JECFA
review or has received the data on which the summaries are based, either from the owner of the data or from a second
party that has obtained permission from the owner of the data for this purpose.
World Health Organization, Geneva, 2023

Safety evaluation of certain contaminants in food: prepared by the ninety-first meeting of the Joint FAO/WHO Expert
Committee on Food Additives (JECFA)
(WHO Food Additives Series, No. 82)
ISBN (WHO) 978-92-4-006076-0 (electronic version)

ISBN (WHO) 978-92-4-006077-7 (print version)
ISBN (FAO) 978-92-5-137666-9
ISSN 0300-0923
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CONTENTS
Preface v
Safety evaluations of specific contaminants in food 1
Cadmium: dietary exposure assessment 3
Ergot alkaloids 57
Previous cargoes – solvents and reactants 229
Annex 1
Reports and other documents resulting from previous meetings of the Joint FAO/WHO
Expert Committee on Food Additives 337
Annex 2
Abbreviations used in the monographs 349
Annex 3
Participants in the ninety-first meeting of the Joint FAO/WHO Expert Committee
on Food Additives 351
iii
PREFACE
The monographs contained in this volume were prepared at the ninety-first meeting of
the Joint Food and Agriculture Organization of the United Nations (FAO)/World Health
Organization (WHO) Expert Committee on Food Additives (JECFA), which met online in
November 2020. These monographs summarize the data on selected food contaminants
reviewed by the Committee.
The ninetieth report of JECFA has been published by WHO as WHO Technical Report
No. 1036. Reports and other documents resulting from previous meetings of JECFA are listed
in Annex 1. The participants in the meeting are listed in Annex 3 of the present publication.
JECFA serves as a scientific advisory body to FAO, WHO, their Member States and the Codex
Alimentarius Commission, primarily through the Codex Committee on Food Additives, the
Codex Committee on Contaminants in Foods and the Codex Committee on Residues of
Veterinary Drugs in Foods, regarding the safety of food additives, residues of veterinary drugs,
naturally occurring toxicants and contaminants in food. Committees accomplish this task by
preparing reports of their meetings and publishing specifications or residue monographs
and dietary exposure and toxicological monographs, such as those contained in this volume,
on substances that they have considered.
The monographs contained in this volume are based on working papers that were
prepared by WHO and FAO experts. A special acknowledgement is given at the beginning of
each monograph to those who prepared these working papers.
The designations employed and the presentation of the material in this publication
do not imply the expression of any opinion whatsoever on the part of the organizations
participating in WHO concerning the legal status of any country, territory, city or area or
its authorities, or concerning the delimitation of its frontiers or boundaries. The mention
of specific companies or of certain manufacturers’ products does not imply that they are
endorsed or recommended by the organizations in preference to others of a similar nature
that are not mentioned.
Any comments or new information on the biological or toxicological properties of
or dietary exposure to the compounds evaluated in this publication should be addressed to:
WHO Joint Secretary of the Joint FAO/WHO Expert Committee on Food Additives, Department
of Food Safety and Zoonoses, World Health Organization, 20 Avenue Appia, 1211 Geneva 27,
Switzerland ([email protected]).
v
SAFETY EVALUATIONS OF
SPECIFIC CONTAMINANTS IN FOOD
Cadmium: dietary exposure assessment
First draft prepared by
Peter Cressey,1 Polly E. Boon2 and Jean-Charles Leblanc3
1
Risk Assessment and Social Systems Group, Institute of Environmental Science and
Research, Christchurch, New Zealand
2
Department of Food Safety, Centre for Nutrition, Prevention and Health, National
Institute for Public Health and the Environment (RIVM), Bilthoven, the Netherlands
3
Laboratory for Food Safety, French Agency for Food, Environmental and Occupational
Health and Safety (ANSES), Maisons-Alfort, France
1. Explanation 4
2. Food consumption and dietary exposure assessment 6
2.1 Concentrations in food used in the dietary exposure estimates 6
2.2 Food consumption data used in the dietary exposure estimates 7
2.3 National estimates of chronic dietary exposure from the literature 7
(a) Australia 8
(b) Bangladesh 9
(c) Benin 9
(d) Brazil 10
(e) Cameroon 10
(f) Canada 11
(g) Chile 11
(h) China 12
(i) Denmark 14
(j) Europe 14
(k) France 15
(l) French Polynesia 16
(m) Germany 17
(n) Hong Kong Special Administrative Region, China 17
(o) Iran, Islamic Republic of 18
(p) Ireland 18
(q) Italy 19
(r) Japan 19
(s) Korea, Republic of 19
(t) Mali 20
(u) The Netherlands 20
(v) New Zealand 21
(w) Nigeria 21
(x) Poland 22
(y) Serbia 22
(z) Spain 23
(aa) Sri Lanka 24
(bb) Sweden 24
(cc) Thailand 25
(dd) United Kingdom 25
(ee) United States of America 25
3
Safety evaluation of certain contaminants in food Ninety-first JECFA
(ff) Viet Nam 26

(gg) Summary 27
2.4 International estimates of chronic dietary exposure 27
2.4.1 Temporal trends in dietary cadmium exposure 38
2.4.2 Impact of cocoa product source on dietary cadmium exposure 38
2.4.3 Impact of proposed maximum limits for cadmium on cocoa product
rejection rates and dietary cadmium exposure 40
3. Evaluation 43
4. References 46
5. Appendix
Global mean cadmium concentrations used for international estimates of
dietary exposure 48
1. Explanation
Cadmium was evaluated by the Committee at its sixteenth, thirty-third, forty-
first, fifty-fifth, sixty-first, sixty-fourth, seventy-third and seventy-seventh
meetings (Annex 1, references 30, 83, 107, 149, 166, 176 and 202). At the sixty-first
and sixty-fourth meetings, the Committee noted that the estimated total mean
dietary exposure to cadmium from all foods, derived from per capita data from
the five Global Environment Monitoring System (GEMS)/Food Contamination
Monitoring and Assessment Programme regional diets, ranged from 40% to
60% of the provisional tolerable weekly intake applicable at that time of 7 μg/kg
body weight (bw). The seven commodity groups that contributed significantly to
total mean dietary exposure to cadmium were rice, wheat, root vegetables, tuber
vegetables, leafy vegetables, other vegetables and molluscs (40–85% of the total
mean dietary exposure to cadmium across the five regional diets).
At its seventy-third meeting in 2011, the Committee re-evaluated
cadmium and established a provisional tolerable monthly intake (PTMI) of
WHO Food Additives Series No. 82, 2022
25 μg/kg bw, reflecting the long half-life of cadmium in humans. Reported

national estimates of mean dietary exposure to cadmium from all foods for adults
ranged from 2.2 to 12 μg/kg bw per month, or 9–48% of the PTMI. For European
children up to 12 years of age, this estimate was 11.9 μg/kg bw per month or
47% of the PTMI. High percentile dietary exposures to cadmium for adults from
Europe, Lebanon and the United States of America (USA) were reported to range
from 6.9 to 12.1 μg/kg bw per month (28–48% of the PTMI), and from 20.4 to
22.0 μg/kg bw per month (82–88% of the PTMI) for children aged 0.5–12 years
from Australia and the USA. Data on cadmium occurrence and consumption of
foods containing cocoa and its derivatives were included in all 2011 estimates.
Although not all estimates of dietary cadmium exposure evaluated at the
4
seventy-third meeting reported the major contributing foods, for those estimates
that did report this information, cereals and cereal products and vegetables were
consistently reported as major contributors, with seafood and meat, including
offal, also reported in some studies. None of the studies reported cocoa products
as major contributors to dietary cadmium exposure.
At its seventy-seventh meeting in 2013, the Committee conducted an
assessment of dietary exposure to cadmium from cocoa and cocoa products at
the request of the sixth session of the Codex Committee on Contaminants in
Foods (CCCF). The Committee considered the exposure to cadmium from foods
containing cocoa and its derivatives in the context of overall dietary exposure. The
estimates of mean dietary exposure to cadmium from foods containing cocoa and
its derivatives ranged from 0.005 to 0.39 μg/kg bw per month or 0.2–1.6% of the
PTMI across the 17 GEMS/Food cluster diets, assuming a body weight of 60 kg.
Mean dietary exposure estimates for individual cocoa products based on national
food consumption data ranged from 0.001 to 0.46 μg/kg bw per month or 0.004–
1.8% of the PTMI. The cocoa products included were cocoa beverages, cocoa
powder and other cocoa products. The highest high exposure (97.5th percentile,
P97.5) was estimated at 12 μg/kg bw per month for European children 7–11 years
of age, solely due to the consumption of cocoa powder. Combining the highest
P97.5 dietary exposure estimate for adults and children out of the three cocoa
products with the mean dietary exposure estimates for both age groups from the
whole diet, the Committee estimated a total dietary exposure of 7.4–17.2 μg/kg
bw per month or 30–69% of the PTMI for adults and 23.9 μg/kg bw per month
or 96% of the PTMI for children aged 0.5–12 years. The Committee noted that
these estimates of total dietary cadmium exposure very likely overestimated the
exposure, because the estimates from the whole diet also included a contribution
from cocoa and cocoa products.
At the request of the thirteenth session of CCCF for more comprehensive
occurrence data for cadmium in food, the JECFA Secretariat issued a call for data
on cadmium in chocolates and cocoa-derived products in 2019. The submitted
data included a wider geographical range of occurrence data for cadmium
in cocoa products than considered at the seventy-seventh meeting of the
Committee. The occurrence data also showed a higher mean concentration for
cadmium in cocoa products than previously noted by the Committee. As a result,
the JECFA Secretariat considered it appropriate to revise the dietary exposure
assessment of cadmium to include not only chocolate and cocoa products but
the contribution from all food sources. At the present meeting the Committee
reassessed cadmium exposure to include the contribution of all food sources,
particularly cocoa products.
5
2. Food consumption and dietary exposure assessment
2.1 Concentrations in food used in the dietary exposure estimates

The GEMS/Food contaminants database was queried for records relating to
cadmium in any food. The database query was restricted to records submitted
since the previous assessment of dietary cadmium exposure from the whole diet
by the Committee in 2011. Data submitted since 1 January 2011 originated from
27 countries or country groups (WHO African Region, WHO European Region),
representing 10 of the 17 GEMS/Food cluster diets. It should be noted that for
several of the countries or clusters the available data were limited in quantity or
restricted to a narrow range of foods. For example, the sole country providing data
from cluster G09 (Indonesia) submitted analytical results for 30 samples of cocoa
products only. Five clusters (G07, G08, G10, G11 and G15) cover the countries of
Europe; however, most of the contaminant concentration data available for these
countries were only identified at the level of the WHO European Region and it
was not possible to examine differences in contamination profile between these
clusters using these data.
The extracted data set was cleaned by removal of records of aggregate
analyses (summary of multiple analyses), records with no result recorded and
records relating to analysis of animal feed. The units of measurement were
standardized to µg/kg. After consideration of the range of reported limits of
detection (LODs), a cut-off was made at 20 µg/kg. When considering the full
distribution of reported LODs, this value was approximately two standard
deviations above the mean LOD.
The final data set contained 277 292 records, of which 216 373 (78%)
were from the WHO European Region. A considerable body of non-European
data were available for cluster G10, submitted by Canada (n = 21 501), Japan (n =
5332) and the USA (n = 5887). Records were widely spread across different food
types, with the most commonly analysed food types being edible pig offal (7.3%),
marine fish (6.9%) and cattle meat (3.7%).
Cadmium concentrations for most food types were consistent with
expectations; however, the data set contained 714 records for cadmium in “sugar”,
most of which came from the WHO European Region. This data set contained a
subset of records of very high concentrations (up to 12 000 µg/kg). Information
from the literature does not support such high concentrations of cadmium in
sugar and the records under this descriptor were excluded from the analyses of
dietary cadmium exposure.
6
2.2 Food consumption data used in the dietary exposure estimates

The Committee used the GEMS/Food cluster diets to investigate the contribution
of different food types, particularly cocoa products, to dietary cadmium
exposure. The consumption cluster diets provide mean per capita consumption
values based on FAO food balance sheet data for raw commodities and some
semi-processed commodities for 17 clusters of countries (Sy et al., 2013). Clusters
G01 and G06 include primarily Middle Eastern, central Asian and north African
countries; clusters G03, G13 and G16 include primarily African countries; cluster
G02 includes countries in west Asia and the Balkan region of Europe; cluster G04
includes Middle Eastern and Caribbean countries; cluster G09 includes countries
in Africa and Asia; clusters G07, G08, G10, G11 and G15 include European and
North American countries and developed countries from Asia and the Pacific
(Australia, Japan, New Zealand, the Republic of Korea); clusters G05 and G12
consist mainly of South and Central American countries; and clusters G14 and
G17 include Caribbean, Asian and Pacific Island states (see Appendix).
In addition to national estimates of dietary exposure published in the
literature, the Committee may derive national estimates of dietary exposure
using food consumption information from the FAO/WHO Chronic Individual
Food Consumption Database – Summary statistics (CIFOCOss), in combination
with summary concentration data from the GEMS/Food contaminants database.
Previously, national estimates of chronic dietary exposure were derived by the
Committee when:
■■ national food consumption information was available through
CIFOCOss;
■■ suitable concentration data were submitted to the Committee (GEMS/
Food contaminants database); and
■■ no existing recent dietary exposure assessment for cadmium was
available for the country from the literature.
These criteria were not fully met for any country with food consumption
information in CIFOCOss or concentration data on cadmium in the GEMS/
Food contaminants database. Consequently, the Committee decided not to
derive additional national estimates of dietary exposure to cadmium.
2.3 National estimates of chronic dietary exposure from the literature

Since the evaluation of cadmium at the seventy-third meeting of the Committee
in 2011, several national evaluations of chronic dietary exposure have been
published. Studies were identified from the scientific literature (PubMed and Web
7
of Science) or the grey literature using the search terms: cadmium AND (food
OR diet*) AND (exposure OR intake OR “total diet”). The studies summarized
below are those that were considered by the Committee to include most of the
main contributors to dietary cadmium exposure. Studies that only considered
a single food type or a narrow range of food types were not summarized. The
Committee considered evaluations from Australia, Bangladesh, Benin, Brazil,
Cameroon, Canada, Chile, China, Denmark, Europe, France, French Polynesia,
Germany, Hong Kong Special Administrative Region (SAR) China, Ireland,
Islamic Republic of Iran, Italy, Japan, Republic of Korea, Mali, the Netherlands,
New Zealand, Nigeria, Poland, Serbia, Spain, Sri Lanka, Sweden, Thailand, the
United Kingdom, the USA and Viet Nam (Table 1).
(a) Australia
Dietary exposure to cadmium was assessed as part of the twenty-fifth Australian
Total Diet Study (ATDS) (FSANZ, 2019). Foods (n = 88) were sampled in all
Australian states and territories on two occasions. Individual food purchases were
combined to give either four or eight food type composites. Food samples were
analysed for cadmium by inductively-coupled plasma-mass spectrometry (ICP-
MS), following acid digestion. The limit of reporting (LOR) for all sample types
was 5 µg/kg. Median concentrations for each food type were used to estimate
dietary exposure. For foods with a low proportion of results above the LOR,
lower-bound (LB)–upper-bound (UB) estimates of the median were determined,
with results below the LOR either substituted by zero or the LOR, respectively.
Individual food consumption information for the population aged 2 years or
above was taken from the 2011–2012 Australian National Nutrition and Physical
Activity Survey. Food consumption information was only used if respondents (n
= 7735) had completed two 24-hour dietary recall (24HDR) surveys. A model
diet was used for infants (9 months old). Mean (90th percentile) LB–UB estimates
of dietary cadmium exposure for all respondents aged 2 years and over were 2.5–
6.6 (4.8–11) µg/kg bw per month. For 9-month-old infants the corresponding
dietary exposure estimates were 2.8–16 (5.7–33) µg/kg bw per month. Cereals
and cereal products and root vegetables were the main contributors to dietary
exposure for all age groups. Hot chocolate beverages, cocoa, chocolate and fudge
contributed 4–6% of estimated dietary exposure.
A study carried out in Western Australia conducted an initial 24HDR
and food frequency questionnaire with 22 families with children aged 5–6 years,
to identify foods for further study (Callan et al., 2014). Samples of foods and
beverages (n = 253) were purchased from supermarkets between April and
July 2011. Food samples were prepared by normal household methods, but
not cooked. Samples were analysed for cadmium by ICP-MS, following nitric
8
acid digestion. LOD and limit of quantification (LOQ) were 3 and 10 µg/kg for
solid and liquid food samples. Geometric mean concentrations were calculated
for each food group. It is unclear how analytical results below the LOQ were
handled. Food consumption information was derived from the 2008 Child and
Adolescent Physical Activity and Nutrition Survey, a state-based survey involving
administration of a single 24HDR to 653 children aged 8–17 years. Data were
used to calculate mean, 5th and 95th percentile (P95) food consumption for the
food groups. Mean (P95) estimates of dietary cadmium exposure ranged from
0.2 (1.0) for 16-year-old males or females to 0.4 (2.2) µg/kg bw per day (12 and
66 µg/kg bw per month) for 8-year-old males. It is likely that the use of food
consumption data from a single 24HDR will have inflated the P95 estimates
of dietary exposure. The main contributors to dietary exposure were fish and
seafood products (36%), cereals (31%), and vegetable products and dishes (18%).
Although it was reported that cadmium concentrations in cocoa powder and
chocolate were among the highest in the foods analysed, contributions of these
foods to dietary exposure were not explicitly reported. However, they do not
appear to be main contributors to dietary exposure.
(b) Bangladesh
The study from Bangladesh was slightly unusual in that the dietary cadmium
exposure of the Bangladeshi population was estimated based on analysis of
Bangladeshi foods imported into the United Kingdom (Al-Rmalli et al., 2012).
Food samples were collected from Bangladeshi shops during 2008 and 2009.
Samples were analysed for cadmium by ICP-MS, following nitric acid/hydrogen
peroxide digestion. The sensitivity of the method was not reported and there
was no indication of how analytical results below the LOD were handled. Food
consumption information was taken from the literature. Rice consumption was
assumed to be equivalent to 500 g of uncooked rice per day. A default body weight
of 60 kg was used. Estimated dietary cadmium exposure was 34.6 µg/day or 17.3
µg/kg bw per month for a 60-kg Bangladeshi. The main contributors to dietary
exposure were steamed rice (54%) and green vegetables (35%). No information
on dietary exposure from cocoa products was provided.
(c) Benin
As part of a multicountry total diet study in sub-Saharan Africa, foods were
collected at two locations in Benin (Littoral and Borgou) and combined to give
40 food group composites, representing 13 food groups (Ingenbleek, 2019).
Samples were analysed for a range of contaminant elements, including cadmium,
by ICP-MS, following nitric acid digestion. LOQs were in the range 0.2–1.0 µg/
kg. Cadmium was quantified in 63% of composite samples. LB–UB estimates of
9
the mean concentration were derived for each food group. Food consumption
information was obtained from a household budget survey including 2494
households across the two sampling locations in Benin. Household food intake
was converted to intake per adult male equivalent (AME). LB–UB mean (P95)
estimates of dietary cadmium exposure for Littoral and Borgou were 0.05–0.05
(0.10–11) and 0.04–0.05 (0.09–0.10) µg/kg bw per day, respectively. These
exposure estimates correspond to 1.5–1.5 (3.0–3.3) µg/kg bw per month and 1.2–
1.5 (2.7–3.0) µg/kg bw per month, respectively. The main contributors to dietary
exposure were tomatoes, rice, pasta, yams and smoked fish. No information on
the contribution to exposure from cocoa products was reported.
(d) Brazil
Dietary cadmium exposure was determined for a cohort (n = 64) of 1–4-year-
old children from day care centres in São Paulo, Brazil (Leroux et al., 2018). For
each child, a weekday 24-hour duplicate diet sample was collected and analysed
for cadmium by acid digestion followed by ICP-MS. Portion sizes were recorded
at the time of collection of the duplicate diet samples. Mean dietary cadmium
exposure for male and female children was estimated to be 0.08 and 0.09 µg/kg
bw per day, respectively. These exposures are equivalent to 2.4 and 2.7 µg/kg bw
per month, assuming the single day sampled was representative of the children’s
normal dietary patterns. The duplicate diet method does not allow assessment of
the contributions of individual food types to overall dietary exposure.
(e) Cameroon
Individual food samples (n = 1773) of 203 different food items were composited
into 64 analytical samples, representing 10 food categories (Gimou et al., 2014).
Samples were analysed for cadmium by ICP-MS following acid digestion. The
LOQ was 1 µg/kg, with 17% of samples containing cadmium concentrations below
the LOQ. Of these, 11% were below the LOD and the remaining 6% were between
the LOD and LOQ. LB and UB estimates of the mean cadmium concentration
were determined for each food category. Dietary exposure to cadmium was
derived using UB estimates of the mean concentration. Food consumption data
were obtained from the second Cameroonian Household Budget Survey. The
survey recorded expenditure for 11 553 households over a 2-week period. Food
expenditure was converted to food consumption for “adult equivalents”. A default
body weight of 60 kg was used. Mean and P95 UB estimates of dietary cadmium
exposure were 4.7 and 8.2 µg/kg bw per month, respectively. Cereals and cereal
products contributed 54% of overall dietary exposure, with fruits, vegetables and
oilseeds (13%) and fish (11%) being the other main contributors. The category
“sugar and cocoa products” accounted for 1% of dietary cadmium exposure.
10
As part of a multicountry total diet study in sub-Saharan Africa, foods

were collected at two locations in Cameroon (Duala and North) and combined to
give 50 food group composites, representing 13 food groups (Ingenbleek, 2019).
Samples were analysed for a range of contaminant elements, including cadmium,
by ICP-MS, following nitric acid digestion. LOQs were in the range 0.2–1.0 µg/
households across the two sampling locations in Cameroon. Household food
intake was converted to intake per AME. LB–UB mean (P95) estimates of
dietary cadmium exposure for Duala and North were 0.08–0.08 (0.17–0.17) and
0.05–0.05 (0.10–0.10) µg/kg bw per day, respectively. These exposure estimates
correspond to 2.4–2.4 (5.1–5.1) µg/kg bw per month and 1.5–1.5 (3.0–3.0) µg/
kg bw per month, respectively. The main contributors to dietary exposure were
rice, green leaves, wheat and bread, peanuts and maize. No information on the
contribution to exposure from cocoa products was reported for either study
location.
(f) Canada
Dietary exposure to cadmium for First Nations peoples residing on-reserve in
the province of Ontario was determined using a total diet study approach (Juric,
2016). The study was conducted under the umbrella of the 2011–2012 First
Nations Food, Nutrition and Environment Study. Cadmium was determined in
tap water from 20 participating communities and in 419 composite food samples,
representing 141 different traditional food items, by ICP-MS following acid
digestion. The LODs were 0.005 µg/L for water and 1 µg/kg for food samples.
The cadmium concentrations for market foods were taken from the Canadian
Total Diet Study (Health Canada, 2020). Analytical results below the LOD were
substituted by a concentration equal to the LOD (UB). Food consumption
information was taken from a single-pass 24HDR. A total of 1429 individuals,
aged 19 years and over, participated. The overall mean and P95 estimates of dietary
cadmium exposure were 3.9 and 9.6 µg/kg bw per month, respectively. Dietary
cadmium exposure was slightly higher for traditional food consumers (6.0 and
11.1 µg/kg bw per month for the mean and P95, respectively). Consumption of
potatoes accounted for 18% of dietary cadmium exposure, followed by plain
pasta (9%). Contributions to exposure from cocoa products were not reported.
(g) Chile
A total diet study was conducted to assess dietary exposure of the adult (18–
65 years) population of Valdivia, Chile to cadmium (Munoz et al., 2017). Food
11
items from food stores in Valdivia were sampled on three different occasions.
The number of food samples taken was not reported. Samples were analysed for
cadmium by flame atomic absorption spectrophotometry (AAS), following acid
digestion. The LOD was 2 µg/kg. Protocols for handling analytical results below
the LOD were not reported. Food consumption information was derived from a
single-pass 24HDR questionnaire administered to 382 adult residents of Valdivia
during 2012. A default body weight of 70 kg was used. The mean estimate of
dietary cadmium exposure was 18.1 µg/day (7.8 µg/kg bw per month for a 70-kg
adult). Bread (27%) contributed most to cadmium exposure, followed by non-
alcoholic beverages (21%) and cereals (11%). No information on the contribution
of cocoa products was reported.
(h) China
Dietary cadmium exposure was estimated for a cohort of Shanghainese over 40
years of age (He et al., 2013). Concentrations of cadmium in food were taken from
a Chinese national cadmium exposure survey carried out in 2000 and a survey
carried out in Shanghai during 2002 and 2007. Samples were analysed for cadmium
by graphite furnace atomic absorption spectrometry (GF-AAS). Performance
characteristics of the analytical method were not reported in this publication.
Food consumption information for the recruited cohort (n = 267) came from a
food frequency questionnaire (FFQ), with associated assessment of portion sizes.
Mean dietary cadmium exposure was estimated to be 12.8 µg/day (6.4 µg/kg bw
per month, assuming a 60 kg body weight), with a 90th percentile estimate of
20.6 µg/day (10.3 µg/kg bw per month, assuming a 60 kg body weight). The main
contributors to dietary cadmium exposure were vegetables (40%) and rice (38%).
No information on the contribution from cocoa products was reported.
In a large national study, 228 687 individual food samples were collected
in 31 provinces, autonomous regions and municipalities across China between
2001 and 2015 (Song et al., 2017). Samples were analysed for cadmium in
laboratories in the various areas. Although no information was provided on the

analytical methods used, LODs were reported to be in the range of 0.01 to 100 µg/
kg, with 35.7% of analytical results below the LOD. Foods were aggregated into
32 food groups. For calculating mean cadmium concentrations, analytical results
below the LOD were substituted by a value of LOD/2, except for milk and eggs, for
which over 60% of results were below the LOD and a UB estimate of the mean was
derived. Food consumption data were derived from the 2002 Chinese National
Nutrition and Health Survey. This survey involved administration of 24HDR
questionnaires on three consecutive days to 67 608 study participants. Individual
body weights were also determined. Mean and P95 estimates of dietary cadmium
exposure were determined for all participants and for five population subgroups.
12
The overall mean and P95 dietary exposure estimates were 15.3 and 33.0 µg/kg
bw per month, respectively. Rice (56%), wheat flour (12%) and leafy vegetables
(11%) were the main contributors to mean dietary exposure. No information on
the contribution from cocoa products was reported.
A total diet study was conducted in Shenzhen province to estimate adult
dietary exposure to cadmium (Wang et al., 2018). Individual food samples (n
= 276) from 13 food groups were analysed for cadmium by ICP-MS following
acid digestion. The LOD and LOQ were 0.2 and 1.0 µg/kg, respectively. Overall,
12% of samples had analytical results below the LOD. For these results a value of
LOD/2 was substituted. Median cadmium concentrations for each food group
were used for estimating dietary exposure. Food consumption information was
derived from a survey of 662 adult (18+ years) residents of Shenzhen, which took
the form of a 3-day food diary. Individual body weights were recorded and used
for estimation of dietary exposure. Dietary exposure was estimated for mean and
P95 consumers in three age groups (18–39, 40–50 and 60+ years) and separately
for males and females. Mean dietary exposure estimates were in the range of 8.3–
11 µg/kg bw per month, whereas P95 estimates were in the range of 12–14 µg/kg
bw per month. The main contributors to mean dietary exposure were vegetables
(33%), rice and rice products (19%) and fish, seafood and shellfish (19%). No
information on the contribution from cocoa products was reported, although
these products would have been included, along with others, in an “others” food
group that contributed 4% of estimated dietary exposure.
Results of the fifth China Total Diet Study (TDS) were used to estimate
dietary exposure to cadmium for residents of 20 regions of China (Wei et al.,
2019; Xiao et al., 2020). Respondents of the fifth TDS included male residents
aged 18–45 years, with an average body weight of 63 kg. Food consumption
information was obtained from 3-day (2 weekdays and 1 weekend day) 24-hour
household dietary surveys. More than 200 foods were sampled and classified into
13 categories. Foods were prepared for consumption and analysed for cadmium
by ICP-MS. The mean dietary exposure to cadmium for the 20 different regions
ranged from 7.3 to 186.1 µg/day (3.7 to 93 µg/kg bw per month for a 60-kg adult).
The overall average exposure was 32.7 µg/day (16.4 µg/kg bw per month for a
60-kg adult). The main dietary sources of cadmium were cereals and vegetables.
No information was provided on how dietary exposure was calculated, how left-
censored data were managed or on the contribution from cocoa products.
Dietary exposure to cadmium was estimated for residents of Guangzhou
City in China (Zhang et al., 2018). For this purpose, foods were sampled in 12
districts of Guangzhou City during 2013–2015. A total of 4039 single-species food
samples were collected belonging to 11 food groups. Food samples were analysed
for cadmium by GF-AAS following nitric acid/hydrogen peroxide digestion. The
LOD and LOQ were 1 and 3 µg/kg, respectively. Food consumption data were
13
obtained from a survey among urban and rural residents of Guangzhou City in
2011 aged 3–88 years (n = 2976). Data were obtained using a 3-day, 24HDR.
Mean dietary exposure was calculated by multiplying the average amount of
food consumed per kg body weight across the three recording days by the mean
concentration of cadmium in each food group. High exposure was calculated
by multiplying the mean concentration by the P95 food consumption level. The
mean and P95 estimates of dietary cadmium exposure were 14.4 and 41 µg/kg bw
per month. The food groups that contributed most to the mean exposure were
cereals (50.2%; mainly rice), laver (19.2%) and vegetables (13%). No information
on the contribution from cocoa products was reported.
(i) Denmark
The Danish National Food Institute (DTU Food) carries out periodic risk
assessments of chemical contaminants in the diet (DTU Food, 2015). Food
samples were collected during the period 2004–2011, on a project-by-project
basis. Analyses were carried out in two regional laboratories, with foods prepared
according to normal household practices, but not cooked. Samples were analysed
for cadmium by ICP-MS, following nitric acid digestion. While not specifically
stated, it appears that LODs were in the range 1.2–8.6 µg/kg. Between 1 and 384
samples of individual food types were analysed. Results below the LOD were
included as indicative values in the calculation of mean concentrations of food
types. Food consumption data were collected as a part of DANSDA (DAnish
National Survey of Diet and physical Activity) in 2005–2008. Participants (n =
2700, aged 4–75 years) completed a 7-day food diary. Individual estimates of
dietary exposure were derived for each participant in the food consumption
survey. The mean and P95 estimates of dietary cadmium exposure for the Danish
population were 0.18 and 0.38 µg/kg bw per day (5.4 and 11.4 µg/kg bw per
month), respectively. The main contributors to dietary exposure were cereals
and cereal products (49%) and vegetables and vegetable products (34%). No
information on the contribution from cocoa products was reported.
(j) Europe
The European Food Safety Authority (EFSA) calculated dietary exposure to
cadmium in Europe in 2012 (EFSA, 2012). In these calculations, individual food
consumption data from European countries were combined with a merged data
set of cadmium occurrence data in foods from 22 European Union Member
States, three European Economic Area or other countries, and some food business
operators, mainly sampled during the period 2003–2011. Most of the analytical
results were obtained from the Slovak Republic, followed by Germany, France,
Romania, Spain and Denmark between 2003 and 2007. EFSA calculated LB,
14
medium-bound (MB) and UB exposure estimates for infants (<1 year), toddlers
(1–2 years), other children (3–9 years), adolescents (10–17 years), adults (18–64
years), elderly people (65–74 years) and the very elderly (75+ years). In the MB
estimates, concentrations below the LOD or LOQ were substituted by a value of
LOD/2. The MB median mean dietary exposure across dietary surveys within an
age group ranged from 1.62 µg/kg bw per week (6.9 µg/kg bw per month) in the
elderly to 4.80 µg/kg bw per week (20.6 μg/kg bw per month) in toddlers. The
median P95 of exposure ranged from 2.89 µg/kg bw per week (12.4 µg/kg bw
per month) for the very elderly to 6.59 µg/kg bw per week (28.2 µg/kg bw per
month) for infants. The food categories contributing most to the LB estimates
of exposure, being least influenced by left-censored data and LODs, were grains
and grain-based products (27%), vegetables and vegetable products (16%), and
starchy roots and tubers (13%). Cocoa products were included in the assessment
based on cadmium concentrations in cocoa powder (n = 732), chocolate (n =
1558) and cocoa beverages (n = 2196). The contribution of cocoa products to
the total dietary exposure distributions ranged from 0.2% for infants to 9.4%
for other children, with the greatest component of this contribution from the
consumption of chocolate.
(k) France
The second French TDS involved collection of 1319 food samples, covering 41
food groups, between 2007 and 2009 (Arnich et al., 2012). Samples were prepared
for consumption and analysed for cadmium by ICP-MS, following acid digestion.
The method LOQ was 1 µg/kg. Analytical results below the LOD or LOQ were
reported for 21% of samples. Food consumption information was derived from the
second individual and national food consumption survey (INCA2). The survey
involved completion of a 7-day food diary by 3362 individuals (1918 adults aged
18–79 years and 1444 children aged 3–17 years). Mean concentrations of cadmium
for the 41 food groups were calculated as MB. Mean (P95) estimates of dietary
cadmium exposure for children and adults were 0.24 (0.45) and 0.16 (0.27) µg/
kg bw per day, respectively. This equates to 7.2 (13.5) and 4.8 (8.1) µg/kg bw per
month, respectively. For adults, the main contributors to mean dietary exposure
were bread and dried bread products (22%), potatoes and potato products (12%),
pasta (6%) and crustaceans and molluscs (6%). Chocolate contributed 1–2% of
dietary cadmium exposure, depending on the age group considered.
A study was carried out to determine dietary and non-dietary exposure
to inorganic species, including cadmium, for French children aged 3–6 years
(Glorennec et al., 2016). Concentration data on cadmium in foods were taken
from the second French TDS and food consumption data from the INCA2 study.
Mean and P95 estimates of dietary cadmium exposure were 0.31 and 0.52 µg/
15
kg bw per day, respectively (9.3 and 15.6 µg/kg bw per month, respectively). The
main foods contributing to cadmium exposure were potatoes and similar (14%),
bread and dry bread (10%), other vegetables (8%) and pasta (8%). Chocolate
contributed 2% of dietary cadmium exposure.
A further study was carried out to assess dietary exposure for French
children <3 years of age (Jean et al., 2018). Food samples (n = 291), including
infant foods (n = 219) common foods and bottled water (n = 72) were collected
during 2011 and 2012 in central France. Samples were analysed for cadmium
by ICP-MS, following acid digestion. LOD and LOQ were 0.3 and 0.5 µg/kg,
respectively. Cadmium was detected in 65% of samples. LB–UB estimates of
the mean concentration were calculated for each food type. Food consumption
information was taken from three consecutive 1-day food diaries completed
for 705 children, collected by the Syndicat Français des Aliments de l’Enfance
et de la Nutrition Clinique. Dietary cadmium exposure was estimated for four
age subgroups: 1–4 months, 5–6 months, 7–12 months and 13–36 months. LB–
UB estimates of mean (90th percentile) dietary cadmium exposure ranged from
0.39 to 0.67 (1.4 to 1.4) µg/kg bw per week for 1–4-month-old infants to 2.3–2.4
(3.8–4.0) µg/kg bw per week. The dietary exposure estimates were converted to
monthly estimates by multiplying by 30/7 (Table 1). Infant formula was the main
contributor to dietary exposure for the 1–4-month cohort (58–72%), whereas
for the 13–36-month cohort potatoes and potato products (23–24%), vegetables
(17–18%) and pasta (10%) were the main contributors. Cocoa products were not
among the foods considered.
(l) French Polynesia

As part of a case–control study to examine associations between thyroid cancer
and exposure to heavy metals, dietary exposure to several heavy metals, including
cadmium, was estimated (Zidane et al., 2019). Locally produced foods were
sampled at seven locations in French Polynesia between 2011 and 2013, with five
individual subsamples composited to give 124 food samples for analysis. Samples
were lyophilized and analysed for cadmium by ICP-MS, following digestion.
Measures of method performance characteristics and the technique for handling
analytical results below the LOD were not reported. Food consumption was
determined for 229 cases and 373 controls by application of an adapted version
of the European Prospective Investigation into Cancer and Nutrition (EPIC)
questionnaire, which collected information on frequency of consumption of 66
foods in the previous year as well as on serving sizes. The age distribution of cases
and controls was not reported, but it is likely that they were adults. Mean dietary
cadmium exposure did not differ between cases and controls and was estimated
to be 0.07 µg/kg bw per day (2.1 µg/kg bw per month). The maximum estimated
16
dietary exposure to cadmium was 1.6 µg/kg bw per day (48 µg/kg bw per month).
No information on the contribution of foods, including cocoa products, to dietary
exposure was reported.
(m) Germany
As part of the LExUKon project, dietary cadmium exposure was estimated for
the adult German population (aged 14–80 years) (Schwarz et al., 2014). Data
on cadmium in foods were taken mainly from the German Food Monitoring
Programme (GFMP) for the years 1993–2007 supplemented by data from
targeted surveys carried out after 2003. MB estimates of mean concentrations
were calculated. Food consumption information was derived from the German
National Nutrition Survey II (NVS II). The survey included three different
methods: a dietary history interview, two 24HDR questionnaires and two 4-day
food diaries. The mean estimate of dietary cadmium exposure for the full cohort
(n = 15 371) was 1.46 µg/kg bw per week (6.3 µg/kg bw per month). A “high-end”
estimate of dietary exposure was calculated using P95 consumption amounts
for two main food groups and mean consumption amounts for the remaining
food groups. The high-end estimate of dietary cadmium exposure for the general
population was 2.35 µg/kg bw per week (10.1 µg/kg bw per month). The main
contributors to dietary exposure were vegetables and cereals. No information on
the contribution from cocoa products was reported.
Data from the same sources were used to specifically examine dietary
cadmium exposure from consumption of cocoa products (Fechner et al., 2019).
Data from the NVS II were re-examined to extract information on cocoa
consumption. Mean and P95 consumption estimates for all cocoa were 0.028 and
0.098 g/kg bw per day, respectively. Results from analyses of samples of cocoa
powder were available from the GFMP for the years 2008–2012. Combining the
mean concentration of cadmium in cocoa products with the median and P95
estimates of cocoa consumption gave estimates of dietary exposure of 0.019 and
0.131 µg/kg bw per week (0.081 and 0.56 µg/kg bw per month, respectively).
When considered in association with the estimates from Schwarz et al. (2014),
these findings are consistent with other studies in suggesting that, at mean or
median estimates of dietary exposure, cocoa products account for about 1% of
dietary cadmium exposure. Other scenarios were considered (P95 concentration,
cocoa from particular locations), but they do not appear to be relevant to the
current assessment.
(n) Hong Kong Special Administrative Region (SAR), China

Dietary exposure to cadmium was estimated as part of the first Hong Kong SAR
TDS (Chen et al., 2014). A total of 1800 food samples were collected during 2010
17
and 2011 and combined into 600 composite samples of 150 food types, prepared
as consumed. Samples were analysed for cadmium by ICP-MS, following acid
digestion. LODs were 0.4 and 2 µg/kg for water and tea and general foods,
respectively. The corresponding LOQs were 2 and 10 µg/kg. Overall, 58% of
analytical results were above the LOD and an MB approach to mean calculation
was adopted. Information on food consumption was derived from the first Hong
Kong Population-based Food Consumption Survey. The survey included two
24HDR and an FFQ administered to approximately 5000 adults (aged 20–84
years). Mean and P95 estimates of dietary cadmium exposure for the whole adult
cohort were 8.3 and 19 µg/kg bw per month, respectively. The main contributors
to dietary exposure were vegetables and their products (36%), fish and seafood
and their products (26%), and cereals and their products (21%). No information
on the contribution of cocoa products was reported.
(o) Iran, Islamic Republic of

Although this study in the Islamic Republic of Iran did not look at all dietary
sources of cadmium, the likely main contributors to dietary exposure were
included (cereals and vegetables) (Heshmati et al., 2020). Samples (n = 50 per
food) of potatoes, onions, tomatoes, lettuces, leeks, carrots, wheat and rice were
collected from retail outlets in Hamadan Province, western Islamic Republic of
Iran. Samples were analysed for cadmium by GF-AAS, following acid digestion.
LOD and LOQ were 0.22 and 0.68 µg/kg, respectively. It was not stated how results
below the LOD were treated in the calculation of mean cadmium concentrations
for each food. Mean daily consumption for each food was reported, but the
method for deriving these estimates was not. A default body weight of 70 kg was
used. Mean estimated daily intakes were reported for each food type. The sum
exposure to cadmium across food types was 0.49 µg/kg bw per day (14.7 µg/
kg bw per month). The main contributors to dietary exposure were the cereals:
wheat and rice. Cocoa products were not included in this study.
(p) Ireland
A TDS was carried out by the Food Safety Authority of Ireland (FSAI) during
2012–2014 (FSAI, 2016). Samples of 141 food types from 27 food categories were
collected. Foods were prepared for consumption and were analysed for cadmium
by ICP-MS, with typical LOD and LOQ of 5 and 17 µg/kg, respectively. The
food consumption data used for adults were derived from the National Adult
Nutrition Survey, which investigated habitual food and beverage consumption
in a representative sample (n = 1500) of adults aged 18 years and over during
2008–2010. Assessment of children used the National Children’s Food Survey,
which investigated habitual food and drink consumption in 594 children aged
18
5–12 years, during 2003–2004, using a 4-day food diary. LB–UB mean (97.5th
percentile) estimates of dietary cadmium exposure were derived separately for
children and adults; 7.2–9.6 (14.1–17.7) µg/kg bw per month and 4.8–6.6 (9.9–
12.6) µg/kg bw per month, respectively. Cereals and vegetables were the main
contributors to dietary exposure for both adults and children. No information on
the contribution of cocoa products was reported.
(q) Italy
A subnational estimate of dietary cadmium exposure was determined in the
Emilia-Romagna region of northern Italy (Filippini et al., 2018). Foods for
inclusion in the study were selected on the basis of previous studies. Foods
(n = 890) were collected during 2016–2017 and analysed for cadmium by ICP-
MS, following nitric acid/hydrogen peroxide digestion. Very low LOD and
LOQ of 0.007 and 0.02 µg/kg, respectively, were reported. Overall, 25 samples
(3%) contained concentrations of cadmium below the LOD. Food consumption
information was derived by administering a semi-quantitative FFQ of 188 food
items, developed for the European Prospective Investigation into Cancer and
Nutrition (EPIC) study, to 719 adults (aged 18–87 years). The median estimate of
dietary cadmium exposure was 0.5 µg/kg bw per week (2.1 µg/kg bw per month).
The main contributors to dietary exposure were cereals (55%) and vegetables
(19%). No information on the contribution of cocoa products was reported.
(r) Japan
Dietary exposure to cadmium was estimated for a cohort (n = 296) of Japanese
children (aged 3–6 years) from Miyagi prefecture during 2001–2004 (Watanabe
et al., 2013). Twenty-four-hour duplicate diet portions were collected and
analysed by ICP-MS. A limit of determination of 0.1 µg/kg was reported, but it
is uncertain whether this was an LOD or an LOQ. Age, height and weight were
recorded for each child. The geometric mean dietary cadmium exposure was
11.8 µg/day (0.60 µg/kg bw per day or 18 µg/kg bw per month). No discernible
pattern was apparent when dietary exposure was considered by individual age (3,
4, 5 or 6 years) and sex. The duplicate diet approach does not allow information
on the contribution of specific food types to dietary exposure to be determined.
(s) Korea, Republic of

Concentrations of cadmium in 118 foods were obtained from the database
of the Korean Research Project on the Integrated Exposure Assessment of
Hazardous Substances for Food Safety (Kim et al., 2014). In total, 3823 analytical
results, representing the 118 core foods, were considered suitable for inclusion
in the study. The results in the database for cadmium resulted from ICP-MS
19
analysis of food collected from seven cities in the Republic of Korea. Analytical
results below the LOD (0.2 µg/kg) were substituted by a value equal to LOD/2
(MB). Consumption data were obtained from two non-consecutive 24HDR
questionnaires administered to 457 child–caregiver pairs of children aged 0–6
years. Body weight was also recorded. The mean body weight for the participating
children was 16.2 kg. Individual estimates of dietary exposure were calculated for
each of the two 24HDR for each participant. The distribution of habitual dietary
exposures was then derived using the Iowa State University method, through
C-SIDE software. The mean and P95 estimates of dietary cadmium exposure for
the cohort were 0.38 and 0.75 µg/kg bw per day, respectively (11.4 and 22.5 µg/kg
bw per month, respectively). The main contributors to dietary cadmium exposure
were cereals, fish, shellfish and seaweed. No information on the contribution of
cocoa products was reported.
(t) Mali
As part of a multicountry TDS in sub-Saharan Africa, foods were collected
at two locations in Mali (Bamako and Sikasso) and combined to give 50 food
group composites, representing 13 food groups (Ingenbleek, 2019). Samples
were analysed for a range of contaminant elements, including cadmium, by
ICP-MS, following nitric acid digestion. LOQs were in the range of 0.2–1.0 µg/
households across the two sampling locations in Mali. Household food intake
was converted to intake per AME. LB–UB mean (P95) estimates of dietary
cadmium exposure for Bamako and Sikasso were 0.07–0.07 (0.10–0.11) and
0.02–0.02 (0.04–0.04) µg/kg bw per day, respectively. These exposure estimates
correspond to 2.1–2.1 (3.0–3.3) µg/kg bw per month and 0.6–0.6 (1.2–1.2) µg/
kg bw per month, respectively. The main contributors to dietary exposure were
rice, peanuts and millet. No information on the contribution of cocoa products

was reported.
(u) The Netherlands

Dietary exposure to cadmium was estimated for children of 2–6 years (n = 1279)
and people aged 7–69 years (n = 3819) (Sprong & Boon, 2015). Concentrations
of cadmium in food and drinking water were obtained from Dutch monitoring
programmes. Concentrations in cocoa beans were not available from these
programmes and were derived from a study performed by EFSA in 2012 (EFSA,
2012). Information on food consumption was obtained on two non-consecutive
days using a food diary or 24HDR, as part of the Dutch National Food
20
Consumption Survey. Based on LB, MB and UB mean concentrations, median

and P95 dietary exposure estimates were calculated using a statistical model that
corrects for inter-individual variability. The MB estimates ranged from 0.40–
0.55 µg/kg bw per day (12–16.5 µg/kg bw per month) for children aged 2–6 years
and from 0.18–0.47 µg/kg bw per day (5.4–14.1 µg/kg bw per month) for people
aged 7–69 years. Corresponding ranges for the P95 dietary exposure were 0.58–
0.81 µg/kg bw per day (17.4–24.3 µg/kg bw per month) and 0.30–0.77 µg/kg bw
per day (9.0–23.1 µg/kg bw per month), respectively. Overall, mean MB dietary
exposure across all ages was 0.25 µg/kg bw per day (7.5 µg/kg bw per month).
Cereals (38–40%), potatoes (16–18%) and vegetables (11–13%) contributed most
to the exposure in both populations. Cocoa beans contributed a maximum of 1%
to the total dietary exposure distribution.
(v) New Zealand

The eighth New Zealand TDS sampled 132 different food types on two occasions
during 2016 (Pearson et al., 2018). Eight composite samples of each food type
were analysed after preparation for consumption. Samples were analysed for
cadmium by ICP-MS following acid digestion. Limits of reporting (LORs)
ranged from 0.05 µg/kg for drinking water to 2 µg/kg for high-fat samples.
Cadmium was detected at concentrations above the LOR in 68% of samples.
Food consumption information was derived from simulated typical diets for 10
population subgroups. Mean LB–UB estimates of dietary cadmium exposure
ranged from 4.4–4.6 µg/kg bw per month for adult females of Pacific Island
ethnicity to 12.4–12.8 µg/kg bw per month for toddlers (aged 1–3 years). The
main contributors to dietary exposure were vegetables, grains and "additional
meat and shellfish". The contribution from the latter food category is a reflection
of the high concentrations of cadmium in bivalve molluscs (oysters and mussels).
Although the contribution to dietary exposure from cocoa products was not
explicitly reported, it can be calculated from available data and was in the range
of 1% (adult males and females) to 8% (children).
(w) Nigeria
As part of a multicountry TDS in sub-Saharan Africa, foods were collected at
two locations in Nigeria (Lagos and Kano) and combined to give 54 food group
composites, representing 13 food groups (Ingenbleek, 2019). Samples were
analysed for a range of contaminant elements, including cadmium, by ICP-
MS, following nitric acid digestion. LOQs were in the range of 0.2–1.0 µg/kg.
Cadmium was quantified in 61% of composite samples. LB–UB estimates of
21
households across the two sampling locations. Household food intake was
converted to intake per AME. LB–UB mean (P95) estimates of dietary cadmium
exposure for Lagos and Kano were 0.09–0.09 (0.18–0.18) and 0.04–0.05 (0.11–
0.12) µg/kg bw per day, respectively. These exposure estimates correspond to
2.7–2.7 (5.4–5.4) µg/kg bw per month and 1.2–1.5 (3.3–3.6) µg/kg bw per month,
respectively. The main contributors to dietary exposure were rice, beef, and wheat
and bread. No information on the contribution of cocoa products was reported.
(x) Poland
A duplicate daily diet study was carried out for a cohort of 850 university students,
recruited over the period 2006–2010 from the south-east of Poland (Marzec et
al., 2014). This study appears to be a variant on the more usual duplicate diet
approach. Students initially completed a 24HDR questionnaire. Representative
duplicate diets were then assembled from locally obtained foods. Samples were
analysed for cadmium by flame AAS, following ashing and acid dissolution. The
LOD and LOQ were 0.2 and 0.5 µg/kg, respectively. Separate estimates of dietary
exposure were derived per year (2006, 2007, 2008 and 2010), sex (male and
female) and for each of three universities. Estimates of mean dietary exposure
were in the range of 76 (females, Catholic University, 2010) to 330 (males, Medical
University, 2008) µg/week. Based on a conservative mean body weight of 60 kg,
these values equate to 5.4 and 24 µg/kg bw per month.
A similar study was conducted in 2011–2013, with an initial 24HDR (583
participants, aged 19–30 years) used to formulate six “market baskets” for each
combination of year (2011, 2012 and 2013) and sex (Koch et al., 2016). Foods
were prepared for consumption. Analyses for cadmium were performed by ICP-
MS, following nitric acid digestion. The LOD for cadmium was 0.12 µg/kg. Mean
estimates of dietary cadmium exposure across the year–sex cohorts were in the
range of 12.7–21.6 µg/day. Based on a default body weight of 60 kg, this range
equates to 6.4–10.8 µg/kg bw per month. No assessment of contributing foods
was conducted.
Both Polish studies were duplicate diets, an approach that does not allow
derivation of information on the contribution of specific food types to dietary
exposure.
(y) Serbia
Food samples (n = 114) were collected in January 2012 and March 2013 from
supermarkets in Novi Sad, Serbia (Skrbic et al., 2013). Food types were selected
on the basis of a total Serbian market basket prepared by the Statistical Office of
the Republic of Serbia. The market basket was also the source of the consumption
information used in the dietary exposure assessment. Samples were analysed for
22
cadmium by GF-AAS, following nitric acid/hydrogen peroxide digestion. The

LOD and LOQ were reported to be the same; 0.3 µg/kg. However, this is almost
certainly a typographical error, and this is likely to be the LOD. Results below the
LOD were substituted by a value equal to LOD/2 (MB). The estimated dietary
exposure to cadmium from adult consumption of the Serbian market basket was
11.5 µg/day (5.8 µg/kg bw per month, for a default body weight of 60 kg). The
main contributors to dietary exposure were white bread (54%), sugar (16%) and
potatoes (9%). Chocolate contributed 0.7% of dietary exposure to cadmium.
(z) Spain
Dietary exposure to cadmium was estimated for the region of Valencia in Spain
(Marín et al., 2017). In 2010–2011, a TDS was performed including collection
of 8100 food samples. Food samples were aggregated in groups of 10 to give 810
samples for analysis for cadmium by ICP-MS. LOQs were in the range 4–10 µg/
kg. Food consumption data were obtained from 1478 subjects (195 children aged
6–15 years and 1281 adults aged 16–95 years) using a 24HDR on three non-
consecutive days. LB and UB estimates of exposure to cadmium were calculated
for children and adults. Mean LB–UB exposure estimates for children were 1.26–
2.89 µg/kg bw per week (5.4–12.4 µg/kg bw per month) and for adults 0.77–
1.78 µg/kg bw per week (3.3–7.6 µg/kg bw per month). For high consumers
(99th percentile), the corresponding estimates were 5.74–9.10 and 4.62–
6.31 µg/kg bw per week (24.6–39.0 and 19.8–27.0 µg/kg bw per month). Cereals
(38%) contributed most to the exposure to cadmium, followed by fish (29%). No
information on the contribution of cocoa products was reported.
A further Spanish dietary exposure assessment for cadmium was
conducted in the region of Catalonia (Gonzalez et al., 2019). Foods were sampled
in 12 cities in Catalonia. For each food type (approximately 70 food types
aggregated into 15 food groups), three composite samples were prepared from
20 individual samples. Composite samples were analysed for cadmium by ICP-
MS, following nitric acid/hydrogen peroxide digestion. The LOD was reported
as 2 µg/kg, although the method of determining the LOD was not reported.
For calculation of mean concentrations, analytical results below the LOD were
substituted by a value of LOD/2 (MB). Food consumption information was
derived from national dietary surveys (ENALIA and ENALIA-2), which included
1862 children and adolescents, 623 adults, 310 “seniors” and 157 pregnant
women. The surveys included two non-consecutive 24HDRs and an FFQ. The
survey cohort was reclassified into eight age-based subgroups. It should be noted
that the terms “toddlers” and “infants” appear to have been transposed in this
publication, as toddlers are referred to as being aged 6–11 months and infants as
12–36 months. The following summary uses the term infants for 6–11-month-
23
olds and toddlers for 12–36-month-olds. Mean body weights for each subgroup
were derived from the literature. Mean estimates of dietary exposure were only
presented graphically but ranged from (approximately) 3 µg/day (infants) to
10.1 µg/day (adolescents, aged 10–17 years). These dietary exposure estimates
equate to 10.7 (infants) and 5.9 (adolescents) µg/kg bw per month, based on the
body weights given in the publication. For the adult population, with a dietary
exposure of 6.1 µg/day or 2.4 µg/kg bw per month, the main contributors to
dietary exposure were fish and seafood (37%), bread and cereals (30%), and
potatoes (14%). Chocolate contributed 1% of adult dietary exposure, despite
being the food type with the second-highest mean cadmium concentration.
(aa) Sri Lanka

Dietary exposure to cadmium (and lead and arsenic) was estimated for a cohort
of people with chronic kidney disease (CKD) from a region in central northern
Sri Lanka (Jayalal et al., 2019). Food samples (n = 277) were collected from 70
households in the region associated with CKD cases. Food samples were analysed
for cadmium by ICP-MS, following acid digestion. The LOQ was reported as
0.3 µg/L; however, it is uncertain what this refers to as the LOQ was reported
on a volume basis, and only 9% of samples were reported to contain cadmium
concentrations above the LOQ. The minimum reported cadmium concentration
was 31 µg/kg, suggesting a LOQ of about 30 µg/kg, which is quite high. Samples
with cadmium concentrations below the LOQ were substituted by a value of
LOQ/2 for calculation of means (MB). Food consumption information was elicited
from 87 CKD patients using a semi-quantitative FFQ. However, it appears that
this information was not used to estimate dietary exposure and instead a model
diet was used, based on the consumption of 3 kg of rice and 1 kg of vegetables per
week. A default body weight of 60 kg was used. Mean dietary cadmium exposure
was estimated to be 83.7 µg/week (6.0 µg/kg bw per month). No information on
the contributions of specific foods to dietary exposure was reported.
(bb) Sweden
Data on occurrence of cadmium in foods were taken from Swedish national
monitoring programmes or Swedish market basket surveys performed during
1999–2008 (Sand & Becker, 2012). Concentrations below the LOD were substituted
by a value of LOD/2 for calculation of food category mean concentrations. Food
consumption information was taken from a 1997–1998 survey carried out by
the National Food Agency in collaboration with the Swedish Statistical Agency
(Riksmaten 97-98). The survey involved the collection of 7-day food diaries for
1211 Swedish adults (aged 17–80 years). Median and P95 estimates of dietary
cadmium exposure were 0.97 and 1.7 µg/kg bw per week, respectively (4.2 and
24
7.3 µg/kg bw per month, respectively). The main contributors to median dietary
exposure were potatoes (25%) and wheat flour (24%). No information on the
contribution of cocoa products was reported.
(cc) Thailand
A study was undertaken in four locations in Thailand during the period 2011–
2013 (Kluengklangdon et al., 2017). One hundred households were recruited in
each location (total 400 households, representing 1241 people) and duplicate
portions of meals collected on each of 4 days. A sample of drinking water was
also collected from each household. The food and water samples were analysed
for cadmium by GF-AAS or ICP-optical emission spectrometry (ICP-OES). MB
estimate of dietary cadmium exposure across the study population was 7.3 µg/
kg bw per month. The duplicate diet approach does not allow information on the
contribution of specific food types to dietary exposure to be determined.
(dd) United Kingdom

Dietary exposure to cadmium in the United Kingdom was estimated based on
the 2014 TDS (FSA, 2019). In this study, foods belonging to 28 food groups were
sampled and samples were analysed for cadmium by ICP-MS. Food consumption
data were obtained from the National Diet and Nutrition Survey Rolling
Programme (NDNSRP) in which data are collected using a 4-day food diary. No
information on the treatment of samples with non-detect results was reported.
The mean and P97.5 of exposure were estimated for children aged 1.5 to 3 years as
being the age group with the highest exposure. The mean and P97.5 of exposure
were 2.24–3.78 μg/kg bw per week and 3.78–5.95 μg/kg bw per week, respectively
(9.6–16.2 and 16.2–25.5 µg/kg bw per month, respectively). The “miscellaneous
cereals” food group made the highest contribution to total mean exposure and
was followed by bread and potatoes. No information on the contribution of cocoa
products to cadmium exposure was reported.
(ee) United States of America

Dietary exposure to cadmium of children 1–6 years of age was estimated based
on cadmium concentrations from the TDS of the Food and Drug Administration
(FDA) and on food consumption data from What We Eat In America, the
food survey portion of the National Health and Nutrition Examination Study
(NHANES) (Spungen, 2019). Food samples (n = 2923) representing 268 food types
were collected during 2014–2016, representing key food groups in the US diet.
Foods were analysed for cadmium by ICP-MS, following microwave digestion.
LODs and LOQs were in the range of 0.04–1.2 and 0.3–11 µg/kg, respectively.
Cadmium was detected in 65% of food samples analysed. Food consumption
25
data were obtained from a 24HDR administered on two non-consecutive days.

Mean and upper level (90th percentile) 2-day average exposures were estimated
for the total age group (n = 3103) and for two age subgroups of 1–3 years (n =
1717) and 4–6 years (n = 1386). LB, UB and hybrid estimates of dietary exposure
were derived. The hybrid approach assigned a value of zero to analytical results
below the LOD if no cadmium had been detected in that food type during 2009–
2016, whereas a value of LOD/2 was assigned otherwise. Hybrid estimates of
mean dietary exposure were 0.41, 0.43 and 0.38 µg/kg bw per day (12.3, 12.9 and
11.4 μg/kg bw per month) for 1–6-year-olds, 1–3-year-olds and 4–6-year-olds,
respectively. Corresponding 90th percentile hybrid estimates were 0.66, 0.70 and
0.59 µg/kg bw per day (19.8, 21.0 and 17.7 μg/kg bw per month) for the same
three age groups. The main contributors to the mean dietary exposure were grains
(31.8%), mixtures (29.1%; e.g. hamburgers, pizza, lasagna, soups) and vegetables
(21.8%). No information on the contribution of cocoa products was reported.
Dietary exposure was also estimated for individuals aged 2 years and
older (n = 12 523) from the NHANES 2007–2012 (Kim et al., 2019). Cadmium
concentrations were obtained from the TDS from 2006–2013, which included
260 individual foods. The TDS determined cadmium by GF-AAS, at the time
these analyses were carried out, although this was not reported in the publication.
LODs and LOQs were not reported. Food consumption data were collected
with 24HDR during two non-consecutive days. No information on treatment of
samples with non-detect results was reported. The weekly dietary exposure to
cadmium was estimated for six age groups and ranged from 0.43 µg/kg bw per
week (1.8 μg/kg bw per month) for 70+ year-olds to 0.94 µg/kg bw per week
(4.0 μg/kg bw per month) for 2–10-year-olds. Mean weekly exposure for the
whole population was 0.54 µg/kg bw per week (2.3 μg/kg bw per month). The
main contributors to cadmium exposure were cereals and bread (34%), leafy
vegetables (20%) and potatoes (11%). No information on the contribution of
cocoa products was reported.
(ff) Viet Nam

An average diet for residents of Hanoi was determined from previous food
consumption surveys, with foods aggregated into 22 food groups (Marcussen et
al., 2013). Samples of foods from each food group (n = 14 per food group) were
collected during 2007–2009. The edible portion of each food sample was analysed
for cadmium by ICP-MS, following nitric acid/hydrogen peroxide digestion. The
LOD was 16 µg/kg. LB–UB estimates of mean cadmium concentration were
calculated for each food group. Average diets were determined from several
surveys, including a three seasonal period 24HDR household survey (250
households). LB–UB estimates of mean dietary cadmium exposure for the Hanoi
26
population were 0.68–0.70 µg/kg bw per day (20.4–21.0 µg/kg bw per month),
with 90% of exposure due to consumption of rice. No information was provided
on cadmium exposure due to the consumption of cocoa products.
(gg) Summary
The national estimates of dietary exposure to cadmium described above are
summarized in Table 1. The mean dietary exposure to cadmium from the total diet
at a national level ranged from 0.6 µg/kg bw per month for adults in the Sikasso
region of Mali (2.4% of the PTMI) up to 24 µg/kg bw per month in children aged
4–11 years in China (96% of the PTMI). The maximum reported high percentile
estimate of dietary cadmium exposure was 66 µg/kg bw per month in boys aged
8 years from Australia (260% of the PTMI). However, this estimate was based
on a 1-day 24HDR, which may have inflated the high percentile estimate. The
highest high percentile estimate of dietary cadmium exposure based on multiple-
day dietary records was for children aged 4–11 years in China (48.2 µg/kg bw per
month; 190% of the PTMI). High percentile estimates of adult dietary cadmium
exposure were only occasionally above the PTMI and were typically 20–60% of
the PTMI. The main sources of cadmium exposure were grain and grain-based
products, vegetables, and fish and seafood.
Where included in the exposure assessment, the contribution of cocoa
products to the total mean dietary exposure to cadmium ranged from 0.2 to 9%.
Owing to differences in methodologies used to assess the exposure (e.g. typical
diets, FFQ, 24HDR), comparisons between studies should made with caution.
2.4 International estimates of chronic dietary exposure

The GEMS/Food cluster diets that were used for international estimates of chronic
dietary exposure to cadmium are based on food availability, rather than actual
food consumption. Consequently, these estimates of dietary exposure will be less
“refined” than the estimates of dietary cadmium exposure from the literature.
Given the large number of national estimates of dietary cadmium exposure
available from the literature, their coverage of countries across the world, and
their consistency, together with the limitations of the GEMS/Food contaminants
database with respect to detailed data on cadmium concentration from many
regions, the Committee considered that deriving less refined estimates of dietary
exposure was not necessary, and therefore the international estimates are not
reported. However, these estimates were used to provide a suitably consistent
basis to examine the contribution of cocoa products and other food sources to
dietary exposure to cadmium.
27
28
Table 1
Summary of national estimates of dietary exposure to cadmium from the literature
Estimated dietary Contribution from
exposure, mean (high cocoa products
Food concentration Population groups consumer)a in µg/kg (% of dietary
Country data used Consumption data used (age) bw per monthb The main contributors exposure) Reference
Australia Median of composites Individual (2 × 24HDR) Infant (9 months) 2.8–16 (5.7–33)c Cereals and cereal 55 FSANZ, 2019
2–5 years 5.5–14 (9.9–20) products, root vegetables 6
6–12 years 4.3–9.5 (8.2–15) 6
13–18 years 2.9–6.3 (5.6–9.6) 4
19+ years 2.0–5.8 (3.7–8.8) 4
2+ years 2.5–6.6 (4.8–11)
Australia Survey mean 24HDR Males Fish and seafood – Callan et al., 2014
8 years 12 (66) products, cereals,
12 years 9 (42) vegetable products and
13 years 6 (39) dishes
Safety evaluation of certain contaminants in food
16 years 6 (30)
Females
8 years 12 (60)
12 years 9 (39)
13 years 6 (36)
16 years 6 (30)
Ninety-first JECFA
Bangladesh Survey mean Literature values General population 17.3 Steamed rice, green – Al-Rmalli et al., 2012
vegetables
Benin LB–UB survey mean Household budget Adult male equivalents Tomatoes, rice, pasta, – Ingenbleek, 2019
Littoral 1.5–1.5 (3.0–3.3) yams, smoked fish
Borgou 1.2–1.5 (2.7–3.0)
Brazil Duplicate diet Portion size of duplicate diet Children (1–4 years) – – Leroux et al., 2018
Males 2.4
Females 2.7
Cameroon Mean of composites Household expenditure Adult equivalents 4.7 (8.2)d Cereals and cereal 1 Gimou et al., 2014
products
Cameroon LB–UB survey mean Household budget Adult male equivalents Rice, green leaves, wheat/ _ Ingenbleek, 2019
Duala 2.4–2.4 (5.1–5.1) bread, peanuts, maize
North 1.5–1.5 (3.0–3.0)
Canada (First Nations) UB mean of composites 24HDR Adults (19+ years) 3.9 (9.6) Root vegetables, cereals – Juric, 2016
and cereal products
Chile Survey mean 24HDR Adults (18–65 years) 7.8 Bread, non-alcoholic – Munoz et al., 2017
beverages
China (Shanghai) Mean from previous FFQ + portion sizes Adults (>40 years) 0.4 (10.3)d Vegetables, rice – He et al., 2013
survey
China Survey means for 32 food 3 × 24HDR 4–11 years 24.2 (48.2) Rice, wheat flour, leafy – Song et al., 2017
categories 12–17 years, male 17.9 (35.7) vegetables
12–17 years, female 15.7 (31.5)
18+ years, male 13.8 (27.4)
18+ years, female 13.6 (27.8)
General population 15.3 (33.0)
China Food group medians 3-day food diary 18–39 years male 9.9 (14) Vegetables, rice and rice – Wang et al., 2018
18–39 years female 11 (13) products, fish, seafood
40–59 years male 9.9 (12) and shellfish
40–59 years female 9.4 (12)
60+ years male 8.3 (12)
60+ years female 8.3 (13)
China Survey mean – General population 15.6i Rice, vegetables, aquatic – Wei et al., 2019; Xiao
foods and potatoes et al., 2020
China (Guangzhou Survey mean 3-day 24HDR 3–88 years 14.4 (41) Cereals, vegetables and – Zhang et al., 2018
City) laver
Denmark Survey mean 7-day food diary General population 5.4 (11.4) Cereals and cereal – DTU Food, 2015
(4–75 years) products, vegetables and
vegetable products
29
30
Table 1 (continued)
Europe MB mean of monitoring Individual (2–7 days; dietary/ Infants (0–1 years) 11.5 (28) Grains and grain-based 0.2–9.4 EFSA, 2012
data food record; 24HDR) Toddlers (1–2 years) 20.6 (28) products, vegetables
Other children (3–9 years) 16.8 (27) and vegetable products,
Adolescents (10–17 years) 9.9 (18) starchy roots and tubers
Adults (18–64 years) 7.6 (13)
Elderly (65–74 years) 6.9 (12)
Very elderly (≥ 75 years) 7.2 (12)
France Survey MB mean 7-day food diary Children (3–17 years) 7.2 (13.5) Crustaceans and molluscs 2 Arnich et al., 2012
Adults (18–79 years) 4.8 (8.1) (adults only) 1
France Survey MB mean 7-day food diary Children (3–6 years) 9.3 (15.6) Potatoes and potato 2 Glorennec et al.,
products, bread and dried 2016
bread products, other

vegetables, pasta,
France Survey LB–UB mean 3-day food diary 1–4 months 1.7–2.9 (5.9–5.9)c Infant formula, – Jean et al., 2018
5–6 months 6.8–7.6 (12.5–13.4) vegetables and
7–12 months 9.7–10.4 (16.2–17.3) vegetable-based meals,
13–36 months 8.7–9.3 (14.7–15.5) potatoes and potato
Ninety-first JECFA
products, meat/fish-
based meals
French Polynesia Survey, summary statistic FFQ Adult 2.1 – – Zidane et al., 2019
not reported
Germany MB mean of monitoring Various methods General population 6.3 (10.1)e Vegetables, cereals – Schwarz et al., 2014
data (14–80 years)
Germany MB mean of monitoring Various methods General population 0.081 (0.56)f Only cocoa products – Fechner et al., 2019
data (14–80 years) included
Hong Kong SAR, China MB mean of food type 2 × 24HDR Males Vegetables and their – Centre for Food
composites 20–29 years 7.5 (18) products, fish, seafood Safety, 2013
30–39 years 8.6 (25) and their products,
40–49 years 8.0 (18) cereals and their products
50–59 years 8.0 (20)
60–69 years 8.0 (17)
70–84 years 6.8 (16)
All males 7.9 (19)
Females
20–29 years 8.9 (21)
30–39 years 9.4 (21)
40–49 years 9.3 (20)
50–59 years 8.2 (16)
60–69 years 7.6 (18)
70–84 years 7.1 (15)
All females 8.7 (19)
All combined 8.3 (19)
Iran, Islamic Republic Survey mean Not stated Adult 14.7 Wheat, rice – Heshmati et al., 2020
of
Ireland, Republic of Survey mean 4-day food diary Children (5–12 years) 7.2–9.6 (14.1–17.7)g Cereals, vegetables – FSAI, 2016
Adults (18+ years) 4.8–6.6 (9.9–12.6)
Italy (northern) Survey median FFQ Adults (18–87 years) 2.1 Cereals, vegetables – Filippini et al., 2018
Japan Duplicate diet Duplicate diet Children (3–6 years) 18 – – Watanabe et al.,
2013
Korea, Republic of Database mean 2 × 24HDR Children (0–6 years) 11.4 (22.5) Cereals, fish and shellfish, – Kim et al., 2014
seaweeds
Mali LB–UB survey mean Household budget Adult male equivalents Rice, millet, peanuts _ Ingenbleek, 2019
Bamako 2.1–2.1 (3.0–3.3)
Sikasso 0.6–0.6 (1.2–1.2)
Netherlands MB mean of monitoring Individual (2 × food diary, 2 2–6 years 12–17 (17–24)f Cereals, potatoes, 1 Sprong & Boon, 2015
data × 24HDR) 7–69 years 5.4–14 (9.0–23) vegetables
New Zealand Survey mean Simulated typical diets Infant 9.5–12.6 Vegetables, cereals, 2 Pearson et al., 2018
Toddler 12.4–12.8 shellfish 4
Children 12.0–12.4 8
31
32
Table 1 (continued)

Teenage boy 7.7–7.9 6
Teenage girl 6.9–7.1 6
Young male 6.8–7.0 3
Adult male (PI) 5.8–6.0 2
Adult female (PI) 4.4–4.6 2
Adult male 6.5–6.7 1
Adult female 5.1–5.3 1
Nigeria LB–UB survey mean Household budget Adult male equivalents Beef, rice, wheat/bread _ Ingenbleek, 2019
Lagos 2.7–2.7 (5.4–5.4)
Kano 1.2–1.5 (3.3–3.6)
Poland Mean of “analogue” 24HDR University students 5.4–24h – – Marzec et al., 2014
duplicate diets
Poland Mean of “analogue” 24HDR University students 6.4–10.8h – – Koch et al., 2016
duplicate diets (19–30 years)
Serbia MB survey mean Market basket Adults 5.8 White bread, sugar, 0.6 Skrbic et al., 2013
potatoes
Ninety-first JECFA
Spain (Valencia) LB–UB survey 3 × 24HDR 6–15 years 5.4–12 (25–39) Cereals and fish – Marin et al., 2017
16–90 years 3.3–7.6 (20–27)
Spain (Catalonia) MB survey mean 2 × 24HDR and FFQ Infants (6–11 months) 10.7 Fish and seafood, bread 1 (adults) Gonzalez et al., 2019
Toddlers (12–36 months) 11.7 and cereals, potatoes
Children (3–9 years) 10.3 (adults)
Adolescents (10–17 years) 5.9
Young adults (18–39 3.0
years)
Adults (40–64 years) 2.4
Seniors (65–74 years) 2.8
Pregnant women 2.4
Sri Lanka MB survey mean Model diet Adult male 6.0 Rice – Jayalal et al., 2019
Sweden Monitoring data mean 7-day food diary Adults (17–80 years) 4.2 (7.3)f Potatoes, wheat flour – Sand & Becker, 2012
Thailand Duplicate diet Duplicate diet General population 7.3 – – Kleungklangdon et
al., 2017
United Kingdom Survey mean 4 × food diary 1.5–3 years 9.6–16 (16–26)g Cereals, bread, potatoes – FSA, 2019
4–10 years 8.3–12 (15–21)
11–18 years 4.5–6.6 (9.0–12)
19+ years 3.6–5.7 (6.3–10)
United States of Survey mean 2 × 24HDR 1–6 years 12 (20)c Grains, mixtures, – Spungen, 2019
America 1–3 years 13 (21) vegetables
4–6 years 11 (18)
United States of Survey mean 2 × 24HDR 2–10 years 4.0 Cereals and bread, leafy – Kim et al., 2018
America 11–19 years 2.1 vegetables and potatoes
20–30 years 2.1
31–50 years 2.1
51–70 years 1.9
70+ years 1.8
2–70+ years 2.3
Viet Nam LB–UB survey mean Average diet Residents of Hanoi 20.4–21.0 Rice – Marcussen et al.,
2013
24HDR, 24-hour dietary recall; PI, Pacific Island ethnicity; LB, lower bound; MB, middle bound; UB, upper bound.
a
The high consumer estimate is at the 95th percentile unless stated otherwise.
b
Dietary exposure estimates originally reported on a daily basis were converted to a monthly basis by multiplying by 30, estimates originally reported on a weekly basis were converted to a monthly basis by multiplying by 30/7.
c
The high consumer estimate is at the 90th percentile.
d
Upper bound estimates.
e
A “high-end” estimate of dietary exposure was derived based on 95th percentile food consumption for two main food groups and mean food consumption for the remaining food groups.
f
The measure of central tendency was the median (50th percentile).
g
The high consumer estimate is at the 97.5th percentile.
h
The study considered various combinations of sex, year and university. The range given is the range of dietary exposure estimates across the various scenarios.
i
Calculated based on a reported body weight of 63 kg (Xiao et al., 2020).
33
Owing to the widespread nature of cadmium contamination across the

food supply, it is important to include as many foods as possible in the dietary
exposure assessment. Due to the very limited cadmium data from some clusters
and the complete lack of data from others, international estimates of dietary
cadmium exposure were derived using global mean concentrations for all
food types. Cadmium exerts its adverse effects after a long period of exposure.
Therefore, mean concentrations are the most appropriate metric for calculating
dietary exposure, as fluctuations in concentrations are expected to level out to a
mean concentration over time.
Concentration data were either reported as a numerical value above the
LOQ, as an indicative value between the LOD and LOQ, or as undetectable. MB
concentrations were used in the assessments due to the relatively low proportion
of left-censored data (46%). Samples with concentrations reported as undetectable
were assumed to contain cadmium at a concentration of LOD/2. All reported
numerical values were used as such.
Global mean concentrations were derived by considering all available data
in the GEMS/Food contaminants database. This meant that, for many food types,
the mean cadmium concentrations were heavily weighted towards concentration
data provided by the WHO European Region. Global mean concentrations are
summarized in the Appendix.
Given the focus of the current assessment on cadmium in cocoa products,
an overview of these data as included in the dietary exposure assessment is
provided in Table 2. In total, 6957 records for cocoa products were available,
representing 2.5% of all records in the final data set. These records related to five
groups of cocoa products: cocoa beans (n = 108), cocoa beverage (n = 20), cocoa
butter (n = 20), cocoa mass (n = 218), cocoa powder (n = 2583) and chocolate (n
= 4008). As for the whole database, the main source of records for cocoa products
was the WHO European Region, accounting for 2293 records (33%).
Mean levels ranged from 5.4 µg/kg in cocoa butter reported in the WHO
European Region to 1601 µg/kg in cocoa powder reported in cluster G05 (Brazil,
Chile, Colombia, Ecuador and Peru) (Table 2). Across clusters and the two WHO
regions, concentrations ranged from 43 µg/kg in cocoa beverage to 971 µg/kg in
cocoa powder. Note that most of the concentration data from European countries
were identified at the level of the WHO European Region. The concentrations of
two clusters including these countries, namely G07 and G10, may therefore not
be representative for that cluster. The mean cadmium concentrations for cluster
G07 were solely based on data from Australia and for cluster G10 on data from
Canada, Japan and the USA. The mean concentrations of cluster G08 (Austria,
Germany, Poland and Spain) were based on concentrations from Germany and
Spain.
34
Table 2
Summary of the cadmium concentrations in cocoa products from the GEMS/Food contaminants database for the GEMS Food clusters and
two WHO regions
WHO
Cocoa WHO African European
products Statisticsa, b G02 G03 G04 G05 G06 G07 G08 G09 G10 Region Region Global
Cocoa Number of individual records – 31 – – 1 – 18 – – – 58 108
beans Perc < LOD (n) – 0% (0) – – 0% (0) – 0% (0) – – – 14% (8) 7.4% (8)
MB conc (µg/kg)
Mean – 113 – – 20 – 195 – – – 362 260
Median – 110 – – 20 – 80 – – – 166 118
P95 – 155 – – – – – – – – 1427 1151
Cocoa Number of individual records – – – – – 4 – 8 – 2 6 20
beverage Perc < LOD (n) – – – – – 25% (1) – 25% (2) – 0% (0) 33% (2) 25% (5)
MB conc (µg/kg)
Mean – – – – – 7.2 – 64 – 48 38 43
Median – – – – – 6.1 – 65 – 48 40 40
P95 – – – – – – – – – – – 111
Cocoa Number of individual records – – 3 – – 1 10 – – – 6 20
butter Perc < LOD (n) – – 0% (0) – – 0% (0) 0% (0) – – – 100% (6) 30% (6)
MB conc (µg/kg)
Mean – – 21 – – 10 77 – – – 5.4 44
Median 14 – – 10 20 – – – 5.0 20
P95 – – – – – – – – – 73
Cocoa Number of individual records – 27 – 141 11 8 7 3 – – 21 218
mass Perc < LOD (n) – 0% (0) – 0% (0) 0% (0) 0% (0) 0% (0) 0% (0) – – 4.8% (1) 0.5% (1)
MB conc (µg/kg)
Mean – 82 – 1035 350 726 375 373 – – 329 773
Median 73 – 730 297 885 350 330 – – 136 500
P95 187 – 2810 753 – – – – – 1000 2592
35
36
Table 2 (continued)
WHO
Cocoa WHO African European
products Statisticsa, b G02 G03 G04 G05 G06 G07 G08 G09 G10 Region Region Global
Cocoa Number of individual records – 74 – 1345 2 2 20 9 399 – 732 2583
powder Perc < LOD (n) – 0% (0) – 0% (0) 0% (0) 0% (0) 0% (0) 0% (0) 0% (0) – 3.4% (25) 1.0% (25)
MB conc (µg/kg)
Mean – 141 – 1601 471 117 650 609 391 – 228 971
Median – 140 – 1120 471 117 465 650 183 – 155 482
P95 – 191 – 5295 – – 1510 – 1258 – 754 3740
Chocolate Number of individual records 4 44 64 1086 74 294 – 10 962 – 1470 4008
Perc < LOD (n) 100% (4) 0% (0) 13% (8) 2.1% (23) 0% (0) 22% (66) – 10% (1) 0.8% (8) – 12% (170) 7.0% (280)
MB conc (µg/kg)
Mean 10 15 381 388 54 39 – 111 117 – 110 184
Median 10 13 21 259 37 19 – 85 76 – 52 84
P95 – 31 121 878 126 172 – 310 – 337 576
conc, concentration; LOD, limit of detection; MB, medium bound; median: 50th percentile; n, number; P95, 95th percentile; perc, percentage.
a
Concentrations below the LOD were assumed to contain cadmium at a level equal to LOD/2 (MB).
b
The 95th percentile concentrations are only reported where at least 20 records were available.
Ninety-first JECFA
Table 3
Contribution of cocoa products to dietary cadmium exposure
GEMS/Food Contribution from
Cluster Description of countries in cluster cocoa products (%)a
G01 Middle East, Central Asia, North Africa 0.6
G02 Balkans, Central Asia 1.5
G03 Africa, Paraguay 0.8
G04 Caribbean, Middle East, Brunei Darussalam, French Polynesia 2.7
G05 South and Central America, Indian and Indian Ocean island states, Djibouti, South Africa, Macedonia, 0.7
Malaysia, New Caledonia, Tajikistan, Trinidad and Tobago
G06 Middle East, Cuba, Egypt, Greece 0.9
G07 Northern Europe/Scandinavia, Australia, Bermuda, Uruguay 5.9
G08 Austria, Germany, Poland, Spain 4.5
G09 East Asia, South-East Asia, Guinea-Bissau, Sierra Leone 0.2
G10 Balkans, Baltic states, Canada, Italy, Malta, New Zealand, Republic of Korea, Russian Federation, 3.4
United States of America
G11 Belgium, Netherlands 0.3
G12 Belize, Dominica 2.9
G13 Africa, Haiti 0.1
G14 Pacific and Indian Ocean island states 0.9
G15 Europe/Scandinavia 3.7
G16 Gabon, Rwanda, Uganda 0.1
G17 Samoa, Sao Tome and Principe 2.9
a
Cocoa products included in the GEMS/Food cluster diets are cocoa beans, cocoa butter, cocoa mass, cocoa powder and chocolate.
Based on the international estimates of dietary cadmium exposure, the

main contributors (>10% in at least one cluster) to dietary exposure were potatoes
(0.4–18% depending on the cluster), fresh vegetables (10–50%), wheat flour (1.1–
19%), and cattle offal (1.5–11%). The contribution of cocoa products to dietary
cadmium exposure for each GEMS/Food cluster is summarized in Table 3.
The clusters with the highest contribution to dietary exposure to
cadmium from cocoa products were the “westernized” clusters (G07, G08, G10
and G15), including predominantly European and North American countries.
Contributions in these clusters ranged from 3.4–5.9%, with the greatest
contribution from G07. These contributions reflect the higher consumption of
chocolate and, more particularly, cocoa powder in the countries within these
clusters, as the cadmium concentrations in foods were assumed not to differ
between clusters.
The major producers of cocoa are African countries (Cameroon, Côte
d’Ivoire, Ghana and Nigeria; 3.5 million tonnes in 2020), Indonesia (0.65 million
tonnes in 2020) and South and Central American countries (Brazil, Colombia,
37
Dominican Republic, Ecuador and Peru; 0.70 million tonnes in 2020).1 These
countries are represented by the clusters G03, G05, G09 and G13. Interestingly,
cocoa products are generally very low contributors to dietary cadmium exposure
(<1%) in these regions.
Overall, the contribution of cocoa products to the total mean cadmium
exposure was consistent with the estimates based on national dietary exposure
estimates (Table 1).
2.4.1 Temporal trends in dietary cadmium exposure

Owing to differences in study design and study location, it was not possible
to identify any trends in dietary exposure to cadmium across the Committee
evaluations (sixty-first, sixty-fourth, seventy-third and current). Most studies
continue to report estimated mean dietary exposure to cadmium approximately in
the range of 10–40% of the health-based guidance value, and sometimes higher.
Similarly, the major foods contributing to dietary cadmium exposure have not
changed, with cereals, vegetables and seafood, especially molluscs, being consistent
major contributors across evaluations. None of the Committee evaluations have
identified cocoa products as major contributors to dietary cadmium exposure.
2.4.2 Impact of cocoa product source on dietary cadmium exposure

The potential impact on the contribution of cocoa products to dietary exposure
to cadmium of consuming products sourced from a single geographical
region (GEMS/Food cluster) was explored for the cluster diet with the greatest
contribution from cocoa products (G07) to cadmium exposure (see Table
2). Clusters for which suitable cadmium concentration data were available
were G03 (including data from Cameroon, Côte d’Ivoire and Ghana) and G05
(including data from Brazil, Chile, Colombia, Ecuador and Peru). Although
data on cadmium in foods were also submitted for cluster G13 by Nigeria, these
data did not include cadmium concentrations in cocoa and cocoa products. A
limited number of records (n = 30) were submitted by Indonesia (G09). Due to

the importance of Indonesia as a cocoa producer, these data were also included
in the analysis. A summary of the results of the analysis is presented in Table 4.
Given the higher concentrations of cadmium in cocoa powder and
chocolate from countries in cluster G05 (Table 2), the contribution of cocoa
products to the mean dietary exposure to cadmium was highest when using
concentration data from this cluster (Table 4). However, the combination of
food consumption information from the cluster with the highest consumption
of cocoa products (G07) and the cadmium concentration information from the
1
https://www.worldatlas.com/articles/top-10-cocoa-producing-countries.html
38
Table 4
Contribution of cocoa productsa to dietary cadmium exposure for cluster G07,b depending
on source (cluster) of cocoa and cocoa products
Contribution (%) of cocoa products to dietary cadmium
Source of cocoa and cocoa products exposure for cluster G07
Global 5.9
Cluster G03 (African countries) 0.9
Cluster G05 (South/Central American countries) 9.8
Cluster G09 (Indonesia) 3.8
a
b
Cluster G07 includes Australia, Bermuda, northern Europe and Uruguay.
cluster with the highest concentrations of cadmium in cocoa products (G05)

still only resulted in cocoa products contributing approximately 10% of dietary
cadmium exposure.
To examine the impact on dietary exposure to cadmium of cocoa
products from different geographical sources, the national studies reviewed for
this evaluation were considered to determine whether sufficient information was
provided to allow substitution of concentration information from Table 2 and
to examine the impact of such a substitution on dietary cadmium exposure and
the contribution from cocoa products. The Committee concluded that sufficient
information for such an analysis was also available from the European dietary
exposure assessment, carried out by EFSA (EFSA, 2012). In this analysis, the
mean concentrations of cadmium in cocoa powder and chocolate were 183 and
76 µg/kg, respectively. Reported contributions of individual food types to total
dietary exposure were used to calculate the absolute contributions of cocoa
powder and chocolate to dietary exposure. These absolute contributions were
then scaled by the ratio of the concentrations used in the original analysis and
the concentration in Table 2 to give revised absolute contributions from cocoa
powder and chocolate. A revised dietary cadmium exposure and a revised
proportional contribution from cocoa products was then calculated. The results
of this analysis are summarized in Table 5.
If the European population was to consume cocoa products sourced
solely from countries in cluster G05, dietary cadmium exposure might increase
by up to 8 µg/kg bw per month, with the contribution of cocoa products to dietary
exposure increasing approximately fourfold for the most highly exposed age
group. Decreases in dietary cadmium exposure if cocoa products were sourced
solely from countries in cluster G03 were relatively modest. This analysis suggests
that there are a few potential scenarios under which cocoa products would be the
main contributor to dietary exposure to cadmium.
39
Table 5
Impact on dietary cadmium exposure and the contribution of cocoa products to dietary
exposure for European countries, depending on source (cluster) of cocoa products
Source of concentration data for cadmium in cocoa productsb
Original (EFSA 2012) G03 G05 G09
Age groupa DE %cocoa DE %cocoa DE %cocoa DE %cocoa
Infants 11.5 0.2 11.5 0.1 11.6 1.3 11.5 0.4
Toddlers 20.6 4.2 19.9 1.2 24.6 19.7 21.2 7.0
Other children 16.8 9.4 15.8 3.9 25.1 39.4 18.5 17.6
Adolescents 9.9 9.4 9.4 4.2 14.9 39.5 11.0 17.9
Adults 7.6 4.6 7.3 1.4 9.2 21.1 7.8 7.6
Elderly 6.9 2.6 6.8 0.7 7.7 12.6 7.1 4.2
Very elderly 7.2 2.8 7.0 0.8 8.1 13.7 7.3 4.7
DE, dietary exposure in µg/kg bw per month; %cocoa, proportional contribution of cocoa products to dietary cadmium exposure.
a
Infants: 12 weeks – 11 months; toddlers: 12–35 months; other children: 3–9 years; adolescents: 10–17 years; adults: 18–64 years, elderly: 65–74 years; very elderly:
≥ 75 years.
b
Cluster G03 includes African countries, G05 includes mainly South and Central American countries, and G09 includes mainly South-East Asian counties.
2.4.3 Impact of proposed maximum limits for cadmium on cocoa product

rejection rates and dietary cadmium exposure
The Codex Alimentarius General Standard for Contaminants and Toxins in Food
and Feed includes maximum limits (MLs) for cadmium in:
■■ chocolate containing or declaring ≥ 50% to < 70% total cocoa solids
on a dry matter basis (800 µg/kg); and
■■ chocolate containing or declaring ≥ 70% total cocoa solids on a dry
matter basis (900 µg/kg).
At the thirteenth meeting of CCCF in 2019, further MLs were discussed
and it was proposed to take a proportional approach to products with lower
proportions of cocoa solids:
■■ chocolates containing or declaring < 30% total cocoa solids on a dry

matter basis (300 µg/kg);
■■ chocolates containing or declaring ≥ 30% to < 50% total cocoa solids
on a dry matter basis (500 µg/kg); and
■■ cocoa powder (100% total cocoa solids on a dry matter basis, sold for
final consumption) (1500 µg/kg).
Of the 4008 records in the GEMS/Food contaminants database related
to chocolate it is only possible to establish the percentage of cocoa solids for 638
(15.9%). These records are virtually all from countries in cluster G05 (South/
40
Table 6
Proportion of chocolate samples in different cocoa solids content classes and cocoa powder
from different sources exceeding the established or proposed Codex maximum limit (ML)
and the impact on mean cadmium concentration (medium bound)
Chocolate, classified by cocoa solids content (%)a Cocoa powder
< 30 ≥ 30 to < 50 ≥ 50 to < 70 ≥ 70 All G03 G05 G09
ML (µg/kg) 300 500 800 900 1500 1500 1500 1500
Number of samples 114 187 251 86 2583 74 1345 9
Number of samples with cadmium 3 (2.6) 4 (2.1) 27 (10.7) 4 (4.7) 420 (16.3) 0 (0.0) 405 (30.1) 0
concentration > ML (%) (0.0)
MB mean, all samples 121 180 474 318 971 141 1600 609
MB mean, sample ≤ ML only (µg/kg) 110 172 418 255 502 141 814 609
G03, mainly African countries; G05, mainly South/Central American countries; G09, mainly South-East Asian countries; LOD, limit of detection; MB, medium bound,
analytical results below the LOD are substituted by a value equal to LOD/2.
a
Samples for which the cocoa solids content was available were almost all from countries in cluster G05.
Central America). For the records with an identified proportion of cocoa solids,
Table 6 summarizes the potential rejection rates for chocolate and cocoa powder
from application of established and proposed MLs and the impact of applying the
MLs on mean cadmium concentrations.
The proportion of samples that exceeded the established or proposed
ML ranged from 2.1% for chocolate with a cocoa solids content of ≥ 30 to
< 50%, to 16.3% for cocoa powder (Table 6). Virtually all cocoa powder samples
with cadmium concentrations above the ML were from countries in cluster G05
(South/Central America), resulting in a substantially higher potential rejection
rate for cocoa powder samples from this cluster (405 of 1345 samples, 30.1%).
Table 7 provides an analysis of the impact of applying these MLs on the
contribution of cocoa products to dietary exposure to cadmium. The approach
is similar to that presented in Table 4, with global cadmium concentrations
(Appendix) matched to all foods in the cluster diets, except for chocolate and
cocoa powder. For these foods, concentrations without application of the MLs
were initially used, then concentrations with application of the MLs were used. For
chocolate, concentrations used were the means of the four categories summarized
in Table 6, weighted by the number of samples in each category. Resulting mean
concentrations for cadmium in chocolate were 304 µg/kg, without application of
MLs, and 269 µg/kg, with application of MLs. The corresponding concentrations
for cocoa powder are available in Table 6.
Across all clusters, the average contribution of cocoa and cocoa products
was 2.2% without application of the MLs and 1.5% with application of MLs.
Application of the MLs resulted in a mean reduction in dietary cadmium exposure
of 0.7% across all clusters. The reductions ranged from 0.0% (cluster G16) to
41
Table 7
Contribution of cocoa products to dietary cadmium exposure, with and without application
of maximum limits for chocolate and cocoa powder, with cocoa products sourced from all
relevant GEMS/Food clusters
Contribution to dietary cadmium exposure from cocoa products (%)a Reduction in estimated dietary
GEMS/Food cadmium exposure from
cluster Without application of MLs With application of MLs application of MLs (%)b
G01 0.8 0.6 0.2
G02 2.1 1.8 0.2
G03 0.9 0.6 0.3
G04 3.3 2.5 0.9
G05 0.7 0.4 0.3
G06 1.0 0.6 0.4
G07 6.6 4.3 2.4
G08 5.2 3.4 1.8
G09 0.2 0.1 0.1
G10 3.7 2.4 1.3
G11 0.3 0.3 0.1
G12 3.5 2.7 0.8
G13 0.1 0.1 0.1
G14 1.0 0.7 0.3
G15 4.6 3.4 1.2
G16 0.1 0.1 0.0
G17 3.0 1.6 1.3
ML, maximum limit; both proposed and established MLs were applied in this analysis.
a
b
The percentages in this column are the percentage decrease in the estimated dietary cadmium exposure due to application of the MLs, rather than the difference in
the contribution from cocoa products.
2.4% (cluster G07). The contributions to dietary cadmium exposure without

application of MLs differed slightly from the contributions shown in Table 3, as
only a subset of the records concerning chocolate was accompanied by sufficient
data to allow application of the MLs.

Table 8 summarizes an extension of the analysis in Table 7, assuming
that the source of cocoa powder was solely from countries in the three main
cocoa-producing regions; clusters G03, G05 and G09. The concentration data for
this analysis are summarized in Table 6. Note that this analysis relates only to the
source of cocoa powder, as the chocolate samples with associated information on
cocoa solids are virtually all from cluster G05.
Application of the MLs had the greatest impact on dietary cadmium
exposure when it was assumed that cocoa powder was sourced entirely from
countries in cluster G05. This is not surprising as, for clusters G03, G05 and
G09, only cocoa powder samples from cluster G05 had cadmium concentrations
42
Table 8
Contribution of cocoa products to dietary cadmium exposure, with and without application
of maximum limits for chocolate and cocoa powder, assuming cocoa powder is sourced
from countries in a single GEMS/Food clustera
Reference
G03 G05 G09
Species ML− ML+ DEb ML− ML+ DEb ML− ML+ DEb
G01 0.5 0.5 0.1 1.0 0.7 0.3 0.7 0.6 0.1
G02 2.0 1.8 0.2 2.1 1.9 0.3 2.0 1.8 0.2
G03 0.4 0.4 0.0 1.2 0.8 0.5 0.7 0.7 0.0
G04 2.1 1.9 0.2 4.2 2.9 1.3 2.8 2.6 0.2
G05 0.2 0.2 0.0 1.1 0.6 0.5 0.5 0.5 0.0
G06 0.4 0.4 0.0 1.5 0.9 0.6 0.8 0.7 0.0
G07 2.8 2.6 0.2 9.3 5.7 3.8 5.0 4.8 0.2
G08 2.4 2.2 0.2 7.1 4.5 2.8 4.0 3.8 0.2
G09 0.0 0.0 0.0 0.3 0.2 0.1 0.1 0.1 0.0
G10 1.6 1.4 0.1 5.3 3.2 2.1 2.8 2.7 0.1
G11 0.2 0.2 0.0 0.4 0.3 0.1 0.3 0.3 0.0
G12 2.5 2.3 0.2 4.3 3.1 1.2 3.0 2.9 0.2
G13 0.1 0.0 0.0 0.2 0.1 0.1 0.1 0.1 0.0
G14 0.6 0.6 0.0 1.3 0.9 0.4 0.8 0.8 0.0
G15 2.9 2.6 0.3 5.8 4.0 1.9 3.9 3.6 0.3
G16 0.1 0.1 0.0 0.1 0.1 0.0 0.1 0.1 0.0
G17 0.7 0.6 0.0 4.7 2.5 2.2 2.0 1.9 0.0
G03, mainly African countries; G05, mainly South/Central American countries; G09, mainly South-East Asian countries; ML−, without application of the maximum
limits; ML+, with application of the maximum limits, both proposed and established MLs were applied in this analysis; DE, decrease in dietary exposure estimate due
to application of maximum limits.
a
b
The percentages in this column are the percentage decrease in the estimated dietary cadmium exposure due to application of the MLs, rather than the difference in
the contribution from cocoa products.
above the ML (30.1%, see Table 6). For cocoa products sourced from countries in
cluster G03 and G09, application of the MLs had a negligible impact on dietary
exposure to cadmium, as the changes in exposure were only due to changes in the
mean cadmium concentration for chocolate.
3. Evaluation
The Committee assessed information related to exposure to cadmium from all
food sources, with a particular focus on cocoa products. Information assessed
was restricted to the period since the previous assessment of dietary exposure
to cadmium in 2011. The Committee summarized dietary cadmium exposure
estimates from 44 national studies conducted worldwide in 32 countries and a
43
country grouping as reported in the literature. The mean dietary exposure to

cadmium from the whole diet ranged from 0.6 µg/kg bw per month (2.4% of the
PTMI) for adults in the Sikasso region of Mali up to 24 µg/kg bw per month (96%
of the PTMI) in children aged 4–11 years in China. These children from China
also had the highest high percentile estimate of dietary cadmium of 48.2 µg/kg
bw per month (190% of the PTMI). High percentile estimates of adult dietary
cadmium exposure were only occasionally above the PTMI and were typically
20–60% of the PTMI. Consistent with the previous evaluations of the Committee,
the present evaluation identified the main sources of dietary cadmium exposure
in these national studies as cereals and cereal-based products, vegetables and fish
and seafood. Of the 44 studies reviewed, only nine reported the contribution of
cocoa products to the total mean dietary exposure to cadmium, which ranged
from 0.2 to 9%.
Given the large number of national estimates of dietary cadmium
exposure available from the literature, their coverage of countries across the
world, and their consistency, the Committee considered that deriving less refined
international and national estimates of dietary exposure was unnecessary.
Based on data on the concentration of cadmium in foods submitted to
the GEMS/Food contaminants database since 1 January 2011, the Committee
examined the contribution of cocoa products to the mean dietary exposure to
cadmium using the GEMS/Food clusters diets. Analyses using these data showed
that the contribution of cocoa products to the dietary exposure to cadmium was
consistent with the estimates based on national dietary exposure studies, ranging
from 0.1% to 5.9%. The highest contributions were calculated for European and
North American countries, reflecting the higher consumption of chocolate and
cocoa powder in these countries.
The potential impact of consumption of cocoa products from a single
geographical region, as represented by GEMS/Food clusters was examined. For
the cluster with the greatest contribution to dietary cadmium exposure from
cocoa products (G07, mainly European countries, 5.9%) this contribution would
decrease to 0.9% or increase to almost 10% if cocoa products were sourced

only from countries in cluster G03 (Africa) or G05 (South/Central America),
respectively. The Committee carried out a similar analysis using data (mean
concentrations of cadmium in cocoa products, dietary cadmium exposure
estimates and contributions of cocoa products to dietary exposure) for European
countries reported by EFSA (2012). In the EFSA study, the age group with the
greatest contribution to dietary cadmium exposure from cocoa products was
children aged 3–9 years (contribution 9.4%). From the Committee’s analysis, if
this age group were to consume cocoa products sourced solely from cluster G03
(Africa), dietary cadmium exposure would decrease modestly (16.8 to 15.8 µg/kg
bw per month), while the contribution from cocoa products would decrease to
44
3.9%. If this group were to consume cocoa products sourced solely from cluster
G05 (South/Central America), dietary cadmium exposure would increase to
25.1 µg/kg bw per month, with cocoa products contributing 39% of dietary
cadmium exposure.
CCCF has proposed MLs for chocolate with proportions of total cocoa
solids of < 30% and ≥ 30% to <50% on a dry matter basis and for cocoa powder
with 100% total cocoa solids on a dry matter basis. These MLs are proposed in
addition to existing MLs for chocolate with ≥ 50% to < 70% and ≥ 70% total cocoa
solids on a dry matter basis. Cocoa solids content information was available for a
limited subset (15.9%) of the chocolate records in the GEMS/Food contaminants
database. Comparing the cadmium concentrations in chocolate and cocoa
powder in the GEMS/Food contaminants database to the existing and proposed
MLs showed that 2.1–10.7% of the chocolate samples and 16.3% of the cocoa
powder samples had concentrations higher than the MLs and could potentially
be rejected by importing countries through application of the MLs. Applying
these MLs compared to not applying them resulted in an average decrease in the
contribution of cocoa products (including also cocoa beans, cocoa butter and
cocoa mass) to the dietary exposure to cadmium of 0.7% across all clusters.
At its seventy-third meeting in 2011, the Committee established a PTMI
of 25 μg/kg bw, reflecting the long half-life of cadmium in humans. The PTMI
was not reviewed at the current meeting. The national exposure estimates were
predominantly below this PTMI, with some exceptions for young children or
adults living in China. The Committee noted that the current JECFA PTMI for
cadmium is based on long-term bioaccumulation in the kidney, with steady-state
not achieved until after 45–60 years of exposure. The Committee concluded that
dietary exposure above the PTMI for limited periods may be of lesser concern in
younger age groups. However, there may be a health concern in areas where the
cadmium exposure during adulthood exceeds the PTMI.
The Committee concluded that major contributors to dietary cadmium
exposure were cereals and cereal products, vegetables and seafood. The
contribution of cocoa products to dietary cadmium exposure was minor in
comparison (0.1–9.4% for national studies and estimates based on GEMS/Food
cluster diets), even in countries in which the consumption of cocoa products is
relatively high.
Application of both established and proposed MLs for chocolate and
cocoa powder may result in substantial rejection rates (up to 30%) for products
from some regions, but has only a minor impact (mean decrease across clusters
of 0.7%, range 0.0–2.4%) on total dietary cadmium exposure.
45
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5. Appendix
Global mean cadmium concentrations used for international

estimates of dietary exposure
Food N N<LOD N<LOD (%) MB mean
Alcoholic beverage NES 494 464 94 1.9
Almonds 192 54 28 13.2
Anise, badian, fennel, coriander 114 10 9 88.0
Apple 3 020 2666 88 2.7
Apple juice 1 855 1788 96 1.7
Apricot 393 318 81 2.8
Artichoke globe 96 76 79 5.6
Asparagus 772 307 40 11.4
Assorted (sub)tropical fruits 14 8 57 1.4
48

Avocado 366 139 38 19.7
Bacon and ham 185 114 62 3.4
Bamboo shoots 5 1 20 3.1
Banana 465 422 91 2.3
Barley 712 210 29 14.2
Beans (dry) 405 288 71 6.4
Beans except broad bean and soya bean (green pods and immature seeds) 931 588 63 5.8
Beans, shelled (immature seeds) 257 145 56 3.4
Beans-based meals 67 17 25 4.7
Beer and beer-like beverage 931 906 97 1.4
Berries and other small fruits 465 317 68 5.9
Bird meat NES 176 153 87 5.0
Blueberries 243 181 74 2.5
Bottled water 908 804 89 4.2
Brazil nut 81 78 96 4.2
Bread and other cooked cereal products 2 759 335 12 26.1
Breakfast cereals 430 82 19 25.9
Broad bean (dry) 29 12 41 6.3
Buckwheat 442 21 5 38.1
Buffalo meat 141 138 98 2.7
Buffalo milk 39 39 100 3.5
Bulb vegetables 24 5 21 11.3
Butter and other animal fat emulsion 163 140 86 3.5
Cabbages and other brassicas 2 253 720 32 6.9
Camel, edible offal of 1 0 0 14.7
Canned fish 5 0 0 19.4
Caper buds 6 2 33 5.0
Cardoon 6 5 83 4.9
Carob 7 6 86 0.8
Carrots and turnips 2 972 697 23 24.3
Cashew nut 46 41 89 2.1
Cassava 70 31 44 2.5
Cattle fat 5 5 100 2.5
Cattle meat 10 340 9698 94 4.7
Cattle milk 3 347 3093 92 1.3
Cauliflower/broccoli 1 239 453 37 9.5
Celeriac 308 23 7 58.6
Celery (whole) 414 100 24 34.8
Celery leaves 29 5 17 25.8
Cephalopods 1 468 278 19 217
Cereal grains 233 37 16 34.1
Cereal starch 9 7 78 7.7
Cereal-based dishes 185 46 25 13.3
Cereal-based food for infants and young children 1 989 483 24 13.4
Cereals and cereal-based products NES 802 54 7 51.8
49
Table (continued)
Cereals flour 292 58 20 22.7
Cheese and analogues 1 712 1497 87 3.1
Cherries 361 333 92 1.8
Chestnuts 13 9 69 5.2
Chicken eggs 860 775 90 2.3
Chicken fat 39 31 79 2.3
Chicken meat 4 958 4439 90 4.0
Chicken, edible offal of 3 338 555 17 34.0
Chick-pea (dry) 463 364 79 6.3
Chicory, roots 17 11 65 10.6
Chives 113 7 6 23.4
Chocolate 4 008 280 7 184
Cinnamon bark 44 1 2 259
Citrus fruits 44 33 75 3.2
Citrus juice 37 27 73 1.0
Cloves, buds 47 29 62 11.1
Cocoa beans 108 8 7 260
Cocoa beverage 20 5 25 43.2
Cocoa butter 20 6 30 43.8
Cocoa mass 218 1 0 773
Cocoa powder 2 583 25 1 971
Coconut 33 15 45 8.5
Coconut oil, refined 8 8 100 5.3
Coffee (beverage) 45 35 78 2.3
Coffee beans 120 20 17 2 460
Coffee beans, roasted 278 188 68 5.1
Coffee imitates beverage 5 5 100 5.0
Common bean (pods and/or immature seeds) 9 9 100 2.9
Composite food NES 636 68 11 55.1
Concentrated fruit juice 94 81 86 2.5
Confectionery 554 279 50 71.9

Corn salad 221 128 58 3.9
Cotton seed 1 0 190
Cowpea (dry) 1 0 0 56.0
Cranberries 37 18 49 6.7
Cream 143 90 63 4.4
Crustaceans 3 032 1017 34 499
Cucumbers and gherkins 1 073 715 67 3.0
Currants, red, black, white 124 26 21 4.9
Dandelion leaves 5 0 0 265
Date 138 124 90 4.3
Dietetic food for diabetics (labelled as such) 10 6 60 53.8
Dried fish 51 0 0 253
50

Dried fruits 127 62 49 1150
Drinking water 6 614 6 284 95 0.6
Dry apricots 60 40 67 4.0
Duck meat 854 747 87 5.7
Duck, edible offal of 364 17 5 111
Edible offals (mammalians) 5 008 730 15 2 600
Eggplant 436 219 50 6.1
Egg-based meal (e.g. omelette) 1 1 100 3.0
Eggs and egg products NES 711 692 97 3.6
Endive 261 85 33 13.2
Extracts tea, mate, prep 219 70 32 68.1
Fats and oils NES 322 301 93 4.2
Fennel, bulb 82 58 71 5.1
Fermented milk products 366 311 85 3.1
Fig 24 18 75 9.5
Figs dried 24 11 46 13.6
Fish and seafood-based meals 701 264 38 33.2
Fish, sea food, amphibian reptile snail or insect NES 2 429 442 18 264
Fishes 310 218 70 12.4
Food for sports people (labelled as such) 296 170 57 44.4
Food for weight reduction 72 24 33 21.4
Freshwater fishes 3 116 2661 85 3.7
Fruit and fruit products NES 950 638 67 31.1
Fruit juice 2 653 1964 74 20.7
Fruit juice and herbal tea for infants and young children 207 135 65 6.0
Fruit nectar 796 719 90 3.0
Fruit or vegetable juice NES 222 186 84 8.4
Fruit, tropical, fresh NES 14 6 43 2.7
Fruiting vegetables 68 27 40 15.6
Galangal, rhizomes 2 0 0 2.0
Game meat 4 325 3084 71 12.4
Garlic 316 54 17 22.4
Ginger, root 81 8 10 47.4
Goat meat 338 320 95 3.8
Goat milk 276 266 96 1.4
Goose and guinea fowl meat 142 110 77 3.7
Goose, edible offal of 90 2 2 134
Gooseberries 68 25 37 2.9
Grape juice 556 496 89 1.5
Grape leaves 6 3 50 5.2
Grapefruit (inc. pomelos) 304 258 85 2.2
Grapefruit juice 143 136 95 2.8
Grapes 1 117 1018 91 3.8
Hazelnut 78 16 21 10.1
Herb, spice or condiment NES 873 492 56 19.2
51
Table (continued)
Herbs 715 148 21 62.2
Honey 2 676 2082 78 17.3
Hops, dry 12 6 50 8.6
Horse meat 2 184 547 25 38.8
Horseradish 19 0 0 48.2
Ices and desserts 399 67 17 12.2
Indian mustard 28 1 4 165
Infant food 3 891 2 590 67 5.1
Kale 268 27 10 16.9
Kale, curly 35 5 14 46.8
Kiwi fruit 217 189 87 2.7
Lactose 6 6 100 5.0
Lard (of pigs) 8 8 100 4.3
Leafy vegetables 388 107 28 38.5
Leek 419 77 18 16.4
Legume vegetable 57 37 65 6.5
Legumes and pulses NES 21 10 48 116
Lemon juice 105 101 96 1.0
Lemons and limes (including citron) 572 535 94 2.6
Lemons and limes, edible oil refined 1 1 100 5.0
Lentils 876 648 74 6.9
Lettuce 2 505 504 20 29.0
Lima bean (young pods and/or immature beans) 50 48 96 5.3
Liquorice, roots 1 0 0 8.0
Liver product 80 18 23 16.2
Loquat 4 3 75 8.1
Macaroni 913 78 9 31.6
Maize 718 514 72 5.0
Maize flour 511 354 69 7.0
Maize oil, edible 147 147 100 3.9
Mandarin and mandarin-like hybrid 275 256 93 2.9

Mango juice 47 27 57 0.7
Mangoes, mangosteens, guavas 313 255 81 3.4
Maple sugar and syrups 2 1 50 10.0
Margarine 170 154 91 4.4
Marine fishes 19 023 12 270 65 10.7
Meat and meat products NES 705 356 50 30.4
Meat of cattle, goats, horses, pigs and sheep 184 143 78 4.8
Meat of cattle, pigs and sheep 23 12 52 0.8
Meat-based meals 503 131 26 7.5
Medical food (specially formulated and intended for the dietary management 242 175 72 7.6
of a disease that has distinctive nutritional needs that cannot be met by normal
diet alone; intended to be used under medical supervision)
52

Medlar 2 2 100 1.8
Melons, except watermelon 388 166 43 7.0
Milk and dairy products NES 395 208 53 16.0
Milks 358 338 94 2.3
Millet 159 23 14 19.1
Mints 5 3 60 8.4
Mixed fruit and vegetable juice 148 81 55 3.5
Molluscs, excluding cephalopods 5 153 300 6 219
Mushroom-based meals 1 1 100 5.0
Mushrooms and truffles 5 718 2 003 35 64.8
Mustard greens 3 2 67 17.2
Non-alcoholic beverage NES 1119 898 80 51.2
Nutmeg, mace and cardamoms 127 0 0 97.9
Oats 955 76 8 24.4
Offal of cattle, edible 7 430 250 3 765
Offal of pigs, edible 20 298 716 4 383
Offal of sheep, edible 1 277 167 13 138
Oilseeds 971 171 18 108
Okra 56 1 2 13.7
Olive 55 40 73 5.4
Olive oil, refined 225 212 94 3.6
Olive oil, virgin 204 202 99 5.1
Olive, processed 16 7 44 1.4
Onion 1 520 545 36 10.5
Orange juice 804 782 97 1.5
Orange juice, concentrated 20 17 85 4.3
Orange, sweet, sour + orange-like hybrid 838 774 92 2.7
Other foods (foods which cannot be included in any other group) 470 282 60 35.4
Palm hearts 4 2 50 1.6
Palm kernels 2 1 50 0.6
Palm oil, edible 56 56 100 4.1
Papaya 21 20 95 1.3
Parsley 444 85 19 24.7
Parsley, turnip-rooted 166 33 20 17.9
Parsnip 225 35 16 48.7
Passion fruit 4 0 0 37.5
Peaches (including nectarines) 983 790 80 2.9
Peanut 339 12 4 77.4
Peanut butter 131 1 1 55.1
Peanut oil, edible 101 100 99 4.9
Pear 686 481 70 3.6
Peas (dry) 366 134 37 11.9
Peas (green pods and immature seeds) 94 56 60 3.0
Peas, shelled (immature seeds) 456 289 63 4.8
Pepper (black, white) 269 64 24 15.3
53
Table (continued)
Peppermint 46 9 20 47.1
Peppers and chillies, dried 283 19 7 76.4
Peppers and chillies, green 1246 566 45 13.1
Persimmon 31 31 100 1.7
Pig meat 13 566 11 605 86 6.8
Pineapple 497 406 82 2.0
Pineapple juice 441 316 72 1.3
Pistachio nut 93 40 43 10.8
Plum (incl. dried) 546 464 85 2.8
Pome fruits 3 3 100 4.2
Poppy seed 516 2 0 501
Potato 5 508 774 14 33.7
Potato-based dishes 65 2 3 18.1
Poultry meat 841 652 78 19.9
Poultry offal 915 157 17 39.0
Poultry, fats 5 2 40 17.5
Prepared salads 132 20 15 28.1
Prickly pear 3 3 100 3.3
Products for special nutritional use NES 492 302 61 23.0
Pumpkins, squashes and gourds 624 319 51 4.0
Quince 24 15 63 2.5
Rabbit meat 749 651 87 4.7
Raisins 120 72 60 2.7
Rape seed oil, edible 208 187 90 3.6
Raspberries, red, black 273 95 35 9.8
Ready-to-eat soups 153 4 3 6.5
Ready-to-eat meals for infants and young children 2 391 1 209 51 7.5
Rhubarb 126 14 11 9.4
Rice 3 606 819 23 24.2
Rice bran oil 3 3 100 2.5
Rice flour 230 17 7 38.3

Rice, polished 5 2 40 4.9
Rice-based meals 172 38 22 26.3
Root and tuber vegetables 1 080 183 17 62.8
Rosemary 79 32 41 7.4
Rye 1 133 284 25 13.2
Safflower seed oil, edible 1 1 100 5.0
Sage and related Salvia species 5 1 20 40.8
Sauce and condiments 501 188 38 16.2
Sausages and related products 1 052 636 60 3.0
Sesame seed 394 61 15 23.9
Sesame seed oil, edible 56 55 98 4.7
Shaddock or pomelo and shaddock-like hybrid 5 5 100 2.5
54

Sheep meat 2 508 2 331 93 3.5
Sheep milk 214 157 73 2.0
Snack food 272 51 19 27.3
Snack or dessert NES 30 17 57 35.8
Sorghum 9 7 78 0.9
Soya bean (dry) 1 771 74 4 83.3
Soya bean oil, refined 61 61 100 4.5
Soya curd 292 55 19 18.5
Soya sauce 115 24 21 7.8
Spices 454 52 11 98.8
Spinach 1 219 58 5 85.3
Stalk and stem vegetables 10 4 40 32.6
Stone fruits 73 55 75 2.5
Strawberry 1 166 530 45 6.8
String beans 5 0 0 0.9
Sugar and confectionery NES 126 77 61 12.2
Sugar crops 20 0 0 18.7
Sunflower seed 456 5 1 219
Sunflower seed oil, edible 181 176 97 3.2
Swede 61 5 8 20.8
Sweet corn 340 209 61 3.5
Sweet potato 648 26 4 17.0
Taro 33 2 6 25.7
Tarragon 6 0 0 113
Tea, green, black (black, fermented and dried) 6 591 2 360 36 37.4
Herbal teas (solid) 7 025 1 777 25 213
Thyme 51 2 4 53.0
Tomato 1 748 886 51 9.3
Tomato juice 223 56 25 9.8
Tomato paste 2 0 0 45.1
Tree nuts 202 41 20 70.7
Tree tomato 5 0 0 1.9
Turkey meat 1 271 1 166 92 3.8
Turkey, edible offal of 791 18 2 129
Turmeric, root 115 14 12 38.3
Vanilla, beans 5 0 0 0.3
Vegetable juice 197 26 13 16.7
Vegetable-based meals 251 71 28 9.9
Vegetables and vegetable products NES 1 762 236 13 502
Walnut 173 134 77 3.2
Watercress 2 0 0 28.5
Watermelon 335 282 84 2.9
Wheat 5 961 809 14 37.1
Wheat flour 2 735 161 6 23.0
Whey and whey products 31 25 81 9.8
55
Table (continued)
Wine 1 229 904 74 1.7
Wine-like drinks (e.g. cider, perry) 135 131 97 3.0
Yams 10 0 0 49.3
Yoghurt 221 159 72 4.0
Total 277 292 128 146 46
N, number of samples; N<LOD, number of samples with cadmium concentrations less than the limit of detection; MB mean, medium bound mean; NES, not elsewhere
specified.
56
Ergot alkaloids
Nathalie Arnich,1 Antonio Agudo,2 Peter Cressey,3 Lutz Edler,4 Mark
Feeley,5 Benoit G.J. Gnonlonfin,6 Ellen F. Kirrane,7 Jean-Charles Leblanc,8
David Lovell,9 Isabelle Oswald,10 Gordon Shephard11 and Stephan G.
Walch12
1
Risk Assessment Department, French Agency for Food, Environmental and
Occupational Health and Safety (ANSES), Maisons-Alfort Cedex, France
2
Unit of Nutrition and Cancer, Catalan Institute of Oncology, Barcelona, Spain
3
Institute of Environmental Science and Research Limited (ESR), Christchurch, New
Zealand
4
Division of Biostatistics, German Cancer Research Center, Heidelberg, Germany
5
Ottawa, Canada
6
Department of Industry and Private Sector Promotion and Directorate of Agriculture
and Rural Development, ECOWAS Commission, Abuja FCT, Nigeria
7
United States Environmental Protection Agency’s Center for Public Health and
Environmental Assessment, Research Triangle Park (NC), United States of America
8
Laboratory for Food Safety, French Agency for Food, Environmental and Occupational
Health and Safety (ANSES), Maisons-Alfort Cedex, France
9
Population Health Research Institute, St George's Medical School, University of
London, London, United Kingdom
10
Toxalim (Research Center in Food Toxicology), Université de Toulouse, INRA, ENVT,
INP-Purpan, Toulouse, France
11
Cape Town, South Africa
12
Chemisches und Veterinäruntersuchungsamt (CVUA) Karlsruhe, Karlsruhe, Germany
1. Explanation 59
2. Biological data 61
2.1 Biochemical aspects 61
2.1.1Absorption, distribution and excretion 61
2.1.2 Biotransformation 72
2.1.3 Effects on enzymes and other biochemical parameters 77
2.1.4 Physiologically based pharmacokinetic (PBPK) modelling 78
2.1.5 Transfer from feed to food 78
2.2 Toxicological studies 79
2.2.1 Acute toxicity 79
2.2.2 Short-term studies of toxicity 81
2.2.3 Long-term studies of toxicity and carcinogenicity 92
2.2.4 Genotoxicity 94
2.2.5 Reproductive and developmental toxicity 97
2.2.6 Special studies 105
2.3 Observations in domestic animals/veterinary toxicology 119
2.4 Observations in humans 126
2.4.1 Biomarkers of exposure 126
2.4.2 Biomarkers of effects 127
2.4.3 Clinical observations 127
57
2.4.4 Epidemiological studies 132

3. Analytical methods 138
3.1 Chemistry 138
3.2 Description of analytical methods 139
3.2.1 Introduction 139
3.2.2 Screening tests 141
3.2.3 Quantitative methods 141
4. Sampling protocols 144
5. Effects of processing 145
5.1 Sorting, cleaning and milling 145
5.2 Thermal and chemical food processing 146
5.3 Fermentation 147
6. Prevention and control 147
6.1 Preharvest control 147
6.2 Postharvest control 148
6.3 Decontamination 149
7. Levels and patterns of contamination in food commodities 150
7.1 Surveillance data 150
7.1.1 African region 154
7.1.2 Region of the Americas 155
7.1.3 Eastern Mediterranean and South-East Asia Regions 155
7.1.4 European Region 155
7.1.5 Western Pacific Region 157
7.2 Conclusions 158
8. Food consumption and dietary exposure estimates 159
8.1 Concentrations in food used in the dietary exposure estimates 159
8.2 Food consumption data used in the dietary exposure estimates 159
8.3 Assessments of dietary exposure 160
8.3.1 National or regional estimates of chronic dietary exposure from the
published literature 160
8.3.2 National or regional estimates of chronic dietary exposure derived
by the Committee 165
8.3.3 National or regional estimates of chronic dietary exposure derived
by the Committee 165
8.3.4 Dietary chronic exposures summary 166
8.4 Assessments of acute dietary exposure 171

8.4.1 National estimates of acute dietary exposure from the published
literature 171
8.4.2 National estimates of acute dietary exposure derived by the
Committee 172
8.5 Global dietary exposure estimates summary 172
9. Dose-response analysis and estimation of toxicity/carcinogenic risk 173
9.1 Identification of key data for risk assessment 173
9.1.1 Pivotal data from biochemical and toxicological studies 173
9.1.2 Pivotal data from human clinical/epidemiological studies 176
9.2 General modelling considerations 177
9.2.1 Selection of data 177
9.2.2 Measure of exposure 177
9.2.3 Measure of response 178
58
Ergot alkaloids
9.2.4 Selection of mathematical model 178

9.3 BMD analysis 179
10. Comments 184
10.3 Observations in domestic animals/veterinary toxicology 191
10.4.1 Biomarkers 192
10.4.2 Clinical observations 192
10.4.3 Epidemiology 193
10.5 Analytical methods 194
10.6 Sampling protocols 196
10.7 Effects of processing 196
10.8 Prevention and control 197
10.9 Levels and patterns of contamination in food commodities 197
10.10 Food consumption and dietary exposure estimates 201
10.10.1 Transfer from feed to food 202
10.11 Dose–response analysis 202
10.11.1 Dose–response data in humans 202
10.11.2 Dose–response data in animals 202
11. Evaluation 205
11.1 Recommendations 207
12. References 207
1. Explanation
Ergot is a disease of plants caused by fungi of the genus Claviceps. Ergot also refers
to the typically elongated fungal structure, technically known as a sclerotium
(plural sclerotia), which replaces kernels on grain ears or seeds on grass heads
of diseased plants. There are 40 known species of Claviceps; the species infecting
hosts relevant for the food chain are primarily C. purpurea (ubiquitous, infects
grasses and cereals such as rye, wheat and triticale), C. africana (infects sorghum)
and C. fusiformis (infects only pearl millet).
Ergot alkaloids (EAs) are a group of toxic fungal metabolites (mycotoxins)
produced by Claviceps spp., in sclerotia. Structurally, EAs are closely related to
biogenic amines such as norepinephrine, dopamine and serotonin. The EAs are
characterized by the ergoline ring system consisting of four fused rings in which
position N6 carries a methyl group, and there is a double bond at either C8,9 or at
C9,10. Substitution at C8 gives rise to the naturally occurring range of alkaloids. Of
those considered in this evaluation, ergometrine (ergonovine) is a simple lysergic
acid derivative and the others are peptide alkaloids (known as ergopeptines or
ergopeptides), in which the substituent at C8 is a cyclized tripeptide (Fig. 1).
59
Fig. 1
Chemical structures of lysergic acid, ergometrine and selected peptide ergot alkaloids
Based on the EAs identified in sclerotia of Claviceps spp. and occurrence data,
the Committee concluded that the assessment should focus on the (R)-epimers:
ergometrine (also known as ergonovine), ergotamine, ergosine, ergocristine,
ergocryptine (a mixture of alpha (α)- and beta (β)-isomers), ergocornine and the
corresponding (S)-inine epimers. Ergotamine and ergometrine have been used
in human medicine for the treatment of migraine headache, management of the
third stage of labour and postpartum blood loss.
EAs have not previously been reviewed by JECFA. The Committee
evaluated EAs at the present meeting in response to a request from the Codex
Committee on Contaminants in Foods (CCCF) in 2016.
The search for biological data followed JECFA guidance on conducting
a comprehensive literature review (WHO JECFA Secretariat, 2017). For animal
60
Ergot alkaloids
data, the search was performed in PubMed and Scopus (first search in May 2020,
updated in October 2020 and January 2021). For human data, PubMed was
searched from 2010 to December 2020 to identify studies of the adverse effects
in humans associated with the main EAs used in medicine. Additional studies
were identified from previous assessments or reports and drug references (for
example, European Food Safety Authority (EFSA), 2010 and Martindale, 2010).
For analytical methods, sampling, processing and decontamination, the search
was performed on Web of Science, PubMed and Scopus as well as official sources
and Google Scholar. The literature search on occurrence and dietary exposure to
EAs was performed using PubMed and Scopus from January 2000 to December
2020.
2. Biological data
Kinetic studies for the majority of the naturally occurring EAs (for example,
ergosine, ergocristine, ergocryptine or ergocornine) are not available. However,
for ergotamine and ergometrine, some human and limited animal data were
available.
Most of the available data on the biochemical aspects in humans are
based on studies with semi-synthetic derivatives of EAs. A brief description of
these data has been included; however, as their kinetics may be different to those
of natural EAs present in food, these data should be interpreted only as supportive
and not definitive evidence.
2.1 Biochemical aspects

2.1.1 Absorption, distribution and excretion
(a) Absorption
Absorption is a measure of the disappearance of a compound from the
gastrointestinal tract and entrance into the systemic circulation, either as
unchanged compound or as metabolite(s). The extent of bioavailability, however,
is a measure of the amount of unchanged compound that reaches the systemic
circulation following an oral dose (Nimmerfall & Rosenthaler, 1976).
Most of the available data are from studies conducted with salts of EAs.
It is assumed that the salts dissociate fully in the gastrointestinal tract. When the
compound is radiolabelled, the radioactivity has been measured with regard to
the base which is the site of the radiolabel.
61
Animal data
Nimmerfall & Rosenthaler (1976) reported that, following oral administration
of 1 mg/kg bw [3H]-dihydroergocristine mesylate to seven Wistar rats, the mean
absorption from the gastrointestinal tract was 38.8%. By contrast, the absorption
of orally administered [3H]-dihydroergotamine mesylate or [14C]-ergotamine
tartrate (both 0.25 mg/kg bw) to six Rhesus monkeys was 31.5 ± 5.2% and 10.3 ±
1.5%, respectively.
After oral administration of [3H]-ergotamine tartrate, dihydro-(9,10-
[ H])ergotamine mesylate or dihydro-(9,10-[3H])ergocristine mesylate to groups
3
of six or seven rats at a dose of 1 mg/kg bw, intestinal absorption was estimated at
41.4 ± 9.5%, 12.3 ± 3.3% and 15.0 ± 2.5%, respectively (Eckert et al., 1978).
Human data
Plasma and urinary radioactivity were measured in six healthy male volunteers
after oral intake of [3H]-labelled ergotamine tartrate (2 mg), either alone or
combined 1:100 with caffeine (Schmidt & Fanchamps, 1974). Their mean age
was 41.5 years (39–49 years) and their mean body weight was 73.3 kg (64–76 kg).
Volunteers fasted overnight and for 4 hours after dosing. The interval between
the two treatments was 10 days. The plasma levels of radioactivity were low; the
maximum plasma concentrations corresponded to drug levels of 1–2 ng/mL. No
radioactivity could be detected at 15 and 30 minutes after administration of [3H]-
ergotamine alone, whereas after administration of the combination with 200 mg
caffeine, radioactivity was found after 30 minutes. Peak plasma concentration was
reached 2 to 4 hours after both treatments. Plasma level was higher after taking
the combination than after ergotamine alone and the difference was significant
at certain times. A detectable plasma level was reached 30 minutes after taking
the combination but only 1 hour after ergotamine alone. These results indicate
faster and more complete intestinal absorption of ergotamine administered in
combination with caffeine.
After oral administration of 0.2 to 2 mg of [3H]-labelled ergotamine (in a

capsule) or [3H]-labelled dihydroergotamine (in a solution) to different groups of
six healthy volunteers, Meier & Schreier (1976) reported an absorption half-life
of 0.38 hours and 0.29 hours, respectively.
Aellig & Nüesch (1977) carried out pharmacokinetic studies in six
men of nine tritium-labelled EAs: dihydroergotamine, dihydroergotoxine,
dihydroergostine, dihydroergocornine, dihydroergovaline, dihydroergonine,
ergotamine, 1-methyl-ergotamine and bromocriptine. Each compound was
administered to six subjects in a randomized cross-over design as single oral and
intravenous doses, with an interval of at least 2 weeks between the administrations.
The mean age of the subjects was between 58 and 71 years (range: 37–85 years)
62
Ergot alkaloids
and the mean body weight was between 55 and 71 kg (range: 36–89 kg). Oral
doses ranged from 0.2 to 3.0 mg and intravenous doses ranged from 0.1 to 1 mg
depending on the compound tested. The compounds were administered in the
morning after a 12-hour overnight fast. For ergotamine, the doses tested were
0.2 mg (intravenous) and 1.0 mg (oral).
All compounds showed the highest plasma concentration about
1.8 hours after oral administration (range 1.0–2.7 hours). The mean maximum
plasma concentration in ng-equivalents per mL, standardized to a 1-mg oral
dose, was 0.56 (range: 0.42–0.77) for hydrogenated and about 2.0 (range: 1.5–2.3)
for nonhydrogenated EAs. For ergotamine, the Tmax was 2.1 ± 0.8 hours and the
Cmax was 1.52 ± 0.09 ng eq/mL.
Cumulative urinary excretion data after oral and after intravenous
administration were used to calculate a quotient of absorption. Values between
25 and 30% were found for most dihydrogenated EAs (dihydroergotamine,
dihydroergotoxine, dihydroergostine and dihydroergocornine), the only
exceptions being dihydroergovaline and dihydroergonine, which were less well
absorbed. For ergotamine, the quotient of absorption was 62 ± 3%. Aellig &
Nüesch (1977) concluded that all the EAs investigated were rapidly absorbed
with an absorption half-life of around 0.5 hours, the peak plasma concentration
being reached approximately 2 hours after oral administration.
Plasma ergotamine levels were measured by radioimmunoassay in 11
volunteers (five females and six males) after a single oral dose of ergotamine
tartrate (2 mg in tablet form, mean dose of 0.031 mg/kg bw) (Ala-Hurula et al.,
1979a). The mean age of the volunteers was 40 years (range: 18–56 years) and
the mean body weight was 65 kg (range: 49–75 kg). Blood was collected from
30 minutes to 48 hours after administration. Wide variation in plasma ergotamine
levels was observed between subjects. A mean peak concentration of 0.36 ±
0.08 ng/mL was found 2 hours after administration. The level decreased below
the detection limit (0.1 ng/mL) beyond 6 hours in most of the participants, but in
7/11 of them, a second rise occurred between 10 hours and 48 hours.
After oral administration of ergotamine tartrate to migraine patients,
no ergotamine could be detected in plasma by high-performance liquid
chromatography (HPLC) with fluorescence detection (detection limit: 0.1 ng/
mL) in two studies (Ekbom et al. 1981; Ibraheem, Paalzow & Tfelt-Hansen, 1983).
Ekbom et al. (1981) studied nine male patients with cluster headache 15–600
minutes after oral therapeutic doses of ergotamine tartrate (+ caffeine). The mean
age of the subjects was 36 years (range: 29–45 years) and the mean body weight
was 70 kg (range: 61–84 kg). Five patients received a constant dose of 2–4 mg
daily for at least seven days. Four patients were given 1 mg five times on one day
and three patients received a single oral dose of 2 mg. Ibraheem, Paalzow & Tfelt-
Hansen (1983) studied seven migraine patients (outside attacks) who received
63
ergotamine tartrate intravenously and, at least 14 days later, 2 mg of ergotamine

tartrate in tablet form after fasting from midnight. After 2 hours, a light meal
was served. The mean age of the patients was 43 years (range: 37–54 years) and
the mean body weight was 57 kg (range: 51–67 kg). Blood was collected from
10 minutes up to 54 hours after administration. The authors estimated that the
maximum possible bioavailablity of ergotamine tartrate would be less than 1%
(Ekbom et al., 1981) or 2% (Ibraheem, Paalzow & Tfelt-Hansen, 1983) and,
according to Ibraheem, Paalzow & Tfelt-Hansen (1983), most of the radioactivity
detected in blood by Aellig & Nüesh (1977) would be in the form of metabolites.
Ala-Hurula et al. (1979b) reported a very low bioavailability of ergotamine
tartrate (plasma level below the limit of detection (LOD) by radioimmunoassay,
1 hour after oral administration) in 7 of 18 migraine patients who used the drug
daily compared with healthy volunteers.
Dihydroergotamine and its metabolites have been measured in plasma
after a single oral administration of 3 mg [3H]-labelled dihydroergotamine
mesylate (as a tablet) to six healthy male volunteers (Maurer & Frick, 1984). They
fasted for 12 hours before dosing and for 4 hours afterwards. Blood samples were
taken up to 60 hours after dosing. Absorption was rapid and the peak plasma
concentration was reached within 3.2 ± 0.8 hours. The plasma concentration
of 8'-hydroxy-dihydroergotamine, the main metabolite, was 5–7 times higher
than the concentration of unchanged dihydroergotamine. The concentrations of
dihydrolysergic acid, its amide and a non-identified metabolite were comparable
to that of the parent drug, present at very low concentration. The plasma level of
non-volatile radioactivity declined biphasically with alpa- and beta-phase half-
lives of 2.1 ± 0.5 hours and 32.3 ± 6.2 hours, respectively.
Eight healthy male volunteers received single doses of dihydroergotamine
of 5 mg, 10 mg and 20 mg orally and 0.1 mg and 0.5 mg intravenously, with an
interval of at least 1 week between administrations (Wyss et al., 1991). The mean
age was 26 years (range 22–31 years) and mean body weight was 78 kg (range 60–
89 kg). Dihydroergotamine in plasma was measured by specific and polyvalent
radioimmunoassay (LOD: 2–14 pg/mL). The maximum plasma levels were

observed 1 hour after administration and were below the detection limit after 48
hours except for two volunteers in whom the polyvalent assay found detectable
amounts for up to 96 hours. After oral administration, rapid absorption was
noted, with a half-life of 8.4 minutes after a lag time of about 10 minutes. From
the individual area under the plasma drug concentration-time curve (AUC)
values (oral and intravenous) a mean absolute bioavailability of 1% ± 0.6% was
calculated, whereas the absolute bioavailability derived from the AUCs after oral
administration of 20 mg (using the polyvalent radioimmunoassay (RIA)) was
6.2%. The first-pass extraction of dihydroergotamine was calculated to be 97%.
64
Ergot alkaloids
de Groot et al. (1994) assessed the pharmacokinetics and bioavailability

of ergometrine in six men. Their mean age was 41 years (range: 33–50 years) and
mean body weight was 72 kg (range: 67–79 kg). A single oral dose of ergometrine
maleate 0.2 mg (= 0.147 mg ergometrine) was taken after a standard breakfast.
One month later, 0.075 mg ergometrine maleate (= 0.055 mg ergometrine) was
injected intravenously in the same volunteers after a similar standard breakfast.
Ergometrine in plasma was measured using HPLC with fluorescence detection
(LOD: 75 pg/mL). After oral administration, the absorption lag time was subject-
dependent and ranged between 0.0073 hours (0.4 minutes) and 0.47 hours (28
minutes). A maximum plasma concentration of 1.16 ng/mL was reached after
54 minutes. The absorption half-life was 0.19 ± 0.22 hours. A large variation
in bioavailability was observed in the six participants, between 34% and 117%
(mean: 76% ± 32%).
The pharmacokinetics of dihydroergotamine in plasma were examined in
six healthy men after single doses of dihydroergotamine: 10 µg/kg intravenously
and 10, 20 and 30 mg orally (Little et al., 1982). Tracer amounts of [3H]-
dihydroergotamine were used. The mean age of the participants was 23.5 years
(range: 18–36 years) and mean body weight was 64.7 kg (range: 60–71 kg). Each
participant received the four treatments, 2 to 4 days apart. Dihydroergotamine in
plasma was measured by radioimmunoassay. Peak concentrations were apparent
within 30 minutes to 1 hour. Mean apparent absorption from the 10-mg dose
was 26.6 ± 10% and ranged from 8.9 to 60.3%. The oral bioavailability after
the 10, 20 and 30 mg doses averaged 0.47 ± 0.07%, 0.59 ± 0.13% and 0.52 ±
0.14% respectively. Inter-patient variability in bioavailability was sixfold. The low
bioavailability was attributed to extensive metabolism during the first passage
through the liver.
In six healthy male volunteers who were given a single oral dose of 20 mg of
[3H]-α-dihydroergocryptine, rapid absorption into the general circulation occurred
with an average rate K01 of 0.99 ± 0.73/hour. Mean age was 28 years (23–36 years)
and mean body weight was 74 kg (59–85 kg). Subjects fasted for 12 hours before
and for 4 hours after dosing. Time to maximum concentration (Tmax) was reached
in approximately 3 hours with a mean of the individual maximum concentration
(Cmax) from the six volunteers of 8.78 ± 5.9 ng eq/mL (Ronca et al., 1996).
In vitro data
Shappell & Smith (2005) showed in vitro that ergovaline in a mixture with its
naturally occurring epimer ergovalinine (60:40) readily moves across human
intestinal cells (Caco-2 cells). After 6 hours of exposure, 25% and 40% of the
administered ergovaline/ergovalinine (6.6 and 25 μM, respectively) crossed the
intestinal cell layer.
65
(b) Distribution
Little is known about the distribution of EAs in the tissues and organs. The
information available is summarized below.
Animal data
No information is available on administration of EAs by the oral route in laboratory
animals. Very limited data are available on intravenous or intraperitoneal
administration, and only for ergotamine. Given the extensive pre-systemic
metabolism, information from studies of non-oral routes of exposure is
considered to be of limited relevance.
After intravenous administration of [3H]-ergotamine (1 mg/kg bw) to rats,
higher radioactivity in liver, lungs, kidney and heart was measured 2 hours after
the injection, in comparison to blood, whereas a low concentration of radioactivity
was observed in the brain (Kalberer, 1970 cited in: Orton & Richardson, 1982;
Eckert et al., 1978 and EFSA, 2012).
In groups of eight male C57Bl/6 J mice, doses of ergotamine (98% pure) at
0, 0.025 or 0.05 mg/kg bw in 1% lactic acid were administered by intraperitoneal
injection (Reddy et al., 2020). Tissues were collected 50 minutes post-treatment
(liver, kidney and brain: dissected into cerebral cortex, thalamus, cerebellum
and brainstem). Quantitation by liquid chromatography coupled with mass
spectrometry (LC-MS) showed high levels of ergotamine in the kidney and
relatively low levels in the liver. Concentrations of ergotamine in the kidney after
the high (1.632 ± 0.289 ng/g) and low dose (0.438 ± 0.157 ng/g) treatment were
11 times (0.148 ± 0.121 ng/g) and 4 times (0.108 ± 0.028 ng/g) higher than in the
liver, respectively, with a clear dose-dependent effect. Ergotamine was consistently
below detectable limits in the cerebral cortex, cerebellum and thalamus (LOD not
mentioned). Following treatment of mice with the high dose, ergotamine was
identified in the brainstem tissue (6.909 ± 5.596 ng/g; n = 4). Although there was
significant variation, levels were 4.2 and 46.5 times higher than in the kidney and
liver, respectively.
In pregnant rats, 60 minutes after intravenous injection of [3H]-
ergotamine (2.5 mg/kg bw), radioactivity was detected in the blood (0.3 mg eq./
kg) and three times greater concentrations were found in the uterus, placenta and
yolk-sac (1.0 to 1.2 mg eq./kg) (Leist & Grauwiler, 1973). Very low radioactivity
was detected in the amniotic fluid (0.05 mg eq./kg) and fetal tissues (0.07 mg eq./
kg). The transplacental passage of ergotamine was estimated to be 2.8%.
Human data
After oral administration of [3H]-dihydroergotamine to six healthy men, peak
concentrations were apparent within 30 minutes to 1 hour. The pharmacokinetic
66
Ergot alkaloids
profile was described by a two-compartment model. The mean volume of

distribution was 14.5 ± 3.1 L/kg (Little et al., 1982).
After oral administration of 2 mg ergotamine tartrate to 10 healthy male
and female volunteers (following an overnight fast), peak plasma concentrations
were achieved about 1 hour after administration as a solid tablet (with 100 mg
caffeine) and 45 minutes following administration as an effervescent tablet (with
50 mg caffeine) (Orton & Richardson, 1982).
After intravenous administration of ergometrine to six men (0.075 mg
ergometrine maleate), the pharmacokinetic profile was described by a two-
compartment model (de Groot et al., 1994). The distribution half-life was 0.18 ±
0.20 hours, and the steady-state volume of distribution was 73.4 ± 22.0 L. After
oral administration of ergometrine maleate (0.2 mg), the pharmacokinetic profile
was described by a one-compartment model.
Following oral administration of 5 or 10 mg of dihydroergotamine
to eight healthy men, a very large volume of distribution (33 L/kg) indicated
extensive tissue distribution (Wyss et al., 1991). The multi-exponential decline
of dihydroergotamine in plasma, with a long terminal half-life, suggested
distribution into a deep compartment. Plasma protein binding was about 94%.
In six healthy male volunteers who were given 20 mg of [3H]-α-
dihydroergocryptine, distribution from the central compartment to the
peripheral compartment occurred with a mean rate constant (K12) of 0.330 ± 0.22/
hour. The mean volume of distribution was 33.9 ± 22.3 L/kg. The rate constant
(K21) of radioactivity washout from the tissue to the central compartment was
0.250 ± 0.130/hour. Plasma radioactivity declined biexponentially with an
overall elimination constant (K10) of 0.029 to 0.146/hour (i.e. half-lives of 23.9–
4.75/hour) (Ronca et al., 1996).
Ala-Hurula et al. (1979b) detected ergotamine tartrate in the cerebro-
spinal fluid of four subjects 1 or 2 hours after oral administration of 2 mg (+
caffeine). The concentrations were comparable to those detected in plasma.
However, Hovdal, Syversen & Rosenthaler (1982) failed to detect ergotamine
in the cerebrospinal fluid after intramuscular and rectal administration to 18
hospitalized patients, but these results could be explained by the detection limit
of the radioimmunoassay method (0.1 ng ergotamine/mL).
The plasma levels in three healthy male and three healthy female
volunteers given 0.25 mg ergotamine tartrate intravenously declined rapidly
over the first 15 minutes after injection (Orton & Richardson, 1982). Thereafter,
ergotamine concentrations fell more slowly and in five of the six subjects no
ergotamine tartrate was detected in the plasma at 240 minutes. The data for each
subject were analysed by computer using a 2-compartment model with first-
order kinetics, and the mean pharmacokinetic parameters were derived. The
mean distribution half-life was 2.43 minutes (range: 1.72–4.55). The apparent
67
volume of distribution was 140.9 L (range: 68–254), greater than the total body
water content, indicating that ergotamine tartrate is concentrated in the tissues.
In vitro data
In an in vitro model using primary porcine brain capillary endothelial cells, Mulac
et al. (2012) observed that ergometrin(in)e, ergotamin(in)e and ergocristin(in)e
(1 or 10 µM) were able to cross the endothelial cell barrier formed to mimic the
blood–brain barrier. For ergotamin(in)e and ergocristin(in)e, the permeability
was high, comparable with the permeability of the amino acid L-leucine in the
same model system. Ergometrin(in)e showed a 10 times lower permeability.
Active transport was identifed for ergometrin(in)e as a substrate for the BCRP/
ABCG2 transporter. No active transport was observed for ergotamin(in)e and
ergocristin(in)e. Furthermore, for the pure 8-(S) isomer, ergocristinine, no
transcellular diffusion was observed, indicating that the transport could be
selective for the 8-(R) form (-ine epimers). In addition, ergocristin(in)e decreased
the integrity of the barrier (reduction of initial transendothelial electrical
resistance). Ergotamin(in)e and ergocristin(in)e had no impact on barrier
integrity. The impact on barrier integrity of ergocristin(in)e was also reflected by
[14C]-sucrose permeability and visualized after staining of tight junction protein
and cell nuclei. Small parts of the cell monolayer were destroyed, resulting in
areas without cell nuclei, and disruption of occludin-staining.
(c) Excretion
Animal data
Nimmerfall & Rosenthaler (1976) investigated the excretion of [3H]-dihydro-
ergocristine mesylate (1 mg/kg bw) in seven male Wistar rats and of [3H]-
dihydroergotamine mesylate (0.25 mg/kg bw) and [14C]-ergotamine tartrate
(0.25 mg/kg bw) in six Rhesus monkeys after oral and intravenous administration.
After ingestion, excretion in the bile was the most important route of elimination
in the rat (13.8 ± 2.4% of the radioactivity on average) and in the monkey (7.6 ±
1.3% for dihydroergotamine and 24.1 ± 5.7% for ergotamine). In contrast, urinary
excretion occurred only to a small extent (mean excretion: 1.2 ± 0.3% in rats,
2.8 ± 0.7% and 7.4 ± 1.6% in monkeys for dihydroergotamine and ergotamine,
respectively). Rat faeces contained 88.6 ± 6.4% of the radioactivity. Monkeys’
faeces contained 91.9 ± 5.0% of the radioactivity from dihydroergotamine and
68.2 ± 6.4% from ergotamine. After intravenous administration to rats, 91% of
the radioactivity was found in the bile and 3.9% in the urine.
After oral administration of [3H]-ergotamine tartrate (1 mg/kg bw) to
six or seven male Wistar rats, excretion was 32.8 ± 9.3% in the bile, 8.6 ± 2.1% in
urine and 63.9 ± 6.7% in the faeces (Eckert et al., 1978). After oral administration
68
Ergot alkaloids
of [3H]-dihydroergocristine mesylate (1 mg/kg bw) to six male Wistar rats,

excretion was 10.4 ± 3.3% in the bile, 1.9 ± 0.2% in urine and 93.2 ± 1.6% in the
faeces (Eckert et al., 1978).
In studies in livestock, no EAs were detected in the milk of eight Holstein
dairy cows collected after 4 weeks of exposure to contaminated diets (Schumann
et al., 2009). The alkaloid exposure varied between 4.1 and 16.3 μg/kg bw per day.
Milk was sampled for four consecutive morning and evening milkings in week
four and pooled for analysis by HPLC with fluorescence detection (LOD: 5 or
10 μg/kg, depending on the EA).
Milk does not appear to be a major excretion route for unmetabolized
ergovaline in goats (Durix et al., 1999). Four lactating goats received ergovaline
intravenously at a dose of 32 µg/kg bw. Milk was sampled after 8, 24, 32 and
48 hours and analysed by liquid chromatography coupled with tandem mass
spectrometry (LC/MS-MS) (LOD, 0.2 µg/L and limit of quantification (LOQ),
0.7 µg/L). Ergovaline was found only in milk sampled 8 hours after dosing, at a
concentration of 0.71 ± 0.17 µg/L.
Human data
Radioactivity measured in the urine of six healthy male volunteers after oral intake
of [3H]-labelled ergotamine tartrate (2 mg) showed a low cumulative excretion
over a 24-hour period of 1.96% ± 0.50% of the dose administered (Schmidt &
Fanchamps, 1974). Meier & Schreier (1976) also reported a low excretion in
urine, after oral administration of 0.2 to 2 mg of [3H]-labelled ergotamine (in a
capsule) or [3H]-labelled dihydroergotamine (in a solution) to different groups of
six volunteers: 4.23% and 3.06% of the administered dose, respectively. The half-
lives of elimination were 34.3 and 30.3 hours, respectively.
The mean elimination half-life for nine tritium-labelled EAs
(dihydroergotamine, dihydroergotoxine, dihydroergostine, dihydroergocornine,
dihydroergovaline, dihydroergonine, ergotamine, 1-methyl-ergotamine and
bromocriptine) in six healthy male volunteers was 2.3 hours (range: 1.4–
6.2 hours) for the alpha-phase and 23 hours (range: 13–50 hours) for the beta-
phase, the longest times being observed with bromocriptine. For ergotamine, the
mean elimination half-life was 2.7 ± 0.9 hours for the alpha-phase and 21 ± 4
hours for the beta-phase (Aellig & Nüesch, 1977). The mean cumulative excretions
measured during the period of urine collection were 4.1% (range: 2.0–12.7%)
and 12.0% (range: 6.2–8.6%) after oral and after intravenous administration,
respectively. Extrapolating to infinity, the values are 4.4% (range: 2.0–13.3%) and
12.3% (range: 6.7–19.1%). After intravenous administration, between 80 and
90% of the EAs are therefore excreted in the faeces (Aellig & Nüesch, 1977).
69
Orton & Richardson (1982) reported a mean elimination half-life

of 96.53 minutes (range: 63.8–154.1) in six volunteers (three males and three
females) given 0.25 mg ergotamine tartrate intravenously. During the 4 hours
of the study, 4.9% of the dose was excreted in the urine, more than 50% of this
during the first hour. In a study of 10 volunteers, 2 mg ergotamine tartrate was
administered orally as a solid tablet (with 100 mg caffeine) or as an effervescent
tablet (with 50 mg caffeine) after an overnight fast. Six hours after taking the solid
tablet, 0.11% of the oral dose was excreted in the urine, and 0.08% after taking
the effervescent preparation. In both cases, 50% of the total amount excreted at
6 hours was excreted in the first 2 hours of the study (Orton & Richardson, 1982).
After intravenous administration of [3H]-dihydroergotamine (10 µg/kg)
to six healthy men, the half-life of the initial alpha-phase of decline averaged
0.23 hours and that of the slower phase 2.37 hours (Little et al., 1982). Total plasma
clearance ranged from 488 to 1379 mL/minute (mean 1002 ± 169 mL/minute).
Excretion of the tritiated dose was rapid and nearly complete by 6 hours. By
24 hours an average of 11.1 ± 1.0% of the administered [3H]-dihydroergotamine
had been excreted in the urine. Following oral administration of a 10-mg dose,
urinary excretion of the tritiated dose was nearly complete by 12 hours and
by 24 hours 3.0 ± 1.1% of the tritiated dose had been excreted. The fraction of
apparently absorbed dose excreted in the urine would correspond to a range of
7.3 to 13.5%.
Dihydroergotamine and its metabolites have been measured in urine
after a single oral administration of 3 mg [3H]-labelled dihydroergotamine
mesylate to six healthy male volunteers (Maurer & Frick, 1984). The volunteers
fasted for 12 hours before dosing and for 4 hours afterwards. Urinary excretion
of total non-volatile radioactivity was low, amounting to 1.01 ± 0.36% of the
dose in the urine samples collected during the 72 hours afer dosing (70% of this
amount was found in the 0–8-hour urine fraction). The parent drug and four
metabolites could be quantified in urine and plasma samples. The concentration
of 8'-hydroxy-dihydroergotamine, the main metabolite, was several times higher
than the concentration of unchanged dihydroergotamine. Dihydroergotamine

was intensively metabolized and only 0.01% of the dose was found in the 0–
24-hour urine fraction.
Following oral administration of 5 or 10 mg of dihydroergotamine to
eight men, the plasma concentrations declined in a bi-exponential manner (Wyss
et al., 1991). A short alpha-phase with a half-life of 1.45 hours was followed
by the beta-phase with a long terminal half-life of 15 hours for unchanged
dihydroergotamine and a terminal half-life of 34.7 hours with the polyvalent
assay (considered to be due to metabolites). The beta-phase contributed 70%,
78% and 87% to the total AUC of unchanged dihydroergotamine after oral
administration of 5 mg, 10 mg and 20 mg, respectively. Over a 48-hour period,
70
Ergot alkaloids
the mean cumulative urinary excretion amounted to 6.7% and 8.8% of the dose
after intravenous administration of 0.5 and 0.1 mg, respectively, whereas, after
oral administration of 20 mg, only 0.04% of the dose was excreted as unchanged
dihydroergotamine. A similar terminal elimination half-life was also calculated
from the urine data alpha-phase of 1 hour and the beta-phase of 11.8 hours.
Renal clearance contributed only 1% to the high total plasma clearance. After oral
administration of 5 mg and 10 mg, the amount of dihydroergotamine excreted in
the urine was too low to be measured reliably.
After oral administration of 0.2 mg of ergometrine maleate to six men,
the elimination half-life was 1.90 ± 0.16 hours (de Groot et al., 1994). In the same
study, after intravenous administration of ergometrine (0.075 mg ergometrine
maleate), the elimination half-life was 2.06 ± 0.90 hours, and the total body
clearance was 35.9 ± 13.4 L/hour.
In six healthy male volunteers who were given 20 mg of [3H]-α-
dihydroergocryptine, total radioactivity recovery in urine and faeces was good,
with 82.78 ± 6.44% of the dose eliminated in faeces and 3.01 ± 0.65% in urine.
The latter concentration was too low to detect metabolites or unchanged drug
by radioactivity image scanning. However, the liquid scintillation count of silica
gel that had been scraped off the thin layer chromatography plates indicated the
presence of metabolites in urine. The terminal elimination half-life was 25.8 ±
7.5 hours (Ronca et al., 1996).
There is no information on the likely presence of ergometrine in the milk
of lactating women (EMEA, 1999). However, after oral dosing with 0.25 mg/
day of the analogue, methylergometrine, up to 1.3 µg/L was present in the milk
of lactating women (Erkkola et al., 1978; Vogel et al., 2004). Vogel et al. (2004)
measured methylergometrine in the milk of 10 lactating women on postpartum
days 3 to 6 at 0.5, 1, 2, 3, 4 and 5 hours postdose. After administration of a single
oral dose of 250 µg of methylergometrine, the breast milk Cmax, determined by
high-performance liquid chromatography coupled with mass spectrometry
(HPLC-MS), was 0.66 µg/L at a Tmax of 1.8 hours, then it steadily declined to
0.20 ng/mL at 5 hours. The milk/plasma ratio was 0.18. These results are in
agreement with an earlier study by Erkkola et al. (1978). After administration
of a single oral dose of 250 µg of methylergometrine to eight lactating women,
measurable amounts of the drug were found in only 5 of 16 milk samples, as
determined by radioimmunoassay 1 and 8 hours after administration (LOD,
0.5 µg/L). The breast milk Cmax was 1.3 µg/L at a Tmax of 1 hour, with levels
becoming undetectable after 8 hours. The milk/plasma ratio was 0.3.
71
2.1.2 Biotransformation
There are limited data available relating to the metabolic pathway of EAs either in
humans or in laboratory animals. Ergotamine metabolism occurs largely through
undefined pathways but is likely to involve cytochrome P450 3A4 (CYP3A4), an
important phase I drug-metabolizing enzyme in humans. The evidence comes
from co-administration of therapeutic compounds known to be potent CYP3A4
inhibitors, such as clarithromycin (Horowitz, Dart & Gomez, 1996) and ritonavir
(Liaudet et al., 1999). Both are reported to be associated with ergotism following
co-administration with ergotamine.
The most common biotransformation of the ergopeptine alkaloids
occurs by opening the tricyclic amino acid ring structure at the proline moiety
(Eckert et al., 1978).
(a) In vivo studies

In their review, Eckert et al. (1978) described the work of a PhD student (Mau-
rer, 1977) on partly [3H]-radiolabelled dihydro-β-ergocryptine administered in-
traperitoneally to male Wistar rats. Sixteen bilary metabolites were isolated and
identified. Oxidation at the C8 position of the proline was a major route of bio-
transformation since most of the metabolites had either a hydroxyl or a carboxyl
group in this position. Two types of dihydroxylated compounds were isolated:
metabolites hydroxylated at C8′ and C9′ and metabolites with hydroxyls at C8′
and C10′, the former occurring in greater quantities. Apart from a glutamic acid
derivative with the acid group at C8′, hydroxyl-glutamic acid derivatives were
also isolated with OH-groups at C9′ and C10′. The glutamic acid derivative and
the dihydroxylated derivatives were the metabolites produced in the greatest
quantities; administration of low doses favoured production of the dihydroxy-
lated derivatives and administration of high doses favoured production of the
glutamic acid derivatives. A metabolite pathway was proposed, with a central
role of the stereo-isomeric metabolites hyroxylated at C8′. The glucuronides of
the monohydroxy (C8′) and dihydroxy (C8′ and C9′) derivatives were mentioned
as minor metabolites, as well as a metabolite hydroxylated at C9′ and conjugated
with glutathione. An epoxide may be formed as an intermediate. Eckert et al.
(1978) noted that the isolated metabolites did not include any demethylated de-
rivatives or N-oxides.
Ergometrine maleate at a dose of 3 mg/kg bw was administered
intravenously to male and female albino rats and the bile was collected for
6 hours (Slaytor & Wright, 1962). The major route of biotransformation was
hydroxylation at position 12 of the ring system, leading to the metabolites
12-hydroxy-ergometrine and its isomer 12-hydroxy-ergometrinine, which are
then conjugated to glucuronic acid and excreted in bile. Following a larger dose
72
Ergot alkaloids
(45 mg/kg bw), some unchanged parent compound and its isomer ergometrinine
as well as glucuronides of ergometrine and ergometrinine (thought to be modified
in the propanolamide side-chain) were observed. Trace amounts of two further
isomeric pairs of metabolites less polar than ergometrine (not further identified
but considered likely to be N-demethylation products) were observed (EMEA,
1999).
Reddy et al. (2020) used electrospray ionization (ESI) + LC-MS
quantitation analysis to determine the presence of ergotamine and its
metabolites in the brain, liver and kidney of C57Bl/6 J male mice 50 minutes
after intraperitoneal administration of ergotamine (98% pure) at 0, 0.025 or
0.05 mg/kg bw in 1% lactic acid. The previously reported metabolic products of
ergotamine (denoted as E1 and E2, in vitro by Rudolph et al., 2019) were present
in relatively high concentrations in the liver compared to the kidney. E1 (mass
divided by charge number (m/z) 598.2670) and E2 (m/z 614.2640) were the result
of ergotamine hydroxylation indicated by the +15.9958 Da (+O) and +31.9928 (+2
O) Da mass shift from the parent ion. As for ergotamine, the fragmentation of the
metabolic products E1 and E2 resulted in neutral loss of water m/z 580.2569 and
596.2489. The most intense signals produced by E1 (m/z 223.1231 and 208.0759)
and E2 (m/z 223.1230 and 208.0758) were consistent with ergotamine fragment
ions of the lysergic acid components. Presence of the fragment ion m/z 308
suggests that hydroxylation of E1 occurs in the peptide group and the fragment
ion m/z 567 ion suggests that hydroxylation is confined to the tetrahydropyrrole
ring. This is further supported by the unique m/z 308 ion, which results in the
fragmentation at the aromatic ring and peptide group containing the hydroxy
group attached to the tetrahydropyran ring. The fragmentation spectrum of the E2
biotransformation product produced the m/z 524 ion resulting in the opening of
the tetrahydropyrrole ring and loss of the dihydroxylated moiety. The m/z 513 ion
also indicates loss of the tetrahydropyrrole ring, further confirming the position
of the hydroxyl group. Together these data suggest that the tetrahydropryrrole
ring is the primary site of metabolism for the ergopeptide alkaloids (Reddy et al.,
2020).
(b) Data from studies in humans

Limited data are available from studies in humans and these are only for
dihydrogenated forms of EAs (semi-synthetic derivatives that do not occur
naturally). They are expected to have a higher bioavailability than the natural
compounds, but the information is considered to be of limited relevance.
Maurer & Frick (1984) found that dihydroergotamine was intensively
metabolized in humans and only 0.01% of the dose of the parent compound was
found in urine collected during 0–24 hours. 8'-Hydroxy-dihydroergotamine was
73
the main metabolite in humans (plasma and urine). The authors characterized
hydroxylation at carbon 8' of the proline structure of dihydroergotamine.
According to the nuclear magnetic resonance spectroscopy (NMR)-integrals
of Hα-C8' and Hβ-C8', they found that the metabolite was a mixture (30/70) of
8'α- and 8'β-hydroxy-dihydroergotamine. In receptor-binding studies performed
with rat brain preparations, 8'-hydroxy-dihydroergotamine had half-maximal
inhibitory concentration (IC50)-values at six monoaminergic binding sites
similar to those of dihydroergotamine. Maurer & Frick (1984) proposed that the
biotransformation of dihydroergotamine in humans is essentially an oxidation of
its peptide moiety.
This metabolite, 8'-hydroxy-dihydroergotamine, showed about the same
potency as dihydroergotamine for venoconstrictor activity. A placebo-controlled
study in seven healthy male volunteers measured changes in venous diameter at
an occlusion pressure of 45 mmHg, after direct local infusion of 0.08 and 0.4 µg
into superficial hand veins (Aellig, 1984).
After a single oral administration of 20 mg dihydroergotamine mesylate
to 16 healthy volunteers (no details provided on the volunteers), levels of 8'-OH-
dihydroergotamine in human plasma were already several times greater than
the parent compound in the first hour after dosing and throughout the 16
hours of the study (Chen et al., 2002). Bicalho et al. (2005) found similar results
with dihydroergocristine mesylate after administration of a single oral dose of
18 mg to 12 healthy male volunteers. The mean peak plasma level of the 8'-OH-
derivative was 20 times greater than the parent compound (5.63 ± 3.34 µg/L and
0.28 ± 0.22 µg/L, respectively).
After administration of a single oral dose of 27 mg dihydroergotoxine
mesylate (a mixture of semi-synthetic EAs: 35% dihydroergocornine, 33%
dihydroergocryptine (2:1 alpha/beta isomers) and 32% dihydroergocristine)
to a healthy volunteer (male, age 45 years, weight 103 kg) Bicalho et al. (2008)
found that mono-hydroxylated metabolites were one order of magnitude higher
in concentration than the parent compounds in human plasma. The plasma
concentration of the three parent compounds remained around the LOQ

(0.02 mg/L) up to 4 hours following dosing. After that they fell below the
LOQ where they remained up to the final blood sampling time at 8 hours. In
contrast, the OH-metabolites were determined in all samples collected. Their
peaks occurred at about 0.5 hours after dosing and were approximately 1 mg/L
hydroxy-dihydroergocornine, 0.5 mg/L hydroxy-dihydroergocryptine and
0.3 mg/L hydroxy-dihydroergocristine.
74
Ergot alkaloids
(c) In vitro studies

In human liver microsomal incubates, 8'-hydroxy-dihydroergotamine was
shown to be the primary metabolite of dihydroergotamine (Maurer & Frick,
1984). 8'-hydroxy-dihydroergotamine was also isolated from incubates of rat
and monkey liver microsome preparations. The biotransformation product of
8'-hydroxy-dihydroergotamine was a metabolite that contains glutamic acid
instead of the initial proline group.
When incubated with bovine liver microsomes, ergotamine was
hydroxylated to the more hydrophilic metabolites, M1 and M2. Similarly, its
isomer was hydroxylated to M1-Iso and M2-Iso (8-hydroxy-derivatives). Further
incubation resulted in a second hydroxylation of M1 and M2 to metabolites
M3 and M4 (8,9-dihydroxy derivatives). A similar metabolite profile (M1, M2,
M1-Iso and M2-Iso) was produced when ergotamine was incubated with liver
microsomes of dexamethasone (an inducer of CYP3A) treated rats (Moubarak &
Rosenkrans, 2000).
Supernatants of alkaloid incubations (ergocristine, ergocryptine,
ergotamine and ergovaline) with equine and human liver S9 fractions were
analysed by reversed-phase liquid chromatography coupled to high-resolution
tandem mass spectrometry with full scan and MS acquisition (Rudolph et al.,
2019). Although various phase I metabolites could be identified, no phase II
metabolites were detected. Metabolite structures were postulated based on
their MS spectra in comparison to those of the parent alkaloids. All compounds
were extensively metabolized yielding nor-, N-oxide, hydroxy and dihydro-
diol metabolites with largely overlapping patterns in equine and human liver
S9 fractions. However, some metabolic steps, for example, the formation of
8'-hydroxy metabolites were unique for human metabolism, while formation of
the 13/14-hydroxy and 13,14-dihydro-diol metabolites were unique for equine
metabolism. Incubations with equine whole liver preparations yielded less
metabolites than the S9 fractions.
Mulac et al. (2011) investigated the metabolism of ergometrine,
ergotamine and ergocristine in human colon and liver cell lines (HT-29, HepG2),
as well as in human primary renal cells (RPTEC), using Fourier transformation
mass spectrometry. Different mono- and di-hydroxylations at the proline partial
structure could be identified.
Dihydroergotoxine (a mixture of semi-synthetic EAs: 35%
dihydroergocornine, 33% dihydroergocryptine (2:1 alpha/beta isomers) and 32%
dihydroergocristine) at 30 µm was added to incubates of rat liver microsomes,
and the resulting major metabolites were identified as mono-hydroxylated
derivatives of the parent compounds (Bicalho et al., 2008). The major metabolite,
75
8'-hydroxy-dihydroergocristine, was produced in incubates of a bovine liver

preparation using dihydroergocristine mesylate as substrate (Bicalho et al., 2005).
Dihydroergotamine was submitted to an in vitro metabolism assay using
human and rat liver microsomes (Bauermeister et al., 2016). Besides the formation
of the known hydroxylated metabolite 8'-OH‑dihydroergotamine, two new major
hydroxylated metabolites were isolated and characterized by MS/MS and proton
NMR analysis as 5-OH‑dihydroergotamine and 11-OH‑dihydroergotamine.
Owing to the asymmetric carbon at the C8 position, EAs exist in two
forms known as the (R)- and (S)-epimers. The (R)-epimers have the suffix “ine”
whereas the (S)-epimers have the suffix “inine”. Merkel et al. (2012) studied
the effect of digestion in vitro on the ratio of (R)- to (S)-isomers of six EAs
(ergometrine, ergosine, ergotamine, ergocornine, ergocryptine and ergocristine)
in cookies baked for 13 minutes. With respect to epimerization, ergometrine
demonstrated minimal effects, with a 4% change in the amount of ergometrine
compared to the total ergometrin(in)e content. For the ergotoxine group
(ergocornine, α-ergocryptine, β-ergocryptine and ergocristine), an intense shift
towards the (S)-epimers was observed. In contrast, digestion of the ergotamine
group (ergosine and ergotamine) resulted in a shift towards the (R)-epimers. The
initial percentage of the (R)-epimers compared to the total content increased
noticeably after digestion (ergosine: 35 to 55%; ergotamine: 32 to 51%). No
change in epimerization was observed when cookies were incubated in saliva
alone or in saliva followed by gastric juice. For the ergotoxine group, digestion
in duodenal juice led to a shift towards the (S)-epimers regardless of whether all
enzymes (bile, trypsin and pancreatin) or only one enzyme was added. For the
ergotamine group, epimerization towards the (R)-epimers occurred with each
enzyme. The experiments including all enzymes, bile and pancreatin caused a
more pronounced effect.
The in vitro metabolism of α-dihydroergocryptine has been studied
in human hepatocytes and two sets of metabolically competent cell lines
expressing one single human cytochrome P450 (1A1, 1A2, 1B1, 2A6, 2C8,
2C9, 2C18, 2C19, 2D6, 2E1 and 3A4). Mono- and dihydroxy- metabolites
could only be detected in the culture media of the cell line expressing human
cytochrome CYP3A4. The same metabolites were found in the media of
cultured human hepatocytes derived from three different donors. After
24 hours of incubation with 1 µM α-dihydroergocryptine, approximately 60%
mono- and approximately 20% dihydroxy- metabolites were detected, i.e.
approximately 80% of α-dihydroergocryptine was metabolized. The data suggest
that α-dihydroergocryptine metabolism in humans is primarily mediated by the
CYP3A4 isoform (Althaus et al., 2000).
Duringer et al. (2005) studied the in vitro metabolism of ergotamine in
mouse liver microsomes and investigated the impact of sex and previous exposure
76
Ergot alkaloids
of mice to ergovaline (1381 µg/kg diet for 2 weeks). Microsomal incubations

produced nine predominant peaks in the HPLC assay. The peaks were confirmed
by LC-MS/MS to be ergotamine, ergotamine epimer, monohydroxylated
metabolites (M1, M2, M1e and M2e) and dihydroxylated metabolites (M3–5).
Hydroxylation of ergotamine took place on the peptide ring structure. A sex
difference for metabolite formation was observed in the non-exposed mice, in
that females produced a greater amount of M1, M1e and M3–5 than males. When
challenged with the ergovaline diet, mice showed differences in concentration
of M3 for line (endophyte-resistant > endophyte-susceptible mice) and sex
(female > male) and of M4 and M5 for sex (female > male). Sex differences in the
metabolism of ergotamine have not been shown before in these lines of mice or
other species used to study metabolism of EAs. This adds a potential source of
variation in the susceptibility to EA toxicity not explored previously.
2.1.3 Effects on enzymes and other biochemical parameters

α-Dihydroergocryptine showed an inhibitory effect on CYP3A4-mediated
testosterone metabolism and additionally could induce CYP3A4 and CYP2E1
mRNA when added at 10 µM to cultured human hepatocytes (Althaus et al.,
2000).
A significant increase in gene expression was observed for various CYP
isoforms, i.e. CYP1A1, CYP2C9, CYP2E1 and CYP3A1 in the livers of male
Wistar rats fed with a diet contaminated with ergovaline (91.5 µg/kg bw per day)
compared to a control group (n = 12 per group). A significant reduction in hepatic
gene expression for CYP3A7 was observed in the rats fed the contaminated diet.
Hepatic gene expression of CYP2C13, CYP1A2 and CYP2C6 was not affected.
Genes for the CYP-inducing nuclear receptors pregnane X receptor and retinoid
X receptor were upregulated in the livers of rats fed the contaminated diet,
compared to the controls. The protein expression of the major CYP isoform,
CYP3A1, was induced to a greater extent in the livers of the treated rats than
in the livers of the controls. Primary hepatocellular cultures from healthy rats
exhibited an ergovaline dose-dependent induction of CYP3A1 protein expression
compared to hepatocytes not exposed to ergovaline (Settivari et al., 2008).
Moubarak et al. (2012) studied the effect of ergotamine on the induction
of CYP3A and the interaction in vivo and in vitro. Five Sprague-Dawley rats
were treated intraperitoneally for 4 days with 100 mg/kg bw ergotamine (in corn
oil). The liver was collected and liver microsomes were prepared. The results
showed that ergotamine and its isomer are substrates for the isozyme CYP3A,
but that these compounds have no effect on the induction of CYP3A after
4 days of treatment. An earlier study on dihydroergotamine in rats showed similar
77
results (no induction of CYP3A4 after 4 days of intraperitoneal administration)

(Moubarak, Rosenkrans & Johnson, 2002).
Using an in vitro assay based on luminescence, Rosenkrans & Ezell (2015)
showed that CYP3A4 activity was inhibited by ergotamine and dihydroergotamine
(20 or 40 µM), but not by ergonovine. The urine of Angus-sired crossbred steers
(n = 39) was collected after grazing tall fescue pastures contaminated with EAs
for 105 days. Urine inhibition of CYP3A4 activity and total alkaloids (determined
using enzyme-linked immunosorbent assay (ELISA)) were correlated. Steers were
genotyped at CYP3A4 single nucleotide polymorphism, C994G. Steer genotype
affected inhibition of CYP3A4 activity by urine; heterozygous steers had the least
CYP3A4 inhibition.
2.1.4 Physiologically based pharmacokinetic (PBPK) modelling

No data available.
2.1.5 Transfer from feed to food

The information regarding a potential carry-over of EAs into products of animal
origin is scarce, as reported in the reviews by EFSA (2005 and 2012).
Broiler chickens were fed diets containing up to 2.03 mg EAs/kg feed
for 35 days. At the end of the experiment, EA residues in liver and breast meat
were lower than 5 ng/g (Dänicke, 2017). Similarly, when ducks received a diet
contaminated with up to 16.4 mg EAs/kg feed for 2 weeks, residues in liver and
breast meat were below 5 ng/g (Dänicke, 2015). When pigs with a body weight of
30 to 115 kg were administered feed containing EAs (up to 4.66 mg EAs/kg), no
residues were detectable in meat and back fat (Mainka et al., 2005).
For dairy cattle (approximately 400 kg bw), the carry-over following
exposure to 50 g ergot sclerotia per animal resulted in milk concentrations
(total alkaloid content) reaching 0.086 mg/L (Parkheava, 1979). Two studies
were performed by Schumann et al. In the first, Holstein Friesian bulls were
fed diets with up to 421 μg/kg dry matter (DM) of total alkaloids for a period
of approximately 230 days and no carry-over into tissues could be detected
(Schumann et al., 2007). In the second study (Schumann et al., 2009), dairy
cows were fed for 4 weeks with EA-contaminated diets (resulting in an alkaloid
concentration of 504.9 and 619.5 μg/kg DM per day) and no EAs were detected
in the milk produced (LOD, 5–10 μg/kg).
In conclusion, the very limited data on tissue distribution and residual
concentrations in edible tissues, milk and eggs, provide evidence of negligible
accumulation of EAs in edible tissues.
78
Ergot alkaloids
2.2 Toxicological studies

2.2.1 Acute toxicity
LD50 values for some natural EAs following oral or intravenous administration
are reported in Table 1 (Griffith et al., 1978). Oral LD50 values were always higher
than intravenous LD50 values in the same species, reflecting the low absorption
and high pre-systemic metabolism subsequent to oral administration. Moreover,
marked differences in sensitivity were observed between species, with the rabbit
being the most sensitive, followed by the rat and the mouse as the less sensitive
species. Oral LD50 values range from 150–3200 mg/kg bw for mice, rats and
rabbits, with the exception of ergometrine in rabbits (27.8 mg/kg bw).
Based on the oral LD50 values in rabbit (mg/kg bw), the natural EAs can
be ordered as follows: ergometrine (27.8) > ergonine (150) > ergotamine (550) >
ergostine (~ 1000). Based on the intravenous LD50 values in rabbit (mg/kg bw),
the natural EAs can be ordered as follows: ergocryptine (both isomers, 0.34) >
ergonine (1.1) ~ ergostine (1.2) ~ ergostine (1.23) > ergovaline (1.7) ~ ergocristine
(1.9) > ergotamine (3.0) ~ ergometrine (3.2) > ergostinine (5.3).
The clinical signs of acute sublethal poisoning relate to neurotoxicity,
including restlessness, miosis or mydriasis, muscular weakness, tremors and
rigidity. Piloerection and tachypnoea were described in mice, rats, rabbits and
guinea-pigs after treatment with ergotamine and ergobasine and, with higher
doses of ergotamine, ergotoxine and ergometrine, convulsions were seen in all
three species. Tail gangrene was observed in 20% of rats 5–7 days after a single
intraperitoneal exposure to a mixture of EAs (ergocornine, α- and β-ergocryptine
and ergocristine) at 25 mg/kg bw.
Administration of ergotamine tartrate to Rhesus monkeys in single
intravenous doses of up to 1 mg/kg did not cause any marked adverse effects.
A dose of 10 mg/kg bw of ergotamine maleate (purity not indicated) was
administered by intraperitoneal injection to a group of six Wistar rats of both
sexes weighing 100–200 g. The treatment induced the 5-HT2A receptor-mediated
behavioural syndrome, namely head and whole body shakes, reciprocal forepaw
treading, lateral head weaving, flat body posture and hind limb abduction (Thorat
et al., 2019).
Reddy et al. (2020) investigated the effects of ergovaline and ergotamine
on blood pressure, heart rate (using non-invasive tail cuff plethysmography) and
motor coordination (latency of falling using accelerating rotarod) after a single
intraperitoneal administration of ergotamine (98% pure) at 0, 0.025 or 0.05 mg/
kg bw or ergovaline (> 97% pure) at 0, 0.015 or 0.025 mg/kg bw in 1% lactic acid
to groups of eight male C57Bl/6 J mice. According to the authors, these doses
correspond to subclinical (low) and potent (high) doses calculated based on
levels of feed intake of ruminants, concentrations in pasture and pharmacological
79
Table 1
LD50 values for some natural EAs following oral or intravenous administration (Griffith et
al., 1978)
Substance Species Oral LD50 (mg/kg bw) Intravenous LD50 (mg/kg bw)
Ergometrine Mouse 460 160
Rat 671 120
Rabbit 27.8 3.2
Ergotamine Mouse 3200 265
Rat 1300 38
Rabbit 550 3.0
Ergosine Mouse ND 33.5
Rat ND 30
Rabbit ND 1.23
Ergocristine Mouse ND 110
Rat (male) ND 64
Rat (female) ND 150
Rabbit ND 1.9
Ergocornine Mouse 2000 275
Rat > 500 95
α-Ergocryptine Mouse ND 275
Rat ND 140
Rabbit ND 0.95
β- Ergocryptine Mouse ND 210
Rat ND 49
Rabbit ND 0.78
Ergocryptine (both isomers) Mouse 870 300
Rat ND 58
Rabbit ND 0.34
Ergostine Mouse 1700 125
Rat > 1000 47
Rabbit ~ 1000 1.2
Ergonine Rabbit 150 1.1
Ergostinine Mouse ND 180

Rat ND 180
Rabbit ND 5.3
Ergotoxine (mixture of ergocristine, Mouse ND 90
ergocornine and ergocryptine)
Rat ND 76
Ergovaline Mouse ND 175
Rat ND 1.7
ND: not determined; number of animals tested not reported by Griffith et al. (1978).
80
Ergot alkaloids
aspects such as bioavailability of the toxins. All treatments (ergovaline high and
low dose, ergotamine high and low dose) induced bradycardia and elevated
systolic and diastolic blood pressures compared to those in the control mice.
High and low doses of ergotamine led to sustained increases in blood pressure
and reduced heart rate, which did not return to baseline during the 50-minute
testing period. No significant impairment in motor coordination by accelerating
rotarod test, 50 minutes post-treatment, was observed.
2.2.2 Short-term studies of toxicity

Griffith et al. (1978) identified repeated-dose toxicity studies in animals treated
with ergot derivatives published between 1932 and 1976. Most of them reported
on intravenous, intramuscular or subcutaneous administration in various species
(cat, cock, dog, frog, guinea-pig, minipig, mouse, rabbit, rat, rhesus monkey and
sheep). Only one short-term study was conducted on the effects of ergotamine
tartrate administered in feed to eight mice (0.25 mg/kg diet) and four rats (1 mg/
kg diet) for 2 months (Langecker, 1932). The original paper (in German) only
provides a short description of the experiment. No symptoms were reported in
the four rats and in six of the mice. One of the eight mice died at day 10 and
another mouse showed alopecia. In other trials conducted for up to 90 days in
mice, rats and guinea-pigs, typical symptoms of chronic ergot poisoning were
observed, such as reduced body weight, gangrene on the atrium, abdominal wall
and tail, as well as diarrhoea, incontinence, paresis of the rear extremities and
alopecia.
(a) Mice
No other studies were identified.
(b) Rats
(i) Four-week studies
Ergotamine tartrate at concentrations of 0, 4, 20, 100 or 500 mg/kg diet was
given to five groups of six Sprague-Dawley rats per group and sex for 4 weeks
(Speijers et al., 1992). The ergotamine tartrate used for the experiment was > 98%
pure. Its stability in the diet was tested during the study. At the beginning of the
experiment, the specific-pathogen-free (SPF) Sprague-Dawley rats weighed 100–
120 g. Urine and blood were collected at the end of the third week of exposure
and after 4 weeks of exposure the animals were killed.
Clinical observations revealed effects of treatment only in the animals
fed the highest concentration (500 mg ergotamine tartrate/kg diet). Redness of
the tail tip was seen in all animals in this group, which in some cases progressed
to necrosis of the tail tip (two of the six males and three of the six females). A
81
significant decrease in body weight and feed intake was observed in both sexes
in the groups fed 100 and 500 mg/kg diet. Slight changes in some haematological
parameters were also seen in these two groups. Biochemical analyses showed
elevated urea concentration (in animals fed 500 mg/kg diet) and decreased T4
(in animals fed 500 mg/kg diet) and thyroid-stimulating hormone (TSH) (100
and 500 mg/kg diet, in males only). A decrease in cholesterol was observed in
the females in the high-concentration group. Urine production was increased
in males at 100 and 500 mg/kg diet. In the females fed 20, 100 and 500 mg/kg
diet, the relative weights of heart, brain and liver were higher than in the control
animals. The relative ovary weights were increased in animals that received 100
and 500 mg/kg diet. In males fed 100 and 500 mg/kg diet the relative weights of
heart and liver were increased. Macroscopic examination of controls and animals
fed the high concentration was performed. In cases of increased histological
findings in animals in the high-concentration group it was also performed on
animals that received 100 mg/kg. Animals in the groups receiving 4 and 20 mg/
kg diet were not examined. Gaseous distension of the duodenum, pale thyroids,
red tail tips and enlarged iliac lymph nodes were observed in the two highest
concentration groups. The histopathological examination revealed only a slight
increase in regenerative and degenerative changes in the kidneys but strong
activation of the iliac lymph nodes in the highest concentration group. The
changes in the thyroids seen in the macroscopic inspection were not confirmed
by microscopic examination. Degenerative changes in the longitudinal skeletal
muscle of the tail were reported in animals of both sexes in the group that received
the highest concentration of 500 mg/kg diet (four out of five males and three out
of six females) and only in males in the group exposed to 100 mg/kg diet (two
out of six males). The tails of animals in the 4 and 20 mg/kg diet groups were not
examined.
Ergometrine maleate at concentrations of 0, 2, 10, 50 or 250 mg/kg diet
was given to six groups of six Sprague-Dawley rats per group and sex for 4 weeks,
equal to approximately 0.2 (range 0.13–0.28), 1 (0.25–1.45), 5 (3.15–6.59) and
25 (16.75–32.74) mg ergometrine maleate/kg bw per day (Peters-Volleberg,

Beems & Speijers, 1996). Two control groups were included: one received the
control diet ad libitum, and the other control group was pair-fed with the highest
concentration group in terms of amount of feed provided, to determine any
effects secondary to a decreased feed intake.
The ergometrine maleate used for the experiment was > 98% pure. Its
homogeneity and stability in the diet was tested during the study. At the beginning
of the experiment, the SPF Sprague-Dawley rats weighed 70–99 g. At week 4 of
exposure, urine and blood were collected. After 4 weeks of exposure, the animals
were killed.
82
Ergot alkaloids
No treatment-related clinical signs were observed during the experiment.

Tail tips were not affected. Body weight was not influenced by ergometrine maleate
treatment, except in females fed 10 mg/kg diet after 4 weeks of exposure, which
showed a significant increase. No treatment-related effects on haematological
and kidney function parameters (creatinine or urea clearance) were seen.
Plasma glucose levels were significantly decreased in females fed 50
and 250 mg/kg diet (but not in males). T4 levels were significantly decreased in
males fed 250 mg/kg diet. Prolactin was determined in serum samples from a
limited number of animals and showed a wide inter-individual variation in all
groups. The authors reported that the levels were markedly decreased in animals
of both sexes in the 50 and 250 mg/kg diet groups (without statistical analysis). In
females that received the high-concentration diet, relative organ weights of heart,
liver and ovaries were increased. In male rats, macroscopic examination revealed
a slight concentration-related increase in the incidence of enlarged mediastinal
lymph nodes. Microscopic examination showed slight reactive hyperplasia in
these enlarged lymph nodes. Treatment-related histopathological changes were
observed in the liver of males and females fed 250 mg/kg diet, which showed
significant evidence of increased glycogen storage.
α-Ergocryptine at concentrations of 0, 4, 20, 100 or 500 mg/kg diet
was given to six groups of six Sprague-Dawley rats per group and sex for 28–
32 days (Janssen et al., 1998, 2000a, b). Two control groups were included: one
received the control diet ad libitum, and the other was pair-fed with the highest
concentration group in terms of amount of feed provided.
The α-ergocryptine used for the experiment was > 99.9% pure. Its
homogeneity in the diet was tested during the study. At the beginning of the
experiment, SPF Sprague-Dawley rats were 23–26 days old. Females weighed on
average 112–115 g and males 151–154 g.
The authors observed that the animals fed 20, 100 and 500 mg/kg
diet were hyperactive (walking constantly in their cages) and had dirty fur. In
addition, the animals in the 500 mg/kg diet group showed a hunched posture and
were readily irritated and aggressive on handling. At week 4 of exposure, urine
and blood were collected. After 4 weeks of exposure, the animals were killed.
Mean body weight, body weight gain, feed intake and feed effciency
were decreased in both sexes in a non-monotonic manner (in the order 100 <
20 = 500 < control = 4 mg/kg diet). Significant changes in animals that received
concentrations higher than 4 mg/kg diet were observed in some haematological
parameters (decreased mean corpuscular volume (MCV) and mean corpuscular
haemoglobin (MCH)), serum enzyme activities (slightly increased/decreased
alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT) and
γ-glutamyl transferase (GGT)), serum urea concentrations (increased), glomular
filtration (decreased creatinine and urea clearances). U-shaped changes
83
were observed for some parameters, which might be caused by the U-shaped
concentration–response relationship for feed intake, due to the dopaminergic
properties of α-ergocryptine. This could be related to inhibition of feed intake
at an intermediate concentration due to activation of satiety mechanisms in
the lateral hypothalamic area and/or due to the activation in the forebrain of
behaviours incompatible with feeding (Janssen et al., 1998).
Relative organ weights were increased (brain, thyroid, heart, liver, kidney,
adrenals, testis and ovary) or decreased (thymus, uterus). For some organs, the
changes were significant in the animals fed 20 and/or 100 mg/kg diet but not
those given 500 mg/kg diet. Most of the changes are considered as indirect effects
of decreased feed intake.
Microscopic examination at autopsy revealed treatment-related findings
in the kidneys (nephrosis), liver (atrophy, glycogen storage), thymus (atrophy), tail
(muscular degeneration), ovaries (atrophy) and uterus (atrophy). In the tail, the
muscular degeneration was characterized by vacuolation, increased eosinophilia,
thinning of muscle fibres and necrosis of individual muscle fibres (Table 2). There
was only a minimal inflammatory reaction. The degenerative muscular changes
were restricted to certain muscle bundles, adjacent to the vertebrae, whereas
other muscles of the tail appeared to be unaffected. The changes are assumed to
be due to the vasoconstrictive properties of ergocryptine.
In a second article (Janssen et al., 2000b), the authors reported the
metabolism and hormonal changes observed in the animals (plasma and
serum). Ergocryptine influenced carbohydrate metabolism and affected thyroid
and pituitary function. Total cholesterol and high-density lipoprotein (HDL)-
cholesterol were decreased in females fed 20, 100 and 500 mg/kg diet but the ratio
of HDL-cholesterol/total cholesterol was only decreased in animals fed 20 mg/kg
diet. In males, only HDL-cholesterol at 20 mg/kg diet was significantly decreased.
Triglycerides and glucose concentrations were decreased in animals of both sexes
in the highest concentration groups. Insulin and glucagon were not changed at
week 4 but had increased in the highest concentration group at the end of the
study, when the animals were allowed to eat prior to blood sampling and necropsy.
Prolactin was decreased in animals of both sexes in the 20, 100 and 500 mg/kg
diet groups (Table 3). T4 was decreased in females fed 500 mg/kg diet and in
males fed 20, 100 and 500 mg/kg diet. TSH was decreased in males fed 500 mg/
kg diet. Follicle-stimulating hormone (FSH) was concentration-dependently
decreased in females but, owing to wide variations, the difference was significant
only for animals in the 500 mg/kg diet group. In males, FSH was increased but
was significant only in the 20 mg/kg diet group. Luteinizing hormone (LH) was
increased in males fed 20, 100 and 500 mg/kg diet.
84
Ergot alkaloids
Table 2
Microscopic findings in the tail of rats exposed to α-ergocryptine for 4 weeks (incidence
report) (Janssen et al., 2000a)
Females Males
0 4 20 100 500 0 4 20 100 500
mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg
diet diet diet diet diet 0pf diet diet diet diet diet 0pf
Animals 6 6 6 5 6 6 6 6 6 6 6 6
examined
Focal muscular 0 0 0 0 4 1 0 0 0 2 6 0
degeneration
Minimal 0 0 0 0 1 1 0 0 0 2 1 0
Slight 0 0 0 0 2 0 0 0 0 0 4 0
Moderate 0 0 0 0 1 0 0 0 0 0 1 0
pf: pair-fed.
Table 3
Prolactin and T4 levels of rats exposed to α-ergocryptine for 4 weeks (Janssen et al., 2000b)
Females Males
0 4 20 100 500 0 4 20 100 500
mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg
diet diet diet diet diet 0pf diet diet diet diet diet 0pf
Prolactin 33±9 27±15 6±3a 11±14a 4±2ab 20±13 9±3 7±3 5±6a 2±1a 2±1ab 11±4
(µg/L)
T4 (nM, 51±10 50±16 41±5 36±10 27±11a 43±13 61±4 62±15 35±7a 29±4a 35±7ab 55±9
week 4)
T4 (nM, 42±8 42±5 37±8 46±11 22±4 ab 40±16 59±8 59±4 37±4a 35±4a 37±8ab 56±12
autopsy)
Free T4 (pM, 12±2 11±2 9±2 10±4 11±11 12±2 14±1 15±1 9±1a 8±1a 9±3ab 13±2
autopsy)
pf: pair-fed with the highest concentration group.
a
Groups significantly different from the control group (P<0.05) according to Dunnett.
b
Groups significantly different from the pair-fed control with the highest concentration group (P<0.05) according to Dunnett.
Serum T4 concentrations were also found to be reduced in subacute

toxicity studies with ergotamine (Speijers et al., 1992) and ergometrine (Peters-
Volleberg, Beems & Speijers, 1996).
(ii) 13-week study

Four groups of 10 Sprague-Dawley rats per group and sex were fed ergotamine
tartrate (EAT) at concentrations of 0, 5, 20 or 80 mg/kg diet and observed for
85
13 weeks (Speijers et al., 1993). The concentrations and the parameters were
selected based on an earlier subacute toxicity study (Speijers et al., 1992).
The EAT used for the experiment was > 98% pure. Its stability and
the homogeneity in the diet were tested during the study. At the beginning
of the experiment, the SPF Sprague-Dawley rats weighed 50–70 g (and were
approximately 4 weeks old). Feed and water intakes were measured twice a week.
Body weight gain was recorded weekly. After 7 weeks and 12 weeks, urine and
serum were sampled to measure the kidney function. After 10 weeks, blood was
sampled for haematological examination. At the end of the experiment, blood
and a small portion of the liver were sampled for biochemical analyses. After
necropsy, organ weights were recorded, and tissues were fixed for a complete
histopathological examination.
After 13 weeks, both body weight gain and feed intake were significantly
decreased in female rats (not in male rats) fed the high concentration of 80 mg
EAT/kg diet compared to the controls.
The haematological examinations showed a signifiant increase in
packed cell volume and erythrocyte (RBC) count in the animals (both males
and females) in the high-concentration group (80 mg EAT/kg diet) only. The
other blood parameters were not significantly different from those of the controls
(haemoglobin concentration (Hb), white blood cells (WBC), thrombocytes
(PLT), MCV and MCH concentration (MCHC)).
Biochemical examination showed increased alkaline phosphatase
activity, LDL-cholesterol and decreased glucose concentrations in the serum of
female rats in the high-concentration group (but not in male rats) after both 12
and 13 weeks. Urine analyses revealed increased urine volume in the females fed
the high (80 mg EAT/kg diet) and low (5 mg EAT/kg diet) concentration diet
after 12 weeks. A significant decrease in the urine volume was observed in the
males in the high-concentration group. No effects were seen on the other urine
parameters (creatinine concentration and clearance, or osmolarity). Relative
spleen and brain weights in the females in the group fed the high concentration
were significantly higher than in the controls and relative pituitary weight was
significantly lower in the high-concentration group (but not in male rats).
Macroscopic examination revealed pale thyroids in animals (9/10
females and 3/10 males) in the high-concentration group and, in males,
haemorrhagic changes were seen in the lymph nodes (2/10). No changes in the
thyroids were observed in the microscopic examination. The only treatment-
related histopathology finding was muscular atrophy in the caudal longitudinal
muscles of the tail of animals in the high-concentration group (0/10 females and
1/10 males in the control group and the group exposed to 5 mg EAT/kg diet, 2/10
females and 1/10 males in the group exposed to 20 mg EAT/kg diet, and 7/10
females and 7/10 males the group exposed to 80 mg EAT/kg diet) (Table 4). This
86
Ergot alkaloids
Table 4
Microscopic findings in the tail of rats exposed to ergotamine tartrate (EAT) in the diet for
13 weeks (incidence report) (Speijers et al., 1993)
0 mg EAT/kg 5 mg EAT/kg 20 mg EAT/kg 80 mg EAT/kg
f m f m f m f m
Animals examined 10 10 10 10 10 10 10 10
No abnormality detected 10 9 10 9 8 9 3 3
Muscular atrophy: slight 0 0 0 1 2 0 0 0
Muscular atrophy: 0 1 0 0 0 1 4 4
moderate
Muscular atrophy: severe 0 0 0 0 0 0 3 3
f, female: m, male.
atrophy consisted of partial or total disappearance of fibres, tinctorial changes

and fibrosis. The low incidence in the control and lower concentration group is
considered by the authors as a background level. In addition to the atrophy in
animals in the high-concentration group, degenerative changes of nerve fibres in
that region were also apparent. No vascular abnormalities could be detected that
might be responsible for these putative ischaemic changes.
Ghanem et al. (2005, 2006) exposed groups of three male albino rats to
EAT (purity not mentioned) by gavage for 13 weeks. Doses per body weight were
stated as being close to the doses used in human treatment (0, 0.03, 0.06, 0.09 or
1.25 mg in 0.5 mL, 1.5 mL or 3 mL of solution, respectively). The rats were 12
weeks old and weighed 250 g at the beginning of the experiment. The strain of
rats is not stated. During the study, the rats did not show any marked abnormal
behaviour or signs of illness. Slight changes in haematological parameters were
observed. Histopathological examination showed damage to the kidney and the
liver with a dose–response relationship. In the kidneys of animals that received
the low dose, slight changes in the interstitium and glomeruli, and inflammation
were observed. At the highest dose, destruction of the glomerular structure and
Bowman’s capsule was seen. In the liver of animals given the low dose, minor
changes and a normal structural pattern was observed. Necrosis of the hepatic
cells and degeneration of the cytoplasm were observed at the dose of 0.06 mg. At
the highest dose, the changes were severe, and the normal pattern of hepatocytes
and architecture of the liver tissue disappeared. The epithelium lining the central
vein disappeared completely; necrosis and inflammation of hepatic tissue was
obvious. Blood vessels were damaged: infiltration of macrophages and other
87
inflammatory cells and occlusions of some of the vessels were observed. Sinusoids
were dilated. Necrosis was also observed in the vicinity of the portal tract area
with disintegration of hepatic cords and hepatocytes. The Committee noted that
similar histopathological findings were not observed in other studies with higher
doses.
(c) Pigs
Three groups of 24 weaned piglets were exposed for 28 days to control feed or
feed contaminated with 1.2 or 2.5 g of sclerotia/kg (i.e. at concentrations close to
EU regulatory limits in feed) (Maruo et al., 2018). Based on the mean feed intake
(1163 and 962 g/day after 2 and 4 weeks of exposure, respectively) and body
weight (17.5 and 27.8 kg after 2 and 4 weeks of exposure, respectively), doses of
total EAs were 159 and 83 μg/kg bw per day after 2 and 4 weeks, respectively for
the animals given the low dose.
After preparation, diets were analysed four times and the mean
concentrations of EAs were 2.36 and 5.05 mg/kg, for diets with ergot
concentrations of 1.2 or 2.5 g of sclerotia/kg, respectively (Table 5). The most
abundant EA was ergotamine, followed by ergosine, ergocristine and their
corresponding -inine epimers.
Contamination with other mycotoxins was investigated. Deoxynivalenol
(DON) was naturally present in wheat (19 μg/kg) and corn (171 μg/kg) but
the amounts were insignificant. The amounts of other mycotoxins, including
deoxynivalenol (DON) acetylated, nivalenol, T-2, HT-2, zearalenone and
fumonisin were below the detection limit.
Seventy-two castrated, 21-day-old crossbreed (P76 X Naïma) piglets
(mean weight 10.7 ± 0.9 kg) were housed in the facility with free access to
control starter feed and water. When they were 34 days old, they were housed in
individual pens and assigned to one of three groups of 12 males and 12 females.
The first group was fed the control diet, the second the ergot concentration 1 diet
and the third the ergot concentration 2 diet. Animals received the experimental
diets for 28 days. Weight and feed consumption were measured on the first day
of the experiment, 14 days later and on the last day of the experiment. At the
end of the experiment (when the piglets were 62 days old) the mean weights
were 27.9 ± 3.8 kg, 27.8 ± 2.6 kg, 25.6 ± 3.1 kg for animals in the control group,
ergot concentration 1 and ergot concentration 2 diet groups, respectively. The
animals were observed daily to detect possible signs of ergot intoxication such
as balance disorders or necrosis of the extremities. At the end of the experiment,
blood samples were taken from the external jugular vein of all the animals for
biochemical and haematological analyses. In addition, six male animals from
88
Ergot alkaloids
Table 5
Content of EAs in experimental feed in a study of pigs by Maruo et al. (2018)
Contamination of the diet (g of sclerotia/kg diet)
Ergot alkaloids (mg/kg diet) 1.2 2.5
Ergotamine 0.52 1.03
Ergotaminine 0.24 0.58
Ergosine 0.29 0.58
Ergosinine 0.16 0.34
Ergocristine 0.26 0.47
Ergocristinine 0.18 0.40
Ergometrine 0.17 0.44
Ergometrinine 0.06 0.12
Ergocornine 0.15 0.30
Ergocorninine 0.11 0.29
Ergocryptine 0.13 0.30
Ergocryptinine 0.09 0.21
Total alkaloids 2.36 5.05
each group were euthanized and liver, jejunum and jejunum with Peyer’s patches
were sampled for histopathological analysis.
Assessment of clinical signs revealed no typical symptoms of acute
toxicity, such as convulsions or muscle spasms, or necrosis of the extremities.
Similarly, no vomiting or fever was observed in animals exposed to EAs.
The effects of EAs on feed intake were detected during the second
week in animals exposed to the highest concentration. For animals exposed
to the lower concentration of ergot, this reduction only appeared in the last
14 days of treatment during the experimental period. The daily feed intake of
animals exposed to the higher concentration of EAs was reduced by about 18%
in comparison with the control group. The reduction in feed ingestion led to a
decrease of animal weight gain, which was significant in the group exposed to the
higher concentration of EAs.
Haematology and serum biochemical analysis performed at the end of
the experiment found that only a few parameters were affected. The percentage
of neutrophils was reduced and the decrease was significant in the group
exposed to the lower concentration, but not in the group exposed to the higher
concentration. Animals exposed to EAs showed an increase in the percentage of
lymphocytes and the difference was again significant in animals exposed to the
lower concentration, but not in the group exposed to the higher concentration.
The percentage of haematocrit was significantly increased in animals in the high-
89
Fig. 2
(A) Liver lesion score. Values are means with the standard errors of the mean represented
by vertical bars (n = 6 animals). Mean values with different letters are significantly different
(P < 0.05). (B) Jejunal lesion score; (C) villi height; (D) number of goblet cells.a
A B C D
a
Values are means of the score, villi height and number of goblet cells/field respectively, with standard errors of the mean represented by vertical bars (n = 6 animals).
Source: Maruo et al. (2018).
concentration group. Biochemical analysis showed a significant concentration-

dependent reduction in the levels of creatine kinase in animals exposed to EAs,
but the values were within the normal range for this species. In addition, the
level of cholesterol decreased, but the difference was significant only in the group
exposed to the lower concentration, and glucose levels increased upon exposure
to EAs, but the difference was significant only in the group exposed to the higher
concentration. The level of bilirubin was decreased in the group exposed to the
higher concentration.
The effects of EAs on the liver and the intestine were investigated through
histological analyses. Exposure to EAs led to mild to moderate lesions of the liver
and the jejunum of the pigs. In the liver, tissue disorganization of hepatic cords,
inflammation and vacuolation of hepatocytes, megalocytosis and necrosis were

the main morphological alterations. Furthermore, animals fed the contaminated
diets exhibited a significant increase in the liver lesion score (Fig. 2).
The main histological changes observed in the jejunum were atrophy of
the villi, oedema of the lamina propria and cytoplasmic vacuolation of enterocytes.
Animals exposed to the higher concentration of EAs displayed a significant
increase of the lesion score in the jejunum compared to control animals (3.4-
fold increase, P < 0.005). Morphometrical analysis revealed a significant decrease
in height of the villi (1.2-fold decrease at both concentrations). The number
of goblet cells decreased significantly in the jejunum (mean 1.6-fold decrease,
90
Ergot alkaloids
P < 0. 001) of piglets exposed to EAs. Similar effects were observed in jejunum
areas with Peyer’s patches (Maruo et al., 2018).
The effects of EAs on mRNA expression in the jejunum were also studied.
The expression of 34 genes coding for junctional proteins, inflammatory and
immunological mediators was evaluated by real-time quantitative polymerase
chain reaction (RT-qPCR). The profile of mRNA expression of different toll-like
receptors and cytokines (TLR4, nuclear factor kappa B (NFkB), interleukin (IL)-
6, IL-8 and tumour necrosis factor-α) showed a tendency to downregulation. In
animals exposed to the higher concentration of ergot, analysis of mRNA expression
of junctional proteins revealed a significant increase in claudin-3 (CLDN3),
claudin-4 (CLDN4), occludin (OCLN), zonula occludens-1 (ZO-1), junctional
adhesion molecule (JAM-A) and E-cadherin (ECAD). The expression of genes
coding for mucin-1 (MUC1), alkaline phosphatase (ALP) and proliferating cell
nuclear antigen was also significantly increased. The overexpression of the genes
involved in maintaining the structure of the intestinal mucosa may reflect an
attempt by the tissue to re-establish its function.
Oresanya et al. (2003) investigated the effect of EAs on growth
performance and clinical symptoms in weaned pigs. Wheat ergot sclerotia
(1880 mg alkaloid/kg – ergocristine (755 mg/kg), ergotamine (680 mg/kg),
ergosine (205 mg/kg), ergocryptine (130 mg/kg), and ergocornine (110 mg/kg))
were added to a basal diet at 0, 0.05, 0.10, 0.25, 0.50 and 1.00% (corresponding to
dietary concentrations of total alkaloids of 0, 1.04, 2.07, 5.21, 10.41 and 20.82 mg/
kg respectively) and fed to 192 weaned pigs (20.4 ± 3.4 days; 6.9 ± 1.3 kg), 32
pigs per concentration, for 28 days, beginning 7 days post-weaning. Pigs fed the
1.00% diet gained 82 and 38% less weight than the controls at weeks 1 and 2,
respectively, and body weight on day 28 was significantly reduced. EAs decreased
average daily feed intake and feed efficiency over the entire period, but average
daily feed intake was not affected during the first 14 days. EAs significantly
decreased serum prolactin and urea nitrogen concentrations measured on day
28. The Committee noted that there was no concentration–response relationship
in the prolactin reduction. The authors identified maximum tolerable ergot levels
in the diet of 0.10 and 0.05% based on average daily weight gain and average daily
feed intake, respectively, corresponding to 2.07 mg and 1.04 mg EAs/kg diet.
Ten weaned 28-day-old Lacombe × Yorkshire × Landrace pigs were
arbitrarily paired, one male with one female, and fed one of five experimental
diets (0.0, 0.225, 0.45, 0.9 or 1.8 g ergot sclerotia/kg diet) ad libitum for 50 days
(Dignean, Schiefer & Blair, 1986). Ergot sclerotia from barley contained 2.27 g
alkaloid/kg sclerotia: ergocristine 48%, ergotamine 18%, ergocristinine 9.5%,
ergocryptine 8%, ergometrine 6%, ergosine 4% and ergocornine 3%. Blood was
collected every 14 days. At the end of the trial, the pigs were necropsied. Tissue
samples were taken from liver, kidney, heart, adrenal gland, spleen, lung, thymus,
91
brain, thyroid, duodenum, ileum, mesenteric lymph nodes, pancreas, ovaries and
peripheral skin of the ear margin and coronary band.
Pigs fed the highest concentration of ergot were less efficient at conversion
of feed to weight gain in spite of greater feed consumption, and animals on the
higher levels of ergot were observed to be much more excitable and difficult to
restrain than control animals after 3 weeks of being fed the experimental diet.
However, no other clinical signs were observed. Haematological evaluation
revealed only occasional leukopenia and monocytosis. Hepatic enzymes and
blood urea nitrogen values varied considerably within the normal range but
showed no correlation with ergot concentration. No gross lesions were present in
any pigs. Histopathological changes were confined to the liver, kidney and spleen.
Hepatic changes were most obvious: in ergot-fed animals, periportal hepatocytes
were swollen with only a little stained cytoplasm evident peripherally. Nuclei
varied in size and in staining intensity. As many as four rows of cells on each side
of the portal area were affected. The remaining cells had granular cytoplasm, and
there was moderate centrilobular (or periacinar) vacuolation identified by oil-
red-O staining as lipid deposition. In animals fed the higher concentrations of
ergot, bile ducts were prominent and ductules were more numerous, suggesting
hyperplasia. The extent of these alterations reflected the ergot concentration.
Cellular vacuolation and cytoplasmic disruption were also evident in renal tubular
epithelium. There was also some attenuation of tubular epithelium associated
with tubule dilation. Hyaline droplets were evident in Bowman’s space, and there
was more lipid within tubules than in control sections. Again, the severity was
associated with the level of ergot in the diet. Splenic differences were not as readily
quantitated, but pigs fed high ergot concentrations had proportionally less white
pulp than red pulp and fewer distinct follicles with mature lymphocytes.
2.2.3 Long-term studies of toxicity and carcinogenicity

No long-term toxicity studies of specific naturally occurring EAs (i.e. ergometrine,
ergotamine, ergosine, ergocristine, ergocryptine or ergocornine) were available.

In an early study (Fitzhugh, Nelson & Calvery, 1944), three series of
experiments were conducted with Osborne-Mendel rats (3 weeks of age) fed
powdered crude ergot (composition not known) in a high protein diet composed
of cornstarch 72%, casein 6%, corn oil 6%, brewer’s yeast 5%, whole liver powder
5%, salt mixture 4% and cod liver oil 2%.
In the first experiment, groups of 20 female rats received a diet with 0, 1,
2 or 5% crude ergot for 6 months. In the second experiment, groups of nine male
and nine female rats received a low protein diet with 0, 1, 2 or 5% crude ergot for
6 months. Since it had been learned from the first experiment that some weanling
92
Ergot alkaloids
rats refused to eat the feed containing 5% ergot, this group was started at a lower
dosage (2%) and the level was increased to 5% after 2 weeks.
In these two experiments, the body weight gain was significantly reduced
at week 15 in the groups fed 5% ergot compared to the controls. In the low protein
experiment, the reduction was more severe in males than in females. For the late
growth gain, no significant difference was observed. A few animals died (exact
number not known). The authors only mentioned that nine rats survived in the
groups fed 5% ergot.
In the third experiment, groups of eight male and eight female rats
received a low protein diet that included 0 or 5% crude ergot, 5% defatted ergot,
5% ergot oil or ergotoxine ethanesulfonate at a dose equivalent to 5% ergot for at
least 1 year and up to 2 years. After 6 months, the diet was changed – the whole
liver powder was eliminated and the starch and casein were increased to 2% and
3%, respectively. The body weight gain was significantly reduced at week 15 in the
groups fed 5% crude and defatted ergot compared to the controls. In both groups,
the males grew more slowly than the females. Males in the ergotoxine group were
also significantly smaller than the controls. A large number of the rats from the
groups on diets containing whole ergot and defatted ergot died (exact number
not known). At the end of the year, animals in the groups fed diets containing
ergotoxine were not significantly different from the controls.
The pathological changes observed were classified into three categories:
i) those observed only in the treated animals and not present in the controls, ii)
those present to some extent in the controls but to a greater extent in the treated
animals, and iii) those caused by inadequacies in certain of the diets and present
equally in control and treated animals.
The authors observed three types of lesions in the first category:
neurofibromas of the ears, necrosis and calcification of the lower ends of the
renal pyramids, and enlargement of the ovaries from marked corpus luteum
hyperplasia. These lesions were noted in 46, 45 and 41 of the 218 treated animals,
respectively. The second category of lesions included calcified renal tubular walls
and tubular casts, most numerous around the corticomedullary junction, and
focal hyperplasia of the squamous epithelium of the proventriculus (forestomach).
The tumours of the ears were all neurofibromas of a uniform gross and
microscopic appearance. They were usually multiple, with individual tumours as
large as 1.2 cm in diameter, although the usual diameter was from 4 to 6 mm. In
two animals, these tumours were also observed in the tail. The earliest tumour
was noted after 9 months of exposure.
The tumours regressed when the feeding of ergot was stopped and
resumed growth when it was restarted.
93
Table 6
Number of rats with ear tumours (Fitzhugh, Nelson & Calvery, 1944)
Groups Number of rats in the group Number of rats with ear tumours
Long-term experiment – low protein diet plus apparent vitamin E deficiency
5% crude ergot 16 6
5% defatted ergot 16 3
5% ergot oil 16 0
5% ergotoxine 16 1
Control 16 0
6 months – low protein diet
5% crude ergot 18 17
2% crude ergot 18 8
1% crude ergot 18 2
Control 18 0
6 months – high protein diet
5% crude ergot 20 6a
2% crude ergot 20 3
1% crude ergot 20 0
Control 20 0
a
Only six rats from this group were studied microscopically; the small size of this group is also due to the death of many rats before the end of the experiment.
2.2.4 Genotoxicity
Data from in vitro and in vivo genotoxicity studies are available only for a limited
number of naturally occurring EAs or their salts (Tables 7 and 8). Ergometrine
tartrate showed no mutagenic potential in vitro in Salmonella Typhimurium
strains (Zeiger et al., 1987). α-Ergocryptine did not induce sister chromatid
exchange in Chinese hamster ovary cells (Dighe & Vaidya, 1988). Agroclavine (a
precursor compound) showed a weak mutagenic response when activated with
rat liver S9 in S. Typhimurium strains (Glatt et al., 1987). However, ergometrine
maleate and ergotamine tartrate induced chromosomal damage in human
leukocytes in vitro (Jarvik & Kato, 1968; Roberts & Rand, 1977a; Dighe & Vaidya,
1988). Ergotamine tartrate and ergometrine maleate induced sister chromatid
exchange in Chinese hamster ovary cells (Dighe & Vaidya, 1988). Semi-synthetic
dihydrogenated derivatives (dihydroergocristine and α-dihydroergocryptine)
also provided negative results in S. Typhimurium strains (Dubini et al., 1990;
Adams et al., 1993).
In vivo, ergotamine tartrate showed no genotoxic potential in the
micronucleus test after intraperitoneal injection in mice and Chinese hamster
(Matter, 1976) and provided negative results in the dominant lethal test after intra-
peritoneal injection in mice (Roberts & Rand, 1978; Matter, 1982). Semi-synthetic
dihydrogenated derivatives (dihydroergocristine and α-dihydroergocryptine)
94
Ergot alkaloids
Table 7
Results of in vitro assays for genotoxicity with EAs
Test system Test object Concentration and test substance Results Reference
Reverse mutation Salmonella Typhimurium Agroclavine, up to 2 µM per plate (in DMSO) −S9: negative Glatt et al. (1987)
TA98, TA100 and TA1537 +S9: weak
positive
Reverse mutation Escherichia coli (WP2 Agroclavine, up to 2 µM per plate (in DMSO) Effects were Glatt et al. (1987)
uvrA) extremely
weak and
quantification
was not
possible
Reverse mutation Salmonella Typhimurium 3.3, 10, 33, 100, 333, 667, 1000 or 2000 µg/plate Negative ± S9 Zeiger et al. (1987)
TA98, TA100, TA1535 and ergometrine tartrate (in DMSO)
TA1537
Reverse mutation Salmonella Typhimurium Dihydroergocristine at 10 000 µg/plate on TA98 Negative ± S9 Dubini et al. (1990)
TA98, TA100, TA1535, and TA1538 strains and at 3000 µg/plate on
TA1537 and TA1538 TA1535, TA1537 and TA100
Reverse mutation Salmonella Typhimurium α-dihydroergocryptine at 50 to 5000 µg/plate Negative ± S9 Adams et al. (1993)
TA98, TA100, TA1535,
TA1537 and TA1538 The chromosome aberration assay was conducted
with Chinese hamster ovary K1-BH4 cells with
and without S9, at 3 to 120 µg/mL. The DNA
repair test was conducted in rat hepatocytes, at
0.01 to 100 µg/plate
Mutation V79 Chinese hamster cells Dihydroergocristine between 0.3 and 30 µg/mL Negative ± S9 Dubini et al. (1990)
Mutation Mouse lymphoma L5178Y α-dihydroergocryptine at 20 to 100 µg/mL Negative ± S9 Adams et al. (1993)
TK+/− cells
Sister chromatid Human lymphocytes 0.1, 0.25 and 0.5 µg/mL of dihydroergotoxinea Negative Tsuchimoto, Matter
exchange mesylate & Deyssenroth
(1979)
Sister chromatid Chinese hamster ovary 10−5, 10−6, 10−7 and 10−8 M of ergotamine Positive Dighe & Vaidya
exchange cells tartrate (1988)
10−5, 10−6, 10−7 and 10−8 M of ergonovine Positive
maleate
10−5, 10−6, 10−7 and 10−8 M of methylergonovine Positive
maleate
Ergocristine only at 10−8 M
10−5, 10−6, 10−7 and 10−8 M of α-ergocryptine Positive
Negative
Chromosomal Human leukocytes 0.1 µg/mL of ergometrine maleate Positive Jarvik & Kato
aberration (1968)
Chromosomal Human lymphocytes 0.1, 0.25 and 0.5 µg/mL of ergotamine tartrate Positive Roberts & Rand
aberration 0.1, 0.25 and 0.5 µg/mL of dihydroergotoxinea (1977a)
mesylate Positive
Chromosomal Human lymphocytes 0.1, 0.25 and 0.5 µg/mL of dihydroergotoxinea Negative Tsuchimoto, Matter
aberration mesylate & Deyssenroth
(1979)
95
Table 7 (continued)
Chromosomal Human lymphocytes 1, 3 and 10 µg/mL of dihydroergocristine Negative Dubini et al. (1990)
aberration
Chromosomal Chinese hamster ovary α-dihydroergocryptine at 3 to 120 µg/mL. The Negative ± S9 Adams et al. (1993)
aberration K1-BH4 cells DNA repair test was conducted in rat hepatocytes,
at 0.01 to 100 µg/plate
Unscheduled DNA Rat hepatocytes α-dihydroergocryptine at 0.01 to 100 µg/plate Negative Adams et al. (1993)
synthesis
Reporter assay Human lymphoblastoid up to 1 mM of dihydroergotamine Negative Hastwell et al.
TK6 cells (2009)
DMSO, dimethyl sulfoxide.
a
Hydergine, an ergot derivative consisting of equal amounts of dihydroergocornine, dihydroergocristine and dihydroergocryptine.
Table 8
Results of in vivo assays for genotoxicity with EAs
Chromosomal Mouse bone marrow 25, 50 or 100 mg/kg bw of dihydroergotoxinea mesylate Positive Roberts & Rand
aberration intraperitoneally (1977b)
25, 50 or 100 mg/kg bw of ergotamine tartrate intraperitoneally Positive
Germinal Dominant lethal test 25, 50 or 100 mg/kg bw of dihydroergotoxinea mesylate Positive Roberts & Rand
mutations in mice intraperitoneally (1978)
25, 50 or 100 mg/kg bw of ergotamine tartrate intraperitoneally Negative
Micronucleus Mouse bone marrow Dihydroergotoxinea mesylate given twice intraperitoneally with Negative Matter & Grauwiler
formation an interval of 24 hours at various doses up to 200 mg/kg bw (1975)
Micronucleus Chinese hamster Dihydroergotoxinea mesylate given twice intraperitoneally with Negative Matter & Grauwiler
formation bone marrow an interval of 24 hours at various doses up to 200 mg/kg bw (1975)
Micronucleus Mouse bone marrow Ergotamine tartrate given twice intraperitoneally with an interval Negative Matter (1976)
formation of 24 hours at various doses up to 300 mg/kg bw
Micronucleus Chinese hamster Ergotamine tartrate given twice intraperitoneally with an interval Negative Matter (1976)
formation bone marrow of 24 hours at various doses up to 300 mg/kg bw
Micronucleus Mouse bone marrow Dihydroergocristine at two doses (50% and 16% of LD50) orally Negative Dubini et al. (1990)
formation
Micronucleus Mouse bone marrow α-dihydroergocryptine at 2.8 g/kg (the maximum tolerable dose Negative Adams et al. (1993)
formation in a preliminary study) orally by gavage
Chromosome Dominant lethal test Ergotamine tartrate at 125 mg/kg bw intraperitoneally Negative Matter (1982)
damage in mice
Chromosome Human lymphocytes 8 and 12 weeks after oral intake by 12 healthy adult male Negative Tsuchimoto &
damage volunteers three times per day of 1.5 mg of dihydroergotoxinea Stalder (1976)
mesylate
a
Hydergine, an ergot derivative consisting of equal amounts of dihydroergocornine, dihydroergocristine and dihydroergocryptine.
96
Ergot alkaloids
also gave negative results in the in vivo mouse micronucleus assay after oral
administration (Dubini et al., 1990; Adams et al., 1993). However, ergotamine
tartrate was reported to induce a significant number of chromosomal aberrations
in bone marrow cells after intraperitoneal injection in mice (Roberts & Rand,
1977b). The semi-synthetic dihydrogenated derivative (dihydroergotoxine
mesylate) was also positive in this assay (Roberts & Rand, 1977b) but negative in
the micronucleus test after intraperitoneal injection in mice and Chinese hamster
(Matter & Grauwiler, 1975; Matter, 1976).
Using a combination of quantitative structure activity relationship
(QSAR) platforms (i.e. VEGA, LAZAR, T.E.S.T. and OECD QSAR Toolbox),
the mutagenic and carcinogenic potential for various EAs were estimated
(Glück et al., 2018). Ergotamine, ergotaminine, ergocristine, ergocristinine,
ergosine, ergosinine, ergocornine, ergocorninine, α- and β-ergocryptine, and
α- and β-ergocryptinine would be classified as non-mutagenic substances (low
probability for mutagenicity). Ergometrinine would be classified as mutagenic
with low reliability.
Taking into account all the available information, the Committee
concluded that naturally occurring EAs do not raise concerns for genotoxicity.
2.2.5 Reproductive and developmental toxicity

In animals, EAs induce effects on ovulation, implantation, early pregnancy,
embryonic and fetal development resulting in abortion, high neonatal mortality,
fetal malformations and growth retardation (Carlsen, Zeilmaker & Shelesnyak,
1961; Deanesly, 1968; Carpent & Desclin, 1969; Mantle, 1969; Floss, Cassady
& Robbers, 1973; Grauwiler & Schön, 1973; Schön, Leist & Grauwiler, 1975;
Griffith et al., 1978; Holstege & Traven, 2014). The effects vary according to the
EA (Griffith et al., 1978).
One of the ways in which EAs can affect reproduction in animals
is by reducing the secretion of prolactin from the pituitary. The release of
prolactin from the pituitary is normally inhibited by dopamine from neurons
in the hypothalamus and the activity of these dopaminergic neurons, in turn, is
regulated by prolactin itself, operating in a short-loop negative feedback system.
EAs reduce prolactin secretion from the pituitary through their activity as
dopamine agonists.
In rodents, such as the mouse, rat and hamster, prolactin originates almost
exclusively in the pituitary. In these species, the maintenance of pregnancy after
mating is critically dependent on release of pituitary prolactin, which maintains
the activity of the corpora lutea that secrete progesterone, allowing implantation
to occur. EAs, by inhibiting prolactin release, can cause loss of implants and
termination of pregnancy in rodents. However, in species such as humans, other
97
primates and guinea-pigs, prolactin is not involved in maintenance of the corpus

luteum in early pregnancy. Rather, the corpus luteum is maintained initially by
LH from the pituitary, then, from the time of implantation, by human chorionic
gonadotrophin (hCG) produced by the early trophoblast. In humans, prolactin
levels increase steadily from about week 20 of gestation onwards and prolactin is
clearly involved in promoting growth of the mammary glands and in release of
breast milk during suckling. With respect to lactation, the function of prolactin in
humans is similar to that in rodents. Unlike rodents, however, humans also have
numerous extrapituitary sites of production of prolactin, where it is regulated
locally by transcriptional mechanisms rather than by dopamine (Ben-Jonathan,
LaPensee & LaPensee, 2008).
(a) Mice
Carlsen, Zeilmaker & Shelesnyak (1961) studied the effect of a single subcutaneous
injection of ergocornine methanesulfonate on early pregnancy in mice. The
animals studied were 3- to 4-month-old hybrid mice (♀C57B1 × ♂CBA)F1,
weighing 22 to 24 g. In the first experiment, 16 pregnant mice were divided into
groups of four and given a single injection of 0.05, 0.1, 0.25 or 0.5 mg ergocornine
methanesulfonate in ethanol. A group of eight mice served as untreated controls
to confirm the fertility index. Ergocornine methanesulfonate had no effect at
0.05 mg, but terminated pregnancy in 50% of the mice at a dose of 0.1 mg and
100% of the mice given doses of 0.25 and 0.5 mg. In the second experiment,
32 mice in groups of four were given a single injection of 0.25 mg ergocornine
methanesulfonate from day 0 to day 7 of gestation. Pregnancy was terminated
in all the mice treated on days 3 or 4. Following treatment on all other days, the
animals delivered normal litters. No evidence of any abnormalities among the
offspring was observed.
Mantle (1969) conducted a series of experiments with female BS/VS
mice fed with 2% sclerotia of Claviceps fusiformis in the diet or with agroclavine
added to the diet at 0.5 mg/kg. The total alkaloid content of the sclerotia was 0.3%
expressed as agroclavine (the major alkaloidal components were agroclavine and
elymoclavine and the minor components were penniclavine, setoclavine and
chanoclavine). At the end of a 20-day period of dietary exposure to the sclerotia,
none of the five female mice in the treated group became pregnant whereas all
the female mice in the control group did. In another experiment with mice fed
0.5 mg ergoclavine/kg diet, the author observed that the most effective period
of treatment by the oral route was during the 2 to 3 days before implantation.
A daily intake of 250 μg agroclavine on days 3 and 4 was found to terminate
pregnancy without inducing any signs of toxicity. Interruption of pregnancy was
also observed after a daily intake of 250 μg ergosine:ergosinine (60:40) on days 3,
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Ergot alkaloids
4 and 5. Other experiments with female mice exposed to agroclavine in the diet
for a few days or 8 weeks showed that they were able to have a normal pregnancy
when they returned to a normal diet. These results suggest that ergoclavine
prevents implantation and that the effects do not last beyond the end of the
exposure period. Agroclavine had no effects on conception.
Fertility
Matter, Tsuchimoto & Deyssenroth (1978) found no effects of dihydroergotoxine
mesylate in the total reproductive capacity test in female mice. Forty-nine 8-week-
old virgin female CD mice were injected intraperitoneally with 250 mg/kg bw of
dihydroergotoxine mesylate and another 49 with the solvent only. Six hours after
treatment, each female was caged permanently for 347 days with an untreated
male of the same strain, aged 8 weeks at first mating. Breeding pens were checked
5 days a week for the presence of newborns. These were culled immediately after
being scored for litter size to maximize the number of births for each mother.
No effects of dihydroergotoxine mesylate on fertility were found when assessed
by the following criteria: average number of newborns per female and per litter,
average number of litters per female, and average length of the fertile period.
Ergotamine tartrate (98.3% pure) had no effect on male fertility in CD-1
mice (aged 8–14 weeks) after a single intraperitoneal injection of 125 mg/kg bw
(Matter, 1982). The males were mated with untreated virgin females on days 8–14
post-treatment. This period corresponded to the late spermatid stage, which,
on the basis of the dominant lethal test, was suspected of being the stage most
sensitive to both ergotamine and triethylenemelamine (TEM), used as positive
control. All F1 males resulting from conceptions during this interval – i.e. 220
males in the ergotamine group and 144 males in the TEM group – as well as 233
control males whose fathers were not treated, underwent individual fertility tests.
Each F1 male was allowed to mate with two virgin females per week for 4 weeks
(a total of eight females/male). Matings were detected by daily examination for
vaginal plugs; pregnant females were thereafter kept separately. These females
were killed 13–16 days after plug formation for uterine analysis according to the
standard protocol of the dominant lethal test. The classification of F1 males as to
their fertility pattern was made on the basis of dead implantations only. Thus, F1
females were classified as (i) fertile, i.e. not more than 20% dead implantations,
(ii) partially sterile, i.e. 50% or more dead implantations, (iii) sterile, and (iv)
questionable, i.e. 21–49% dead implantations. The fertility patterns in the control
and ergotamine groups were similar, whereas in the TEM group there was a slight
reduction in the number of total and living implantations, and an increase in
dead implantations.
99
(b) Rat
Ergocornine methanesulfonate was given by subcutaneous injection to pregnant
rats (Carpent & Desclin, 1969). When administered on the fifth day at low
doses (0.18–0.3 mg), ergocornine methanesulfonate did not interfere with
implantation. Doses from 0.35 to 1.0 mg, although they did not always inhibit
implantation, were very deleterious for the maintenance of pregnancy but did
not significantly increase the incidence of embryopathies. The administration of
1 mg of ergocornine on the eighth day seriously disturbed pregnancy and induced
a very significant increase in fetal malformations. When the same treatment was
given on the twelfth day, it had no detrimental effect on pregnancy. Treatment
with progesterone (5 mg daily), beginning on the day when ergocornine was
given, allowed pregnancy to be almost perfectly maintained. Fetal malformations
decreased when the supplementary progesterone treatment was started on the
eighth day. It was concluded that the teratogenic activity of ergocornine seems to
be mediated by the progesterone deficiency that it induces.
In a short communication, Schön, Leist & Grauwiler (1975) reported that
a single oral administration of ergotamine (10 mg/kg bw by gavage) to pregnant
rats led to death in the fetuses. In early pregnancy, from days 4 to 10, mortality was
only 0–11%, and on days 14 and 15, it was 62% and 59%, respectively. Exposure
on days 13–16 resulted in fetal anomalies; no anomalies occurred before day 12
(i.e. during organogenesis). The effects of ergotamine were totally antagonized by
α-adrenergic blockade with phenoxybenzamine (10 mg/kg subcutaneously). The
authors suggested that the effects of ergotamine were attributable to its action on
maternal cardiovascular function and not to direct embryotoxicity.
In their review, Griffith et al. (1978) summarized the studies conducted in
the 1950s by Shelesnyak and collaborators on ergotoxine, a natural EA with three
components: ergocornine, ergocryptine and ergocristine. A single subcutaneous
injection of ergotoxine ethanesulfonate to rats during early gestation (up to
day 7 post coitum) terminated pregnancy within 1–3 days; threshold doses
ranged between 0.1 and 1.0 mg/rat. Rats given ergotoxine after implantation
was established (day 8 post coitum), delivered normal litters. Of the three
components, ergocristine was only weakly active, the other two were equally
potent. Termination of pregnancy was associated with suppressed deciduoma
formation and with the occurrence of proestrus and estrus within 48–98 hours
after administration. Further investigations showed that:
■■ Ergotoxine had no inherent estrogenic or gonadotrophic activity.
■■ Ergotoxine in doses effective in terminating pregnancy did not
modify the gonadotrophin content of the pituitary.
■■ The presence of the adrenal glands was not essential for this action of
ergotoxine.
100
Ergot alkaloids
■■ Exogenous prolactin was effective in stimulating the ovary to produce

enough additional progesterone to reverse the ergotoxine action.
■■ Reserpine was effective in overcoming the action of ergotoxine.
These findings suggested that, after ergotoxine treatment, the uterus

could still respond to progesterone, the ovary to prolactin and the pituitary to
reserpine, i.e. a direct toxic action on these target organs could be excluded.
Griffith et al. (1978) also compared the potency of some EAs to inhibit
implantation in rats after subcutaneous administration. Ergocryptine was the
most potent, followed by ergocornine, DH-ergocornine, ergonine and DH-
ergocryptine. The least potent were ergometrine, ergotamine, DH-ergotamine
and DH-ergonine. Based on the oral median effective dose (ED50) for implantation
inhibition in rats, the order was as follows (in mg/kg per day on days 6–15
post coitum): ergocornine (1.4), ergonine (5.0), DH-ergocornine (14.2), DH-
ergotamine (>30), DH-ergocryptine (>30), DH-ergonine (59), ergotamine (133)
and ergometrine (209). Ergocryptine was not tested (Griffith et al., 1978).
Griffith et al. (1978) reported unpublished results on doses of various
EAs (oral dose in mg/kg daily from days 6–15 post coitum) needed to produce
50% embryonic mortality when administered to female rats. The order was as
follows: ergonine (4.5), ergovaline (9.7), ergostine (11.8), DH-ergovaline (21.4),
ergotamine (27.3), ergometrine (86.9), DH-ergonine (104), DH-ergotamine
(617) and DH-ergostine (1095).
Fertility
In their review, Floss, Cassady & Robbers (1973) reported the results of a study
conducted in the 1950s by Shelesnyak and collaborators on the effectiveness of
ergotoxine and ergocornine as fertility-control agents in rats. In these trials, only
23 pregnancies occurred as a result of 1303 coital contacts. The fertility index was
decreased to between 1.5 and 4% from a normal 96.5% in the same colony. It was
assumed that ergotoxine terminated early pregnancy by preventing nidation.
(c) Mice, rats and rabbits

OF-1 mice (20–30 g), OFA rats (180–200 g) and Silver Fawn rabbits (2.5–
3.5 kg) were treated orally with ergotamine during mid-gestation following the
procedure of teratological drug testing recommended in the FDA guidelines of
1966 (Grauwiler & Schön, 1973). Ergotamine tartrate (purity not mentioned) was
administered by gavage (in 2% gelatin) at doses of 0, 30, 100 or 300 mg/kg bw in
mice; 1, 3, 10, 30 or 100 mg/kg bw in rats, and 0, 1, 3, 10 or 30 mg/kg bw in rabbits.
Groups of 23–29 mice and 12–28 rats received daily doses of ergotamine on days
6–15, and groups of 11–17 rabbits on days 6–18. The control animals received 2%
gelatin solution. A significant reduction in maternal weight gain was observed in
101
mice at all doses, in rats at all doses (except 3 mg/kg bw) and in rabbits only at the
highest dose. Prenatal resorption was significantly increased at the doses of 100
and 300 mg/kg bw in mice, at 10, 30 and 100 mg/kg bw in rats and at all doses
in rabbits. The mean weight of live fetuses was significantly reduced in mice and
rats, but not in rabbits. At the same dose levels, fetal anomalies (retardation of
skeletal ossification) were observed in mice and rats. In rabbits, type B anomalies
(presumably occurring during the early fetal phase of organ differentiation) were
more frequent at a dose of 30 mg/kg bw. In rats, litter size decreased in parallel
with the increase in prenatal mortality. No teratogenic activity was detected in
any of the three species. The authors explained the effects observed in the study
as a result of an impairment of blood supply to the uterus and placenta, mainly as
a consequence of ergotamine-induced vasoconstriction. A uterotonic effect may
also be involved in the mechanism of action (Grauwiler & Schön, 1973).
(d) Rabbit
Morris et al. (1967) investigated the effect of a single subcutaneous injection
of ergocornine methanesulfonate given on day 3–4 of gestation on ovum
implantation and development in five white New Zealand rabbits (body weight
3.5–4.5 kg). No effects on the number of implantations and no lost or abnormal
fetuses were observed at the dose tested (1.5–2.5 mg/kg bw). At higher doses (4–6
mg/kg bw) death of rabbits occurred.
(e) Guinea-pig
Unmated female guinea-pigs received subcutaneous injections of ergocornine
methane sulfate 3 to 5 days after estrus; two animals received 1 mg and three
animals received 2 mg (in two successive daily doses). The cycle length was not
affected and there was no premature return of estrus. Two other females had
three daily injections each of 2 mg, 6, 7 and 8 days after estrus; one was autopsied
1 day after the last injection and the ovary sectioned serially. The corpora lutea
were normal in size and appearance. In the other female, estrus returned at the
normal time, 9 days after the last injection (Deanesly, 1968).
In the guinea-pig, enlargement of the corpora lutea occurs at the
time when ovarian progesterone is most essential for embryonic growth and
differentiation, between days 15 and 20. This is, therefore, the best time at which
to test substances, such as ergocornine, which may be expected to interfere with
luteal cell secretion.
Six mated guinea-pigs were injected subcutaneously with ergocornine
methane sulfate. One female exposed to 2 mg on day 6 was normally pregnant
when killed on day 15. Of the two females exposed to 2 mg on day 10, one was
pregnant when killed on day 14, and the other ovulated before being killed on
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Ergot alkaloids
day 16. Two more females had doses of 2 mg on days 13 and 14 and were killed
on day 18; again one was normally pregnant, while the other showed a fresh
ovulation. The last female was given 4 mg on day 13 and was pregnant on day
20. None of the six females showed signs of pregnancy regression. All pregnant
animals had normal corpora lutea and there was no indication that ergocornine
had effectively checked progesterone secretion at a critical developmental period
(Deanesly, 1968).
(f) Pig
Twenty-five Landrace × Yorkshire sows were selected when they had weaned their
last litter (Dignean, Schiefer & Blair, 1986). The sows had all reared at least two
previous litters and were randomly assigned to receive one of five diets (0.0, 0.25,
0.5, 1.0 or 2.0 g ergot sclerotia/kg diet) at the time of breeding and maintained
on this diet until they weaned their litter. Ergot from barley contained 2.27 g
alkaloid/kg: ergocristine 48%, ergotamine 18%, ergocristinine 9.5%, ergocryptine
8%, ergometrine 6%, ergosine 4% and ergocornine 3%. They were fed 2.0 kg of the
diet per day during gestation and ad libitum during lactation. Feed consumption
was recorded daily and sows were monitored for abortion throughout pregnancy.
The number of piglets born live or dead, as well as individual birth weights, were
recorded. Number of piglets weaned and individual weaning weights (at 21 days
of age) were tabulated. These parameters were compared between groups of sows.
All pigs that died were necropsied and the cause of death determined. Blood
was collected every 2 weeks during pregnancy, then 1 week prior to farrowing,
on the day of farrowing and 4 days afterwards. Collected serum was frozen and
prolactin levels were determined by radioimmunoassay. The mean of the first
five post-breeding serum values was considered as the prolactin baseline. The
peak height was the difference between the highest value, measured on the day of
farrowing, and the baseline value.
Sows on diets with higher ergot concentrations took longer to consume
their daily allotment and were observed to be more irritable and active than control
animals. Abortion was not seen in any of the sows. Farrowings were uneventful
and piglets were vigorous. Two sows on the 0.25 g/kg diet produced abnormal
piglets. The first had two mummified fetuses and one with hydrocephalus; the
second had one piglet with atresia ani. Neonatal mortalities were due to crushing
and there were no intergroup differences in the number of piglets lost. There were
no significant differences between groups with respect to any factor examined
(reproductive performance and piglet performance). All sows had complete
mammary development and piglets grew at rates consistent with established herd
levels.
103
Serum prolactin peaks showed marked variation between individual

sows regardless of ergot concentration group, and no statistically significant
differences between the groups were found. Four sows, however, failed to exhibit
a prolactin peak at parturition, with levels below 50 ng/mL. All these sows were on
contaminated diets, one on 0.5, two on 1.0 and one on 2.0 g/kg ergot. This failure
to peak was not associated with clinical agalactia. Peripheral gangrene was not
observed, but one sow on the 1.0 g/kg ergot diet experienced transitory swelling
of a rear limb. One sow on the diet with the highest ergot concentration became
recumbent after farrowing and was killed when her condition deteriorated. She
had peripelvic abscesses and fractures of the pelvis, but no evidence of cutaneous
gangrenous lesions.
(g) Other species

Ford & Yoshinaga (1975) observed that ergocryptine mesylate terminated
pregnancy in hamsters when administered (subcutaneously) on day 5 at 1 mg (no
females were pregnant on day 11 in a group of seven females treated) and 0.5 mg
(only 2/7 females were pregnant on day 11). When ergocryptine was given on day
6, the response was diminished (3/6 females given 0.5 mg and 3/8 females given
1 mg were pregnant on day 11), and pregnancy continued after treatment on day
7. The abortifacient action of ergocryptine in hamsters on day 5 of pregnancy was
overridden by exogenous prolactin but not FSH and LH.
Sharma et al. (2002) studied the reproductive toxicity of EAs in mink. Four
groups of 12 females were fed diets containing EAs at concentrations of 0, 3, 6
or 12 mg/kg diet from 2 weeks prior to the breeding season until the kits were
approximately 33 days old (133 days in total). The contaminated oats used in the
diets contained 110 mg/kg total EAs (6.8% ergosine, 12.9% ergotamine, 14.7%
ergocornine, 16.0% ergocryptine and 49.6% ergocristine). Females were mated
with untreated males. EAs caused a transient decrease in feed consumption, but
body weights and organ weights were unaffected. The number of mink whelping
was significantly reduced: 9 mink whelping in the control and 3 mg/kg groups,
compared to four mink in the 6 mg/kg group and one in the 12 mg/kg group. The
gestation period of mink in the 6 mg/kg group was longer than that of controls (61
versus 47 days). In the group that received 12 mg/kg, only one female whelped,
after a gestation period of 62 days. EAs had a significant effect on kit survival: 75%
in the control group survived, 33% in the 3 mg/kg group, 6% in the 6 mg/kg and
none in the 12 mg/kg group. However, average litter size was not affected. During
necropsy, one female in the 3 mg/kg group had four live fetuses in her uterus at 78
days of gestation, and two females in the 6 mg/kg group were discovered to have live
fetuses. The female in the 3 mg/kg group and the two females in the 6 mg/kg group
had five and six live fetuses at 68 and 69 days of gestation, respectively. Besides the
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Ergot alkaloids
females that had live fetuses in the uterus, there were three females in the 12 mg/kg
group that had eight, six and two fetuses in the process of being resorbed at 65, 64
and 56 days of gestation, respectively. Serum prolactin (at the end of the trial) was
significantly depressed in the groups fed with EAs compared to the control group.
Histopathological examination of the tissues showed no alterations attributable to
EAs. This study indicated that ingestion of EAs at concentrations of 3 mg/kg or
higher resulted in reproductive toxicity in mink. The authors postulated that these
effects could be due to induction of uterine motor activity, impairment of the blood
supply to the uterus or the placenta, and reduced prolactin concentration.
Poole & Poole (2019) reviewed the effects of EAs on female reproduction
in grazing livestock species, including altered cyclicity, suppressed hormone
secretion, reduced pregnancy rates, early embryonic loss, agalactia and reduced
offspring birth weights. In cattle, reproductive failure following exposure to EAs
can be attributed to altered ovarian follicle development, luteal dysfunction
and reduced circulating steroid hormone concentrations, leading to reduced
pregnancy rates. Conversely, EA exposure has minimal impact during
reproductive cyclicity and a greater impact during pregnancy and postpartum in
sheep and mares.
2.2.6 Special studies

(a) Mode of action
The pharmacological mechanisms associated with ergot toxicity are complex and
have not been fully delineated (Holstege & Traven, 2014). They include peripheral
vasoconstriction, peripheral adrenergic blockade, reduced secretion of prolactin
and stimulation of uterine smooth muscle (Peters-Volleberg, Beems & Speijers,
1996).
EAs are structurally related to biogenic amines such as norepinephrine,
dopamine and serotonin. This structural feature allows the EAs to interact
with evolutionary related G-protein coupled receptors – GPCR (for example,
dopaminergic, noradrenergic and serotoninergic ones) as agonists and/or
antagonists. The receptor affinity and selectivity, as well as the intrinsic activity
(efficacy), of these compounds are highly dependent upon the substituents present
at positions 1, 6, 8 and 10 of the lysergic acid moiety. In addition, the specific
interaction between EAs and monoaminergic receptors appears to be organ-
specific (Zajdel et al., 2015). Ergotamine displayed high affinity for adrenergic
(α1, α2), dopaminergic (D1, D2) and serotoninergic (5-hydroxytryptamine)
(5-HT1A, 5-HT1B, 5-HT1D, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT5A, 5-HT5B and 5-HT6)
receptors. Ergotamine behaves as an antagonist at adrenergic receptors, partially
inhibits the 5-HT1 receptor, modulates neurotransmitter release presynaptically,
and excites the 5-HT2, 5-HT3, 5-HT4, 5-HT6 and 5-HT7 receptors. Because of their
105
structural differences from the physiological monoamine neurotransmitters,

EAs are generally characterized by a low specificity and selectivity with respect
to the above-mentioned neuroreceptors and, depending on the individual
structure, they can display a complex behaviour as receptor agonists, partial
agonists or antagonists (Mantegani, Brambilla & Varasi, 1999). Serotonin (5-HT,
5-hydroxytryptamine) is an important neurotransmitter that mediates many
central and peripheral physiological functions including food intake, sleep,
sexual behaviour, memory and blood pressure. 5-HT achieves this wide variety
of functions by acting on distinct receptor types (Mantegani, Brambilla & Varasi,
1999).
Ergotamine, ergosine, ergocornine, α-ergocryptine and ergocristine
generally have a higher affinity for α-adrenergic receptors than the lower
molecular weight alkaloids such as ergometrine. However, ergometrine is a
more selective antagonist of 5-HT receptors in smooth muscle. Alkaloids of the
ergotoxine group (ergocornine, α-ergocryptine and ergocristine) have the greatest
α-adrenergic blocking activity as well as having the highest prolactin suppression
potency through their agonistic actions on dopaminergic receptors. On the other
hand, ergotamine is the most potent vasoconstrictor of the natural EAs. It also
has high emetic potency through dopaminergic receptors and produces strong
stimulation of the uterus and other smooth muscles through 5-HT receptors
(Goodman & Gilman, 1990).
Ergotamine and dihydroergotamine have small structural variations but
these lead to clinically important differences in their pharmacological profiles.
For example, dihydroergotamine is about 10 times less potent than ergotamine
at binding to the 5-HT1B receptor. Sullivan et al. (2020) investigated the binding
interactions of ergotamine and dihydroergotamine to the 5-HT1B receptor using
molecular dynamics simulations and dynamic network analysis. They reported
that ergotamine bound more tightly to 5-HT1B with fewer fluctuations and that
the 5-HT1B−ergotamine complex was in a more active conformation state than
the 5-HT1B−dihydroergotamine complex.
Previous in silico modelling also enabled a deep understanding of

the binding sites and receptor–ligand contacts of ergotamine with 5-HT1B and
5-HT2B receptors (Wacker et al., 2013; Wang et al., 2013; Marti-Solano et al.,
2014; Rodríguez, Ranganathan & Carlsson, 2014; Rodriguez et al., 2014) and of
dihydroergotamine with 5-HT1B and 5-HT2B receptors (Wang et al., 2013).
Using in silico modelling, Dellafiora, Dall'Asta & Cozzini (2015)
investigated the interaction between 5HT2A and 5HT2B receptors with ergotamine
metabolites, both experimentally detected molecules (n = 10) and predicted
phase I derivatives (n = 22). Six mono- and di-hydroxylated metabolites were
predicted to interact with 5HT2A, and only one hydroxylated metabolite was
predicted to interact with 5HT2B. None of the putative conjugated metabolites
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Ergot alkaloids
were predicted as able to interact with the receptors. As more than two-thirds
of the metabolites studied were predicted as active, Dellafiora, Dall'Asta &
Cozzini (2015) highlighted the need for further data on serotonergic activity of
ergotamine’s metabolites.
(b) Vascular effects

The vascular effects of EAs have been known for centuries. The vasoconstrictor
effect is related mainly to the partial agonistic properties at α-adrenoreceptors
and is secondary to the partial agonistic effects at 5-HT receptors (Goodman
& Gilman, 1990). EAs have an agonist effect on the α-adrenergic receptors of
the peripheral vascularization. Blood vessels with α-adrenergic receptors are
present in the skin, the sphincters of the gastrointestinal system, kidney (renal
artery) and brain. α-Adrenergic receptors primarily mediate smooth muscle
contraction. In the smooth muscle cells of blood vessels, the principal effect of
activation of these receptors is vasoconstriction, which can lead to ischaemia,
and has been associated with the development of gangrene of the extremities
in poisoned individuals (Tran, Montastruc & Montastruc, 1983 in Cornière,
2014). The occurrence of gangrene in various animal species correlates closely
with the vasoconstrictor potential of the EAs (Griffith et al., 1978). Subacute
and subchronic toxicity experiments with ergotamine in rats showed necrosis
and fibrosis in the tail tips of animals exposed to high concentrations, which is
explained by the vasoconstrictive properties of ergotamine (Speijers et al., 1992,
1993).
Decreased blood flow to the reproductive and digestive systems and to the
central nervous system (CNS) affects hormonal control of these systems, nutrient
delivery, metabolism and excretion. Stimulation of the vascular smooth muscle
causes constriction of the blood vessels in the vascular bed, and the resulting
constriction of intracranial arteries is useful in the treatment of migraine. The
sensitivity of the smooth muscle of the uterus to stimulation by ergot increases
along with the stage of gestation, so the resulting contractions at the end of the
third trimester may induce labour. EAs have been administered postpartum to
prevent haemorrhage (Floss, Cassady & Robbers, 1973).
Janssen et al. (1998) reported that ergocryptine and ergocornine
have vasoconstrictor potencies similar to that of ergotamine, the most potent
vasoconstrictor of the natural EAs.
In isolated human mesenteric and crural veins, ergotamine tartrate
induced long-lasting contractions (Mikkelsen et al., 1981). These contractions
were resistant to repeated washout and were not affected by α-adrenoceptor
blockade but could be abolished by removal of extracellular calcium or by the
presence of the calcium-blocker nifedipine. In contrast to its effect on human
107
mesenteric and crural veins, ergotamine had no contractile effect, but a marked
relaxant effect, on mesenteric arteries mediated via blockade of α-receptors.
The ergotamine-induced contraction was not affected by indomethacin (0.28–
2.8 µM) nor was it influenced by serotonin (5-HT). In both mesenteric and crural
veins, the ergotamine-induced contraction was diminished by the 5-HT blocking
agent, methysergide. In veins, development of tachyphylaxis to 5-HT was
demonstrated. The authors concluded that ergotamine has a direct contractile
effect on isolated human mesenteric and crural veins. These effects are dependent
on unhindered influx of extracellular calcium and are at least partly mediated via
5-HT receptors. In mesenteric arteries, ergotamine acted as an α-adrenoceptor
blocker, and had no contractant effect.
In isolated human superficial temporal artery, ergotamine tartrate
(7.6 × 10−9 M) also induced long-lasting contractions refractive to additional
stimulations and resistant to repeated washout. When tested against 5-HT,
ergotamine acted as a non-competitive antagonist (Ostergaard, Mikkelsen
& Voldby, 1981). The maximum response to ergotamine was only 20% of the
maximum response to serotonin.
The maximal contractions induced in vitro by dihydroergotamine were
higher in the human saphenous vein, followed by the meningeal artery and
the proximal coronary artery, and were smaller in the distal coronary artery,
confirming its venous contractile properties (Labruijere et al., 2015).
In isolated perfused middle cerebral artery of rats, ergotamine and
dihydroergotamine induced similar dose-dependent contractions (Tfelt-Hansen,
Nilsson & Edvinsson, 2007). Smooth muscle cells of the rat middle cerebral
artery contain primarily 5-HT2A receptors and only a minor proportion of 5-HT1B
receptors. The artery returned to baseline only after repeated washing, illustrating
the long on and off effects, probably due to a slow dissociation from the receptor.
In rabbit, ergonovine maleate was used to provoke coronary spasm by
intravenous injection (0.45 μmol/kg) during the infusion of norepinephrine
(12 nmol/kg per minute) through a marginal ear vein (Koike et al., 2016).
In male Wistar pithed rats, the vasopressor (systemic vasoconstriction)

responses to intravenous bolus injections of ergotamine tartrate (up to 310 µg/kg
given cumulatively) were determined after administration of vehicle (saline), or
several α1 or α2-adrenoceptor antagonists, and compared with baseline heart rate
and diastolic blood pressure. The results suggested that the vasopressor responses
to ergotamine were mainly mediated by α1A-, α1B-, α1D-, α2A- and α2C-
adrenoceptors. The vasopressor responses to 310 μg/kg ergotamine were long-
lasting, with no recovery (Villamil-Hernández et al., 2013). Further investigations
in male Wistar pithed rats suggested that ergotamine tartrate induced inhibition
of the vasopressor sympathetic outflow by activation of prejunctional 5-HT1A,
5-HT1B/1D, α2-adrenoceptors and D2-like receptors (Villamil-Hernández et
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Ergot alkaloids
al., 2014). González-Hernández et al. (2019) also reported that prejunctional

5-HT1B/1D, D2-like and α2-adrenergic receptors mediate the sensory-inhibition
induced by ergotamine (intravenous injection, 0.31 µg/kg per minute) in pithed
Wistar rats, whereas higher doses may involve other receptors. Rivera-Mancilla
et al. (2017) also used male Wistar pithed rats to show that the vasopressor
responses to intravenous bolus injections of dihydroergotamine mesylate (up to
3100 µg/kg given cumulatively) were mediated by α1- (probably α1A, α1B and α1D)
and α2- (probably α2A, α2B and α2C) adrenoceptors.
A vasoconstrictive response (reduced diameter) was observed in sheep
(ewes’) uterine and umbilical arteries after exposure to EAs in the diet (4.14 mg
ergovaline + ergovalinine per kg) during pregnancy, which would explain the
reduced fetal weights (Britt et al., 2019; Klotz et al., 2019). Empty uterine and total
placentome weights were lower in ewes exposed to EAs during late gestation. Britt
et al. (2019) suggested that EAs impaired vascular development of the placenta.
In vitro, ergotamine and ergovaline induced a contractile response in
uterine and umbilical arteries collected from pregnant ewes (Klotz et al., 2019).
Like the responses to serotonin, both ergotamine and ergovaline were 100-fold
more vasoactive in the umbilical artery than in the uterine artery. This contractile
response is driven by serotonin receptor 5HT2A mediating vasoconstriction in the
umbilical artery. In nonpregnant horses, ergotamine and ergonovine were not
vasoactive in uterine artery but produced a contractile response in the palmar
artery and vein, as did ergocryptine, ergocristine, ergocornine and ergonovine
(Klotz & McDowell, 2017). Ergovaline was the most vasoactive EA in both the
palmar artery and the palmar vein, followed by ergonovine, whereas ergocristine
induced the lowest contractile response. In lateral saphenous vein biopsies
from Angus steers, ergotamine and ergovaline altered the contractile response
to serotonergic and adrenergic agonists (Klotz et al., 2016). These effects were
reversible after a minimum of 35 days free of EAs (Klotz et al., 2016).
Ergometrine, ergotamine, ergocristine, ergocryptine, ergocornine and
ergovaline produced contractile responses in lateral saphenous veins collected
from Holstein steers (Pesqueira et al., 2014). The various classes of alkaloids
differed in the type of contractile response elicited since ergonovine (an ergoline
alkaloid) did not display the sustained contractile response observed with
ergopeptine alkaloids (Pesqueira et al., 2014).
In isolated rat tail artery, ergovaline also induced a sustained contractile
response (very slow dissociation from the 5-HT2A receptor) (Schöning, Flieger &
Pertz, 2001). Ergotamine was equipotent with ergovaline in eliciting contractile
responses. In rat thoracic aorta, ergovaline showed low efficacy in the activation
of α1-adrenoceptors. It was concluded that the constrictor effect of ergovaline
was mediated by activation of vascular 5-HT2A and 5-HT1B/1D receptors.
109
An in vitro study on bovine vascular smooth muscle cells from the dorsal
metatarsal artery showed that ergonovine and α-ergocryptine stimulated cell
growth, supporting the hypothesis that EAs exert their vascular effects through
hyperplasia of the intima (Strickland et al., 1996). Ergovaline exhibited a dual
action on the cell growth, stimulating growth of quiescent cells but inhibiting
growth of growing cultures.
Owing to the asymmetric carbon at the C8 position, EAs exist in two
forms known as the (R)- and (S)-epimers. The (R)-epimers have the suffix “ine”,
whereas the (S)-epimers have the suffix “inine”. The (S)-epimers of EAs are thought
to be biologically inactive and, therefore, harmless. A major mechanism by which
the (R)-epimers of EAs produce their toxic effect is through vasoconstriction.
Therefore, Cherewyk et al. (2020) sought to examine the vasoactivity potential
(contractile response) of four (S)-epimers, namely ergocryptinine, ergocristinine,
ergocorninine and ergotaminine utilizing an in vitro arterial tissue bath system.
Bovine metatarsal arteries were collected from healthy mixed-breed beef steers.
To assess the contractile response of each (S)-epimer, a cumulative contractile
dose–response curve was constructed by incubating arteries with increasing
concentrations (1 × 10−11 to 1 × 10−6 M) of that (S)-epimer. Contractile responses
were recorded as grams of tension and were normalized to an initial contraction
of phenylephrine. Contrary to the widespread belief, all (S)-epimers tested
were found to be vasoactive and produced a concentration-dependent arterial
contractile response similar to that reported for the (R)-epimers. The arterial
contractile response to ergotaminine was the strongest, followed by ergocorninine,
ergocristinine and ergocryptinine.
Several hydroxylated metabolites were found to retain the biochemical
activity and receptor-binding potential of the parent compound (Müller-
Schweinitzer, 1984). The effects of dihydroergotamine mesylate and five of its
main metabolites, namely 8′-hydroxy-dihydroergotamine, 8′,10′-dihydroxy-
dihydroergotamine, 2,3seco,N(1)formyl,3-keto,8′-hydroxy-dihydroergotamine,
dihydrolysergic acid amide and dihydrolysergic acid were investigated on human
and canine veins in vitro, on canine veins in situ, and in the ganglion-blocked
rat in vivo (Müller-Schweinitzer, 1984). Like dihydroergotamine, the metabolites
8′-hydroxy-dihydroergotamine, 8′,10′-dihydroxy-dihydroergotamine and dihy-
drolysergic acid amide caused contriction of human varicose veins and only weak
α-adrenoceptor blockade. On canine femoral vein strips, the same compounds
produced predominantly α-adrenoceptor blockade and only negligible
stimulation. 8′-OH,N(1)formyl-dihydroergotamine and dihydrolysergic acid
were inactive. The same compounds, which were agonists on human vein strips
in vitro, induced dose-dependent reduction of venous compliance when infused
locally into the dog saphenous vein in situ. In the ganglion-blocked rat, besides
the parent compound, only 8′-hydroxy-dihydroergotamine and 8′,10′-dihydroxy-
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Ergot alkaloids
dihydroergotamine produced an increase in diastolic blood pressure when

injected intravenously.
According to Holstege & Traven (2014) ergocryptine and ergocornine
have vasoconstrictor potencies similar to that of ergotamine, the most potent
vasoconstrictor of the natural EAs.
(c) Uterotonic activity and effects during gestation

In the uterus, EAs can play an agonist role on α-adrenergic receptors, leading to
oxytocic effects (promotion of uterine contractions). The activation of receptors
is characterized by an increase in the three parameters of uterine contraction:
frequency, amplitude and basic tone (Tran, Montastruc & Montastruc, 1983; in
Cornière, 2014). Janssen et al. (1998) reported that ergocryptine, ergocornine
and ergocristine display oxytocic activity, cause vascular spasms, and inhibit
ovulation and implantation. Of the EAs, ergometrine has the most oxytocic
activity.
In their review, Griffith et al. (1978) suggested that embryotoxicity
in rats given ergotamine is caused by α-adrenoceptor stimulation producing
hypoxia. This was based on studies showing that the ergotamine effect can be
totally antagonized by α-adrenoceptor blockade (phenoxybenzamine 10 mg/
kg, administered subcutaneously) (Grauwiler & Leist, 1973; Schön, Leist &
Grauwiler, 1975).
Grauwiler & Leist (1973) showed that ergotamine (2.5 mg/kg
intravenously) reduced the transplacental passage of [3H]-L-leucine, used as a
marker for uteroplacental blood supply, for at least 3 hours, and that this effect
could be antagonized by phenoxybenzamine. However, ergometrine (10 mg/
kg administered intravenously on day 14 of gestation) produced no inhibition
of uteroplacental blood circulation and had no adverse effect on the fetuses.
Ergometrine has a uterotonic activity comparable to that of ergotamine but only
very slight vasoconstrictor potential (Leist & Grauwiler, 1974).
In Sprague-Dawley rats, ergometrine given orally (1 mg/rat) induced
a significant increase in maximal intrauterine pressure (denoting uterotonic
effects) in postpartum females but not in pregnant females (Monji et al., 2018).
In vitro, ergonovine maleate (10−10 to 10−5 M) induced contractility in
myometrial samples obtained from women undergoing caesarean delivery. The
combination of oxytocin with ergonovine produced a superior response to that
of oxytocin alone (Balki et al., 2015). Ergometrine also induced uteronic effects
(on amplitude and frequency of contractions) in human myometrium in vitro
(Morrison, Crosby & Crankshaw, 2016; Crankshaw, Crosby & Morrison, 2017;
Fanning et al., 2017; Ryan, Crankshaw & Morrison, 2019). The α-adrenergic
antagonist phentolamine and the more selective α1-adrenergic antagonist
111
prazosin inhibited the ergometrine-mediated increase in motility index,

amplitude and frequency (Fanning et al., 2017).
(d) Lactation
Mice
A few days before parturition, groups of 6 to 18 female BS/VS mice were fed with
a diet containing sclerotia of Claviceps fusiformis at 0, 0.5, 1.0, 2.0, 3.0 and 5.0%,
equivalent to an intake of EAs of approximately 0, 100, 210, 420, 630 and 500 µg
(Mantle, 1968). Chromatographic analysis showed that the major components
were agroclavine and elymoclavine; the minor components were penniclavine,
setoclavine and chanoclavine. In the groups fed the two highest concentrations,
the diet was not well eaten and one and three females, respectively, died before
parturition. Mantle (1968) observed that the females fed on concentrations of
ergot sclerotia higher than 1% failed to raise their litters, and the condition of the
pups suggested impaired lactation.
In another experiment, 10 pregnant BS/VS mice were studied. Three
days after the birth of litters, when pups were growing vigorously and white milk-
filled stomachs were clearly visible through the abdominal wall, the maternal diet
was changed to 2% ergot (equivalent to 350 µg/day of EAs). Within 1 or 2 days
the extent of the abdominal milk lines in all replicates had decreased noticeably
and within 4 to 26 days of commencing the 2% ergot diet all the pups had died.
At postmortem examination the stomachs were almost empty. Similar effects on
pup mortality were observed when seven lactating mice were fed with 70 mg
agroclavine/kg diet (Mantle, 1968).
Pregnant BS/VS mice were fed untreated meal or a meal containing
50 mg/kg agroclavine from 10 to 12 days before parturition. Within a few hours
of birth, five treated and five control mice were dissected to remove mammary
tissue (Mantle, 1968). The weight of mammary tissues was decreased in treated
females compared to the controls, being in the same range as for virgin untreated
mice.
Rat
Zeilmakla & Carlsen (1962) showed that intraperitoneal injection of 1 mg
ergocornine methane sulfonate to lactating rats temporarily inhibited milk
production, the effect being prevented by treatment with prolactin.
Griffith et al. (1978) compared the potency of some EAs to inhibit
prolactin secretion in rats after subcutaneous or intraperitoneal administration:
ergocornine was the most potent followed by ergocryptine, dihydro-ergocryptine
and dihydro-ergocornine. Ergotamine and ergometrine were the least potent.
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Ergot alkaloids
Flint & Ensor (1979) administered α-ergocryptine subcutaneously (2 mg/

kg bw) on day 4 of pregnancy to inbred Wistar rats (n = 6) experiencing concurrent
lactation and pregnancy. The α-ergocryptine induced a marked reduction of
serum prolactin concentration (collected from the tail vein) compared to a group
control (n = 6). To study long-term effects, nine treated females had a subsequent
pregnancy. The growth rate of their young was significantly reduced on days 5, 6,
7 and 8 of lactation. Some deaths of young occurred in five litters, and only four
out of the nine rats maintained their pregnancy compared with all nine in the
group control.
Shaar & Clemens (1972) correlated the effect of a number of EAs on
serum prolactin levels with parameters such as litter weight gain, body weight
gain of mothers and weight of mammary tissue. Ergocornine hydrogenmalienate,
0.5 mg and 1.0 mg; ergonovine maleate, 4.0 mg; dihydroergocornine, 1.0 mg;
ergotamine tartrate, 4.0 mg; and ergocryptine mesylate, 0.5 mg; were administered
subcutaneously once a day to lactating female rats from day 4 postpartum
(delivery = day 0) to day 8 postpartum. Serum prolactin levels as measured by
radioimmunoassay were depressed significantly as a result of treatment with each EA
when compared with corn oil-treated control rats. These compounds significantly
depressed lactation as assessed by litter weight gain. The total mammary tissue
weight determined in each animal in an ergocornine hydrogenmalienate-
treated group was reduced compared to the weight in animals in a control group
receiving corn oil. Hypophysial homografts equivalent to two pituitary glands
from donor litter mates were transplanted beneath the kidney capsules of mature
hypophysectomized female rats 3 days after hypophysectomy. The rats were
treated for 2 days with 0.5 mg ergocornine hydrogenmalienate beginning 10 days
after transplantation. Serum prolactin levels from ergocornine-treated rats were
lower than levels in hypophysectomized, pituitary-grafted rats receiving corn oil
alone. These results indicate that EAs have a direct action on the pituitary, thus
preventing prolactin secretion, and that the inhibition of lactation occurs partly
as a result of this deficiency in prolactin secretion. Floss, Cassady & Robbers
(1973) commented that action on the pituitary might also be mediated through
the hypothalamus.
Comparisons of the ability of various EAs to suppress lactation through
their potential to inhibit nidation showed good correlation for ergotoxine,
ergocornine, ergocryptine and dihydroergocornine but not for ergometrine
and dihydroergotamine (ineffective for both activities). Ergotamine showed
adverse effects on lactation but little activity in inhibiting implantation. Another
possibility is that differences in the pharmacokinetic properties could account
for the differences in the quantitative effectiveness of various EAs in inhibition
of lactation and nidation (Floss, Cassady & Robbers, 1973; Griffith et al., 1978).
113
Pig
Fifty-one pregnant sows were sequentially inducted into an experiment each
week in groups of four to seven, as they approached within 14 days of farrowing
(Kopinski et al., 2007). Diets containing sorghum ergot sclerotia (Claviceps
africana) at 0 (control), 0.3%, 0.6%, 0.9%, 1.2% or 1.5% w/w (equivalent to 0,
1.4, 2.8, 4.2, 5.6 and 7 mg EAs/kg, 86% as dihydroergosine/kg) were randomly
allocated and individually fed to sows. Diets with ergot were replaced with control
diets after farrowing. Postfarrowing milk production was assessed by direct
palpation and observation of udders, and by piglet responses and growth. Blood
samples were taken from sows on three days each week for prolactin estimation.
The three sows fed 1.5% ergot for 6–10 days preceding farrowing
produced no milk, and 87% of their piglets died as a result of agalactia. One sow
in a group of 13 fed 0.6% ergot for 7–14 days produced no milk. No effects were
observed on milk production in the other 12 sows in this group. In the other
groups, reduced milk production was observed in 3/3 sows fed 1.2% ergot for
8 days, 5/9 sows fed 0.9% for 6–10 days and 1/9 sows fed 0.3% for 5–9 days. No
neonatal piglet mortality was observed in these groups. EAs caused pronounced
reductions in blood prolactin, and first-litter sows had lower plasma prolactin
than multiparous sows, increasing their susceptibility to ergot (Kopinski et al.,
2007).
Diets containing 3% sorghum ergot sclerotia (Claviceps africana) (16 mg
alkaloids/kg, including 14 mg dihydroergosine/kg) were fed to 12 sows from
14 days post-farrowing (mid-lactation) until weaning 14 days later, and their
performance was compared with that of 10 control sows (Kopinski et al., 2008).
Ergot-fed sows displayed a smaller weight loss during lactation (24 kg/animal
versus 29 kg/animal in control sows) despite feed consumption being less (61 kg/
head total feed intake versus 73 kg/head by control sows). The litters of ergot-
fed sows had poorer weight gain over the 14-day period (16.6 kg/litter versus
28.3 kg/litter for controls) despite an increase in consumption of creep feed by
the piglets from the ergot-fed sows (1.9 kg/litter compared with 1.1 kg/litter by
the controls). After 7 days, in sows fed ergot, plasma prolactin was reduced to
4.8 µg/L compared with 15.1 µg/L in the control sows, and then, at weaning,
prolactin was 4.9 µg/L compared with 8.0 µg/L in the control sows. Two sows fed
ergot ceased lactation early (after 10 days of ergot feeding), and their feed intakes,
body weight losses with litter weight gains and creep consumption indirectly
indicated an effect of ergot on milk production (Kopinski et al., 2008).
Milk production was not affected in sows fed up to 0.2% ergot from barley
(containing 2.27 g alkaloid/kg; ergocristine 48%, ergotamine 18%, ergocristinine
9.5%, ergocryptine 8%, ergometrine 6%, ergosine 4% and ergocornine 3%;
equivalent to 4.5 mg alkaloid/kg diet) from mating until 21 days of lactation.
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Ergot alkaloids
However, the authors noted that their result was at variance with other reports
showing agalactia produced by lower ergot levels, and it was suggested that
ergocristine might have lower toxicity than other rye EAs such as ergotamine
and ergocryptine (Dignean, Schiefer & Blair, 1986; Kopinski et al., 2007).
(e) Prolactin
The dopaminergic activity of EAs (dopamine agonist) reproduces the effects of
dopamine at the level of the hypothalamic–pituitary pathways and inhibits the
secretion of prolactin. Intoxication with EAs (or their medical use) therefore
decreases lactation or may even lead to agalactia in sensitive species. Ergometrine,
ergocornine and ergocryptine show this activity (Tran, Montastruc & Montastruc,
1983 in Cornière, 2014). Janssen et al. (1998) reported that ergocryptine,
ergocornine and ergocristine have the greatest prolactin suppression potency and
the largest α-adrenergic blocking potency.
Prolactin is a hormone that is essential for mammogenesis and
lactogenesis in mammalian species (Farmer, 2001). It is secreted by the anterior
pituitary, also known as the adenohypophysis. Prolactin plays a dominant role in
several aspects of the breast, including growth and development of the mammary
gland (mammogenesis), synthesis of milk (lactogenesis) and maintenance of milk
secretion (galactopoiesis) (Binart, 2017). Shaar & Clemens (1972) suggested that
EAs have a direct action on the pituitary thus preventing prolactin secretion, and
that the inhibition of lactation occurs partly as a result of this deficiency. Floss,
Cassady & Robbers (1973) commented that the action on the pituitary is also
mediated through the hypothalamus.
EAs significantly decreased serum prolactin in weaned pigs fed with
0, 0.05, 0.10, 0.25, 0.50 and 1.00% wheat ergot sclerotia in the diet (1880 mg
alkaloid/kg; ergocristine, ergotamine, ergosine, ergocryptine and ergocornine
constituting 40, 36, 11, 7 and 6% of the total, respectively) for 28 days, beginning
7 days post-weaning (Oresanya et al., 2003).
In pregnant sows fed diets containing sorghum ergot sclerotia (Claviceps
africana) at 0 (control), 0.3%, 0.6%, 0.9%, 1.2% or 1.5% w/w (equivalent to 0,
1.4, 2.8, 4.2, 5.6 and 7 mg EAs/kg, 86% as dihydroergosine/kg) for 5–14 days,
a pronounced reduction in blood prolactin was observed, and first-litter sows
had lower plasma prolactin than multiparous sows (Kopinski et al., 2007). Sow
plasma prolactin was reduced in animals fed a diet containing 3% sorghum
ergot (16 mg alkaloids/kg, including 14 mg dihydroergosine/kg) after 7 days of
exposure postfarrowing (4.8 µg/L compared with 15.1 µg/L in the control sows).
And, at weaning, plasma prolactin was 4.9 µg/L compared with 8.0 µg/L in the
control sows (Kopinski et al., 2008).
115
(f) Satiety
EAs have an agonist effect on the α-adrenergic receptors. Activation of α1-
adrenergic receptors produces anorexia. Reduced appetite was observed in rat
studies (Speijers et al., 1992,1993; Janssen et al., 2000a,b). and in rabbit studies
(Canty et al., 2014; Solano-Baez et al., 2018) and, subsidiary to this, decreased
body weight was reported. Janssen et al. (1998) suggested the U-shaped pattern
of changes for some parameters observed in the subacute rat toxicity study with
α-ergocryptine in the diet might be caused by the U-shaped concentration–
response relationship for feed intake. This may be explained by the dopaminergic
properties of α-ergocryptine, leading to inhibition of feed intake at an intermediate
concentration due to activation of satiety mechanisms in the lateral hypothalamic
area and/or the activation in the forebrain of behaviours incompatible with
feeding.
Ergotoxines (ergocryptine, ergocristine and ergocornine) are dopamine
agonists and may inhibit feed intake by activating the satiety mechanisms in the
lateral hypothalamic area and/or produce effects in the forebrain incompatible
with feeding behaviour (Opara et al., 1996; Oresanya et al., 2003).
(g) Immunotoxicity
Filipov et al. (1999) investigated whether major splenocyte-derived cytokines
(interleukin 2 (IL-2), interleukin 4 (IL-4), interferon gamma (IFN-γ)) and
macrophage-derived cytokines (interleukin 1b, (IL-1β), interleukin 6 (IL-6), and
tumour necrosis factor alpha (TNF-α)) were affected by ergotamine. Two groups
of male BALB/c mice (n = 5/treatment) were treated with ergotamine tartrate
(subcutaneous injection) for 10 days at doses of 0, 0.4, 2, 10 or 50 mg/kg bw.
Twenty-four hours after the last treatment, splenocytes (S) were isolated from one
group of animals and macrophages (MO) from the other group for determination
of IL-2, IL-4 and IFN-γ, and IL-1β, IL-6 and TNF-α, respectively. Following
activation with 5 µg/mL concanavalin A (S) and 10 µg/mL lipopolysaccharide
(MO), cells were incubated for 48 and 24 hours, respectively, and supernatants
were collected and assayed for cytokines by ELISA. Additionally, differential white
blood cell (WBC) counts were performed and the neutrophil (N):lymphocyte (L)
ratio calculated. Ergotamine treatment significantly increased IL-6 levels at doses
of 2 mg/kg and greater and TNF-α at the highest dose. There was no treatment
effect on IL-1β, IL-2, IL-4 and IFN-γ. Also, no effect was observed upon total and
differential WBC counts or N:L ratio. Filipov et al. (1999) concluded that the IL-6
increased by ergotamine treatment would result in an increased inflammatory
response.
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Ergot alkaloids
A high alkaloid content in the diet fed to rabbits for 4 weeks adversely
affected the immune system. Rabbits that consumed the diets with 5 and 15%
sclerotia developed respiratory problems (Solano-Báez et al., 2018).
(h) Other effects

Janssen et al. (1998) reported a concentration-related decrease in body temperature
in rats exposed to α-ergocryptine in the diet at fluctuating concentrations from
100 to 750 mg/kg for several days. Hyperthermia in rabbits but hypothermia in
rats and mice was reported after ergotoxine exposure. These effects were reported
to be related to a direct action of ergotoxine on the central “heat regulating centre''
(Loew et al., 1978 in Janssen et al., 1998).
Administration of ergotamine tartrate (40 µg/kg bw per day) for
224 days increased rectal temperature in bulls (Schuenemann et al., 2005a).
British-Continental steers grazing endophyte-infected tall fescue had raised
rectal temperatures in spring but not in autumn (Parish et al., 2013). The
vasoconstrictive effects of EAs on bovine vasculature could result in impaired
ability to dissipate heat.
No effect on body temperature was observed in sows fed for 5–14 days
with a diet containing sorghum ergot sclerotia (Claviceps africana) up to 1.5%
w/w (equivalent to 7 mg EAs/kg, 6 mg as dihydroergosine/kg) (Kopinski et al.,
2007). Nor was any effect seen in ewes exposed to endophyte-infected fescue
seed during gestation (4.14 mg ergovaline + ergovalinine per kg, equivalent to
17.9–21.2 μg ergovaline and ergovalinine/kg bw) (Britt et al., 2019) or exposed
to ergovaline in the diet (497 ± 52 µg/kg, equivalent to 6.8 µg/kg bw) for 28 days
during lactation (Zbib et al., 2014).
Intrathecal and intraplantar injection of ergotamine tartrate (15 nmol)
to female Wistar rats blocked formalin-induced nociception (measured by the
mean number of flinches of the injected paw). By use of antagonist compounds,
it was shown that this antinociceptive action took place via 5HT5A and 5HT1B/1D
receptors located at both spinal and peripheral sites (Vidal-Cantu et al., 2016).
After intrapenile injection to groups of 30 male Wistar rats,
dihydroergotoxine mesylate (1 mg/0.1 mL) showed an erectogenic effect and
ergotamine tartrate showed a detumescent effect compared with saline solution
(Radosavljevic et al., 2012).
Ergotamine tartrate (1 µM) tested in vitro on isolated rat distal colon
increased the contractile tension and the frequency of contraction (Dalziel,
Dunstan & Finch, 2013). This result is consistent with effects of serotonin
agonist drugs that increase gastrointestinal motility via specific serotonin (5HT)
receptors.
117
(i) Cytotoxicity
Ergotamine and ergocornine showed moderate cytotoxicity in V79 cells (lung
fibroblasts from male Chinese hamster) with IC50 of 20 and 32 µM, respectively
(Behm, Föllmann & Degen, 2012) compared to IC50 of 3 and 14 nM for the most
cytotoxic mycotoxins tested in the assay, i.e. T-2 and HT-2.
Ergotamine showed no cytotoxic potential in studies on two renal cell
lines in vitro when tested up to 46 µM (HK62, a human immortalized kidney cell
line and SA7K, a renal cell model using pseudo-immortalized human primary
renal proximal tubule epithelial cells) (Li et al., 2017).
Mrusek et al. (2015) investigated the cytotoxicity of six EAs (agroclavine,
ergosterol, ergocornine, ergotamine, dihydroergocristine and 1-propylagroclavine
tartrate) in a panel of cell lines of different tumour origin (ovarian carcinoma,
brain tumour, prostate cancer, lung cancer, melanoma, colon cancer, renal
carcinoma, breast cancer or leukaemia). 1-Propylagroclavine tartrate showed
the strongest inhibitory activity on tumour cells. Ergocornine, ergotamine and
dihydroergocristine showed similar activity (IC50 around 10−4.5 M). Leukaemia
cell lines were more sensitive to EAs.
Dihydroergotamine tartrate (10 µM) reduced survival of human lung
cancer cells (A549, NCI-H226 or NCI-H460) by the induction of apoptosis and
mitophagy suggesting that dihydroergotamine tartrate could be developed as a
therapeutic agent against lung cancer (Chang et al., 2016).
(j) Effects on tumours

In rodent studies, ergocornine and ergocryptine induced regression of carcinogen-
induced mammary adenocarcinomas (Floss, Cassady & Robbers, 1973).
The effect of ergocornine on transplanted D2-mammary tumour growth
was investigated in 28 BALB/c mice, which were divided into two groups and
administered subcutaneous injections for 5 weeks (Singh et al., 1972). The
control group received saline-ethanol. The experimental group received 20 μg
ergocornine/day for the first week, 50 μg/day from the second to the fourth week,
and 100 μg/day for the fifth week. Throughout treatment, tumours in control
mice grew more rapidly than tumours in the ergocornine-treated animals.
Mammary tumour growth was suppressed by ergocornine, but the tumours did
not regress significantly below their initial size. When ergocornine treatment was
terminated at the end of 5 weeks, a prompt renewal of mammary tumour growth
was observed, and the rate of growth paralleled that of tumours in the controls.
Pituitary prolactin levels were decreased about 70% by ergocornine treatment
as compared to the controls. These results indicate that growth of transplantable
D2-mammary tumours in BALB/c agent-free mice is decreased by ergocornine,
and that this is associated with reduced pituitary prolactin levels.
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Ergot alkaloids
Nagasawa & Meites (1970) found that ergocornine inhibited the growth
of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumours in
rats injected daily for 15 days with a dose of 0.6 mg ergocornine/day. Cassell,
Meites & Welsch (1971) reported a study involving a longer treatment period (28
days). Sixty-eight female Sprague-Dawley rats with tumours were divided into
five groups, and three groups were given subcutaneous injections of ergocornine
or ergocryptine every day for 4 weeks; one intact and one ovariectomized group
served as controls. The intact controls showed a significant increase in number and
size of tumours throughout the treatment period, whereas ergocornine produced
a significant reduction in number and size of tumours, paralleling the effects
seen in the ovariectomized controls. Ergocryptine suppressed mammary tumour
growth throughout the treatment period but produced no significant decrease
in number and size of tumours. When ergocornine treatment was terminated at
the end of 4 weeks, prompt renewal of mammary tumour growth was observed.
Inhibition of mammary tumour growth in rats by these compounds is believed to
be due to their demonstrated suppression of pituitary prolactin secretion.
In the study by Clemens & Shaar (1972), administration of ergocornine
at 0.4 mg/rat daily 11 days before and 5 days after treatment with DMBA
significantly inhibited the induction of mammary tumours in rats. Ergocornine
caused rapid and complete (to palpation) regression of 62% of established,
DMBA-induced, mammary tumours. Examination of sites where tumours had
completely regressed to palpation 2 months after ergocornine treatment was
discontinued, revealed that 64% of the tumours did not recur. The effectiveness of
ergocornine in regressing tumours was found to be related to initial tumour size.
Tumours with volumes up to 1.8 cm3 regressed rapidly after administration of
ergocornine; whereas tumours approximately 14.1 cm3 and larger did not respond
to treatment. Tumours of intermediate size showed growth inhibition, and many
showed regression to varying degrees. Therefore, mammary tumours start to lose
their hormonal dependence as they become larger and may eventually lose it
completely.
2.3 Observations in domestic animals/veterinary toxicology

EAs have negative impacts on growth (decreased feed intake and weight gain)
and reproductive performance (decreased prolactin levels, lower conception
rates and birth weights and, in males, reduced fertilization potential) of domestic
animals, such as cattle, horses, pigs and sheep (reviewed by Klotz, 2015).
Gangrenous ergotism (i.e. fescue foot or fescue lameness in livestock) is one of
the most acute and obvious visible effects of exposure to EAs. The effects of EAs
119
on lactation vary with the livestock species. Ingestion of EAs reduces milk yield
in cattle, horses and sheep (Poole & Poole, 2019).
(a) Pigs
In piglets (n = 8 castrated males and 8 females), ergot mixed into cereal–soyabean
meal based diets at concentrations of 0, 0.5, 1, 2 and 4 g/kg for 35 days induced a
significant decrease in feed intake and live weight gain at the highest concentration
tested (Mainka et al., 2005). The total alkaloid content of the ergot was 2775 mg/kg
(ergocristine 14.9%, ergometrine 8.1%, ergotamine 5.4%, ergocornine 3.2% and
α-ergocryptine 1.9%; the remaining 66.5% alkaloid content was unidentified).
No effects on performance were observed in chickens (n = 28 males) exposed for
21 days; however, the heart weights showed a significant linear decrease.
On a farrow-to-finish pig farm, the exposure of sows to 3.49 mg EAs/kg
diet (sum of ergotamine, ergocristine, ergosine, ergocryptine, ergocornine and
ergometrine) for 10 to 15 days before the end of gestation and to 8.06 mg EAs/kg
diet over 3 to 4 days at the beginning of lactation led to agalactia in 13 of 20 sows
in a batch and to high neonatal mortality rates for all litters (79% on average).
No clinical signs associated with vasoconstrictive effects were observed (Waret-
Szkuta et al., 2019).
(b) Cattle
Angus heifers (n = 36) received either endophyte-infected fescue seed (E+) or
noninfected fescue seed (E−; control) in a total mixed ration for 63 days (Poole et
al., 2018). Infected fescue seeds (E+) contained ergovaline 3.9 mg/kg, ergocryptine
1.9 mg/kg, ergotamine 1.4 mg/kg, ergosine 1.4 mg/kg, ergocornine 0.96 mg/kg
and ergocristine 0.67 mg/kg. The authors used a dietary concentration of 500 μg
ergovaline + ergotamine per kg. The heifers were synchronized at the same stage
of estrous cycle by hormone treatment. Average daily weight gain was decreased
in animals in the E+ group (0.8 kg/day) compared to control heifers (1.0 kg/day)
although daily intake was not affected. Heart rate, rectal temperature, respiration
rate and blood pressure did not differ between treatment groups. Using Doppler
ultrasonography, vasoconstriction was observed in the caudal artery, but not the
caudal vein in heifers consuming the E+ diet. No differences were observed in
corpus luteum area or circulating progesterone and prolactin concentrations in
heifers on the E+ diet compared to controls. There was a significant decrease in
the number of medium-sized follicles and in the diameter of arteries and veins
servicing the ovary and uterus on days 10 and 17 of the estrous cycle. Reduction
in blood flow to the reproductive organs during critical times in the estrous
cycle may contribute to the reductions in ovarian function and pregnancy rates
associated with fescue toxicosis.
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Ergot alkaloids
Lactating Hereford cows (n = 4 per group) received a control (<15 μg

total EAs/kg dry matter), low (132 μg/kg diet equal to 0.12 µg/kg bw), medium
(529 μg/kg equal to 0.58 µg/kg bw), or high (2115 μg/kg equal to 2.43 µg/kg
bw) diet (Cowan et al., 2018). Ergotized barley used for the ration contained
ergocristine 18 mg/kg, ergotamine 12 mg/kg, α-ergocryptine 7 mg/kg,
ergocornine 3.4 mg/kg, ergometrine 3.2 mg/kg and ergosine 2.7 mg/kg). The
study included three experimental periods: pretreatment (4 days), treatment
(7 days) and post-treatment (4 days). The caudal, median sacral and internal
iliac arteries were examined daily using ultrasonography in B-mode and
Doppler (colour and spectral) mode. Caudal artery diameter decreased in the
medium- (10%, −0.3 mm) and high-dose (19%, −0.5 mm) groups compared to
pretreatment values. During the post-treatment period, the diameter returned
to the pretreatment values for animals in both groups. The pulsatility index was
increased in animals given all the ergot treatments during the post-exposure
period compared to the control group. Blood volume per pulse (mL) and blood
flow (mL/minute) through the caudal artery during the treatment period were
reduced in the medium- (−1.0 mL) and high-dose (−1.1 mL) groups compared to
pretreatment values but did not differ when compared to the control group. The
median sacral artery diameter decreased in animals given the medium- and high-
dose treatments compared to the control group. No differences were detected
in any haemodynamic end-points for the internal iliac artery except changes in
pulse rate. Prolactin levels, body weight and rectal temperatures were not affected
by the treatment.
A similar study was conducted in periparturient Hereford cows with
an exposure period extended to 9 weeks (Cowan et al., 2019). Beginning before
parturition, cows were fed mixed rations containing <15 μg EAs/kg of dry matter
(control, n = 9), 48 μg/kg (low, n = 9), 201 μg/kg (medium, n = 8), or 822 μg/
kg (high, n = 6). The study included three experimental periods: pretreatment
(2 weeks), treatment (9 weeks), and post-treatment (3 weeks). Caudal artery
diameter decreased by 14% (−0.6 mm) in animals in the high-concentration
group during the treatment period compared to pretreatment values. During
the post-treatment period, the diameter returned to the pretreatment value.
Reductions in caudal artery blood flow (37%, 29%) and blood volume per pulse
(29%, 11%) were recorded during the treatment period in animals in the high-
and medium-concentration groups, respectively, compared to pretreatment
values (and they had returned towards pretreatment values by the post-
treatment period). Internal iliac artery diameter and blood flow decreased by
13% (−1.0 mm) and 40%, respectively, during the treatment period in animals in
the medium-concentration group but were not significantly reduced in those in
the high-concentration group. Animals in the medium- and high-concentration
groups showed moderate reductions (12–25%) in the mean blood velocity
121
during the treatment and post-treatment periods, and decreases (12–17%)

in the peak systolic velocity of both arteries during the post-treatment period
were also detected. Prolactin levels and rectal temperatures were not affected by
the treatment. The study documented moderate vasoconstriction in the caudal
artery and the internal iliac artery in cows fed 201–822 μg EAs/kg of dry matter
for a 9-week period around the time of parturition. The Committee noted that
the authors did not make any distinction between measurements taken during
the prepartum period and the postpartum period and that the data only allow a
qualitative conclusion on the existence of a concentration–response relationship.
Angus cows were fed daily with either 40 µg/kg bw of ergotamine
tartrate (n = 37) or a control diet (n = 38) for up to 65 days (Schuenemann et al.,
2005b). Compared to the controls, prolactin concentration and the percentage
of embryos that developed to compacted morula or greater were decreased in
cows exposed to ergotamine. Pregnancy rates of transferred embryos did not
differ between treatment groups. Thus, administration of ergotamine altered the
developmental potential of embryos, but did not affect uterine competency to
establish pregnancy.
Semen parameters, fertilization and endocrinology were studied in
Angus bulls exposed to ergotamine (Schuenemann et al., 2005a). Bulls were
allotted to a control diet (n = 8) or a diet supplemented daily with 40 µg/kg bw of
ergotamine tartrate (n = 8). Administration of ergotamine tartrate increased rectal
temperature and resulted in lower scrotal temperatures (a sign of vasoconstriction)
compared to control bulls. However, average daily weight gain, prolactin, scrotal
circumference, testosterone, and semen motility and morphology did not differ
between groups throughout the experimental period (224 days). Cleavage rates
of embryos derived from in vitro fertilization with semen of bulls fed ergotamine
tartrate were reduced compared to controls; however, development of cleaved
embryos to blastocyst did not differ between treatment groups. In conclusion,
extended exposure of bulls to ergotamine tartrate appeared to reduce fertilization
potential of sperm.
To study the effects of EAs on male reproduction, Pratt et al. (2015)

exposed Angus bulls to endophyte-infected tall fescue (E+, n = 5, no quantification)
or noninfected tall fescue (E−, n = 7, control) for 155 days. Bulls fed E+ showed
decreased total gain, average daily gain and body weight compared with control
bulls (E−). Sperm concentration and velocity were lower, and the number of
abnormal sperm was higher. In addition, spermatozoa motility and progressive
motility were decreased on thawing in semen samples. No differences were
observed for serum testosterone concentrations.
Pregnant Hereford cross beef cows (n = 10 per group except for the
highest concentration, n = 6) were fed a diet containing 5 (control), 48, 201 or
822 μg/kg of EAs (sum of six EAs: ergosine, ergocornine, ergocristine, ergocryptine,
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Ergot alkaloids
ergotamine and ergometrine) for 9 weeks (Grusie et al., 2018). Concentrations of

EAs up to 822 μg/kg did not alter the weight of peripartum and postpartum beef
cows or nursing calves, rectal temperature or plasma prolactin concentrations.
Ergot did not influence the time or the progesterone concentration at the time
of first postpartum rise, or the size of the first and second follicles to ovulate.
The maximum size of the first postpartum corpus luteum was 4 mm larger in
the group fed 822 μg/kg ergot compared with the controls for the first ovulation
postpartum, but not for the second. There was no effect of ergot exposure on
the number of days until the appearance of the first or second corpus luteum
postpartum. EA concentrations up to 822 μg/kg did not affect pregnancy rates.
In conclusion, exposure to EAs for 9 weeks at concentrations up to 822 μg/kg
did not alter performance in pregnant and postpartum beef cattle at moderate
ambient temperatures.
Holstein Friesian bulls were fed with a diet contaminated with EAs
(ergotamine 25%, ergocristine 15% and ergosine 13% as the main alkaloids) for
230 days. No carry-over of alkaloids was found in serum, abdominal fat, muscle,
liver, kidney, bile or urine (Schumann et al., 2007). No EAs were detected in the milk
of eight Holstein dairy cows collected after 4 weeks of exposure to contaminated
diets (Schumann et al., 2009). The alkaloid exposure varied between 4.1 and
16.3 μg/kg bw per day. Milk was sampled through four consecutive morning and
evening milkings at week 4 and pooled for analysis by HPLC with fluorescence
detection (LOD: 5 or 10 μg/kg).
Angus beef steers grazed a low (LE; 23 μg/kg dry matter EAs mainly as
ergovaline + ergovalinine, n = 9) or a high toxic endophyte (HE; 746 μg/kg of EAs,
n = 10) tall fescue pasture (Brown et al., 2009). After 85 days of grazing, serum
concentrations of alkaline phosphatase (ALP), alanine aminotransferase (ALT),
aspartate aminotransferase (AST), cholesterol, lactate dehydrogenase (LDH)
and prolactin were significantly lower for steers in the HE than in the LE group.
At slaughter, hepatic content of cytosolic phosphoenolpyruvate carboxykinase
was significantly greater in steers that grazed the HE than LE pastures. Hepatic
content of AST was also greater, whereas renal and LM content were not. No
differences were observed for hepatic, renal and LM content of AST, glutamate
dehydrogenase, glutamine synthetase and three glutamate transport proteins.
Longer hair, mud accumulation on hair coats, and decreased average daily weight
gain were observed in steers grazing the HE forage for 85 days. They exhibited
symptoms of the classic summer slump phenomenon associated with tall fescue
toxicosis.
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(c) Sheep
Suffolk ewes (n = 36) were exposed to endophyte-infected fescue seed (E+) or
noninfected fescue seed (E−, control) during two stages of gestation (MID, days
35–85 and LATE, days 86–133) (Britt et al., 2019). Exposure to EAs (4.14 mg
ergovaline + ergovalinine per kg, equivalent to 17.9–21.2 μg/kg bw ergovaline
and ergovalinine) reduced prolactin levels. Exposure during LATE (days 86–133)
gestation had the greatest impact on placental development by reducing uterine
and placentome weights. Total fetal weight per ewe was significantly reduced for
ewes fed E+ during LATE gestation compared with E−, but not in ewes exposed
during MID gestation.
In lactating ewes (n = 8) exposed to ergovaline in the diet (497 ± 52 µg/
kg, equivalent to 6.8 µg/kg bw) for 28 days, serum prolactin concentration was
reduced but no consequences were observed on milk quantity or quality (Zbib et
al., 2014). No ergovaline residues were found in milk collected on day 22 (LOD
0.15 µg/L) or in organs collected at the end of the study (LOD 0.15 µg/kg).
Canadian Arcott ram lambs (n = 8 per group) received experimental
diets for 42 to 91 days. They were fed 34 (control), 930, 1402 or 2447 µg alkaloids/
kg, based on total R and S epimers (ergocristine was the dominant R epimer,
followed by ergometrine, with ergocristinine the dominant S epimer). Initial and
final body weight, dry matter intake and feed efficiency did not differ among
animals given the different treatments. Increasing alkaloid concentration caused
a linear decrease in average daily weight gain. Compared to control lambs, rectal
temperatures were 0.33 °C higher in lambs fed diets with added alkaloids. Serum
prolactin concentrations declined linearly with increasing alkaloid concentration
(Coufal-Majewski et al., 2017).
Vasoconstrictive activity of ergovaline and ergovalinine has also been
reported in ovine uterine and umbilical arteries (Klotz et al., 2019).
(d) Horse
Gravid mares grazing endophyte-infested (E+) tall fescue exhibited increased

gestation lengths, agalactia, foal and mare mortality, tough and thickened
placentas, weak and dysmature foals, increased sweating during warm weather,
reduced serum prolactin and progesterone, and increased serum estradiol-
17β levels (Cross, Redmond & Strickland, 1995). Unlike many other species,
horses consuming E+ tall fescue did not exhibit increased body temperature.
Young horses consuming only E+ pasture did not gain as much weight as those
consuming E− pasture.
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Ergot alkaloids
(e) Rabbit
Korn et al. (2014) reported that EAs may have been the cause of tail necrosis
observed in 14 out of 103 rabbits kept in outdoor group housing, fed with hay
and a commercial pelleted feed. Feed analysis for EAs found a mean content of
EAs of 410 ± 250 μg/kg and and a maximum content of 1700 μg/kg. Faeces of
affected rabbits contained EAs at levels up to 200 μg/kg. The mean and maximum
dietary intake of total EAs were 17 and 71 μg/kg bw, respectively. Fusarium toxins
(trichothecenes, zearalenone and fumonisins) were also found in the feed, but at
levels that did not explain the observed effects.
(f) Poultry
Pekin ducks (day 0–49, n = 54/group) received a diet with 0, 0.6, 7.0, 11.4 or
16.4 mg/kg diet of EAs (sum of 12 compounds dominated by ergotamine 26%,
ergocristine 18%, ergocornine 5%, ergocryptine 5% and ergonovine (=ergo-
metrine 5%) (Dänicke, 2015). At the beginning of the experiment, feed intake
decreased significantly by 9%, 28%, 41% and 47% in treated groups compared
to the control group. The experiment was terminated after 2 weeks for ducks
exposed to the two higher concentrations owing to significant growth retardation.
Residues of EAs in liver, breast meat and serum were below the LOQ (5 µg/kg).
Ergonovine was the only EA detected in the bile, with a mean concentration of
40 ng/g in animals fed 7 mg/kg EAs. Monocyte proportions were significantly
lower in all treatment groups compared to the controls, which might hint at an
effect of EAs in modulation of immune responses. The author identified a lowest-
observed-adverse-effect level (LOAEL) of 0.6 mg/kg diet.
Chickens (male broilers of the strain Lohmann Meat) received a diet
with four levels of EAs up to 6.76 mg/kg (sum of 12 compounds, dominated by
ergotamine 25%, ergocristine 22% and ergosine 10%) for 2 weeks (n = 80 per
group) or 5 weeks (n = 72 per group) (Dänicke, 2017). Feed intake significantly
decreased with increasing dietary ergot content. Feed intake was identified as the
most sensitive end-point suitable for deducing both LOAEL and no-observed-
adverse-effect level (NOAEL). Animals fed diets with the highest ergot level were
partly unable to stand and displayed uncoordinated movements. Residues of
EAs in liver, breast meat and serum were below the LOQ (5 µg/kg). No carry-
over of EAs into egg yolk and albumen, blood, liver and breast muscle was found
in laying hens fed with EAs up to 14.56 mg/kg diet (Dänicke, 2016). Most of
the laying performance and reproductive traits were significantly compromised
during the test period between 22 and 42 weeks of age when the diet with the
highest EA content was fed.
125
Table 9
Biomarkers of exposure to EAs
Analytical Results detection Results detection
Country, year Population method EAs LOD/LOQ (ng/mL) (%) serum (%) urine
EFCOVAL, 600 adults LC-MS/MS Ergocornine 99/296 14 (5.2) 16 (8.5)
2006–2010; (45–65 years); Ergocorninine 99/296 38 (14.1) 22 (11.7)
Belgium, Czech 188 with serum
Ergocristine 99/296 19 (7.1) 14 (7.4)
Republic, France, sample and
the Netherlands, 268 with urine Ergocristinine 99/296 23 (8.6) 18 (9.6)
Norway sample Ergocryptine 99/296 4 (1.5) 9 (4.8)
Ergocryptinine 50/99 22 (8.2) 16 (8.5)
Ergometrine 60/99 12 (4.5) 6 (3.2)
Ergometrinine 83/99 19 (17.1) 10 (5.3)
Ergosine 99/209 32 (11.9) 10 (5.3)
Ergosinine 99/889 30 (11.1) 17 (9.0)
Ergotamine 99/296 14 (4.2) 9 (4.8)
Ergotaminine 99/296 1 (0.4) 10 (5.3)
EFCOVAL, European Food Consumption Validation project; LC-MS/MS, liquid chromatography with tandem mass spectrometry; LOD, limit of detection; LOQ, limit of
quantitation.
Source: De Ruyck et al. (2020).
2.4 Observations in humans

2.4.1 Biomarkers of exposure
Few studies of biomarkers of exposure to EAs were available. De Ruyck et al.
(2020) measured EAs in serum and urine of 600 men and women participating
in the European Food Consumption Validation (EFCOVAL) project (De Ruyck
et al., 2020). In addition, 24-hour dietary recall interviews were conducted
twice, 1 month apart, and matched against the EFSA database on mycotoxin
contamination to estimate exposure to EAs. Participants provided a non-fasting
blood sample approximately 1 week before the first 24-hour dietary recall
interview. Urine samples were collected during the 24-hour period following the
2 days on which participants had been asked for a 24-hour dietary recall. Six EAs
and their -inine epimers were included in the study. Overall, EAs were detected
in 116 (out of 268) serum samples and 106 (out of 188) urine samples (Table 9).
Participants in the EFCOVAL project had a median daily exposure (ng
per kg bw) of 41.4 for EAs, overall. The median exposure ranged from 1.44 for
ergometrinine to 24.0 (ergocryptine plus ergocryptinine). Of 188 participants
with both urine and dietary exposure estimates, the agreement between the two
measurements was 53% for EAs overall, and ranged from 5% (ergocryptine) to 72%
(ergometrine). Spearman correlations were generally not statistically significant,
and most were < 0.1. Of the 268 participants with serum measurements, agree-
ment between the two measurements was 51% for EAs overall, ranging from 1%
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Ergot alkaloids
(ergocryptine) to 74% (ergometrinine and ergocryptinine). Spearman correlation

coefficients were generally < 0.1 with the exception of a significant correlation
reported for ergotamine (ρ = 0.15). Across all mycotoxins evaluated (58 analytes,
including parent compounds and metabolites), Cohen’s κ statistic indicated only
slight (κ < 0.1) agreement between exposure estimates and concentrations in
serum or urine (De Ruyck et al., 2020). These findings suggest that biological
measurements were generally not sufficient for describing chronic dietary
exposure, but they may provide information on single exposures.
In a study describing technical issues pertaining to the use of LC-MS to
measure ergotamine (Favretto et al., 2007), the method was applied to a 40-year-
old woman with a 7-day history of clinical ergotism. The woman had taken an oral
preparation containing 1 mg ergotamine tartrate and 100 mg caffeine per tablet
for prophylaxis and as a rescue treatment. Measured levels of ergotamine in urine,
blood and hair were as follows: urine 100 pg/mL, blood 320 pg/mL, proximal hair
24 pg/mg and distal hair 15 pg/mg. Although the exact dose and time of oral
treatment in relation to sample collection was not reported, it can be assumed
that the patient had been using ergotamine chronically. The authors concluded
that the presence of ergotamine in both proximal and distal hair samples was
consistent with prolonged use of ergotamine (roughly 2 years, considering an
average hair growth rate of 0.6 to 1.4 cm/month), which is consistent with the
patient’s reported clinical history.
2.4.2 Biomarkers of effect

No studies of biomarkers of effect were identified.
2.4.3 Clinical observations

Data pertaining to the use, dose and precautions for the medicinal use of
ergotamine and ergometrine are summarized in Table 10, based on information
in Martindale’s complete drug reference (Martindale, 2010). Ergotoxine esilate and
ergotoxine phosphate were formerly used as an oxytocic and in the treatment
of migraine. Ergotoxine is a mixture of naturally occurring EAs including
ergocornine, ergocristine and ergocryptine. Although semi-synthetic EAs
were screened and considered for inclusion in this monograph, the Committee
concluded that they were not relevant because their kinetic properties are different
from those of the naturally occurring EAs that may be present in food and feed.
2.4.3.1 Ergotamine
Adverse effects
Ergotamine is recommended for treating patients with migraine who experience
infrequent attacks, long duration headaches, and are likely to adhere to the
127
Table 10
Summary of use, dose, and precautions for the medicinal use of ergotamine tartrate and
ergometrine maleate
EA Uses Oral dose Precautions
Ergotamine Migraine and cluster 1 to 2 mg, repeated 30 minutes Contraindicated in people with hypertension, shock,
tartrate headache later; no more than 4–6 mg in severe or persistent sepsis, PVD, IHD, temporal arteritis,
24 hours, no more than 8 mg per hyperthyroidism, hepatic or renal impairment, basilar or
attack, no more than 12 mg per hemiplegic migraine, and pregnant and breastfeeding
week, no more than two courses women.
per month. Increased risk of peripheral vasoconstriction if used with
beta-blockers and CYP3A4 substrates (azole antifungals,
macrolide antibiotics, HIV-protease inhibitors).
Ergometrine Induction of Oral dosage 0.2 mg initially, and Contraindicated in people with hypertension, heart disease
maleate uterine contraction up to 0.4 mg 2–4 times daily. Dose venoatrial shunts, mitral valve stenosis or obliterative
and prevention may be administered for 2–7 days. vascular disease, sepsis, hepatic or renal impairment.
of postpartum Also administered by intravenous Contraindicated in the first stage of labour and in patients
haemorrhage in the and intramuscular routes. with pre-eclampsia, eclampsia, or at risk of preterm birth.
third stage of labour
CYP3A4, cytochrome P450 3A4; HIV, human immunodeficiency virus; IHD, ischaemic heart disease; mg, milligram; PVD, peripheral vascular disease.
Source: The complete drug reference, ergotamine tartrate, ergometrine maleate (Martindale, 2010).
dosing restrictions (Silberstein & McCrory, 2003; Tfelt-Hansen & Diener, 2014).
The adverse effects of ergotamine are related to its effects on the CNS and its
vasoconstrictive properties (Schiff, 2006; Martindale, 2010; Tfelt-Hansen &
Diener, 2014). At therapeutic doses, nausea and vomiting are common (Silberstein
& McCrory, 2003). Weakness, muscle pain in the extremities, and numbness and
tingling of the fingers and toes may also occur.
At greater than therapeutic doses, vasoconstriction, cardiovascular
effects, gangrene, confusion and convulsions have been reported. As summarized
by Martindale (2010), “symptoms of acute overdosage include nausea, vomiting,
diarrhoea, extreme thirst, coldness, tingling and itching of the skin, a rapid and
weak pulse, hypertension or hypotension, shock, confusion, convulsions and
unconsciousness; fatalities have been reported”. Side-effects typically occur as a

result of prolonged use for migraine headaches rather than following acute single
doses. Prolonged use or overuse of ergotamine can result in “severe circulatory
disturbances”. Symptoms of ergotism associated with overdosage or poisoning
include numbness, cold, tingling, pale or cyanotic extremities accompanied
by muscle pain, with the possibility that gangrene may develop (Martindale,
2010). Cardiovascular symptoms including angina, tachycardia or bradycardia,
hypertension or hypotension, and myocardial infarction have been reported and
vasoconstriction of blood vessels in the brain, eye, intestines and kidneys may
also occur (Fisher et al., 1985; Martindale, 2010; Deviere, Reuse & Askenasi,
1987; Galer et al., 1991; Lazarides et al., 1992; Redfield et al., 1992). Symptoms of
128
Ergot alkaloids
Table 11
Case reports of adverse effects associated with ergotamine
Reference Dose and duration Adverse effects
(Sran & Vathsala, 2016) Ergotamine tartrate (1 mg/100 mg A 54-year-old male presented with severe sudden onset left loin
caffeine tablets) intermittently over 20 pain resulting from renal infarction
years. Recent use of daily tablet with three
tablets in 24 hours before admission
(Pérez Baztarrica et al., Ergotamine use for migraine, 8 mg within A 48-year-old female presented with acute coronary syndrome; i.e.
2019) 12 hours before the hospital admission inferior and posterior ST-segment elevation myocardial infarction
(formulation not reported)
(Pakfetrat et al., 2013) Ergotamine tartrate (2–4 mg) daily as A 22-year-old female presented with hypertension, renal
needed for 4 years, for migraine treatment
failure and tubulo-interstitial nephritis on biopsy. Neurological
examination was normal
(Maréchaux et al., 2015) 1–3 mg ergotamine for 30 years Mitral regurgitation during exercise echocardiography in a 67-year-
old female with a history of hypertension and migraine
(Murad, Miller & Glockner, Ergotamine–caffeine (dose not reported) A 43-year-old woman presented with increasing shortness
2011) suppositories used on average three times of breath and bilateral leg oedema of 3 months duration.
per week for 12 years Multi-valvular heart disease and retroperitoneal fibrosis was
demonstrated
(Patel et al., 2013) Extensive use of ergotamine (10 years), A 47-year-old female presented signs and symptoms of
dose not reported decompensated heart failure; echocardiogram showed aortic and
mitral valvulopathy
(Bois et al., 2012) High-dose ergotamine suppositories over Valvulopathy (mitral stenosis and recurrent chylous pleural
34 years (dose not reported) effusion) in a 55-year-old woman
(Küçükalp, Durak & 4–5 ergotamine tartrate (0.75 mg) tablets Vasospasm affecting arteries in the lower limb 10 days post-trauma
Bilgen, 2013) daily for 10 years in a 46-year-old male
peripheral vasoconstriction or of cardiovascular disturbances, as seen in chronic

ergotamine poisoning, may also occur following acute overdosage (Martindale,
2010). Other adverse effects include confusion and convulsions (Martindale,
2010). In rare cases, pleural and peritoneal fibrosis and fibrosis of the cardiac
valves have been reported (Lepage-Savary & Vallières, 1982; Robert et al., 1984;
Damstrup, 1986; Martindale, 2010).
Several case reports involving patients who developed ergotism following
prolonged oral administration of ergotamine tartrate (i.e. 1–6 mg/day for 2 weeks
up to 21 years) were described by the EFSA Panel on Contaminants in the Food
Chain (CONTAM Panel) (EFSA, 2012). Cases of ergotism following rectal and
sublingual administration of ergotamine tartrate were also described (EFSA,
2012). Seminerio et al. (2014) described the potential for ergotamine to induce
localized ischaemia leading to ischaemic colitis. Additional reports of adverse
effects among patients using ergotamine are listed in Table 11.
129
Drug interactions
Because ergotamine is metabolized by CYP3A4, its effects can be altered by co-
administration of other agents that are also metabolized by CYP3A4 such as azole
antifungals, macrolide antibiotics, and HIV-protease inhibitors (Martindale,
2010; EFSA, 2012). Information relevant to interactions of EAs with drugs used
for antiviral therapy in patients with HIV and interactions with stimulants such
as cocaine is discussed by Ayarragaray (2014). Srisuma, Lavonas & Wananukul
(2014) studied a retrospective case series using data from all patients referred to a
poison centre in Bangkok, Thailand due to ergotism from January 2006 to August
2013. Nine of the 12 cases identified involved interactions between ergotamine
(1 mg ergotamine/100 mg caffeine combination tablets) and CYP3A4 inhibitors
(Srisuma, Lavonas & Wananukul, 2014). Most patients presented with symptoms
of vascular insufficiency, such as cooling, numbness, pain and pulse deficits in
distal limbs but recovered following treatment. A 49-year-old female with HIV in
this case series developed acute kidney injury and rhabdomyolysis and died from
cardiac arrest. Additional recent case reports of interactions between ergotamine
and drugs used to treat HIV and macrolide antibiotics are summarized in Table
12.
Pregnancy
Ergotamine is contraindicated during pregnancy because it can cause premature
labour and vasoconstriction (Martindale, 2010). EFSA (2012) reviewed reports
of accidental exposure to ergotamine during pregnancy that resulted in uterine
contraction, fetal tachycardia, arrested cerebral maturation and Moebius
syndrome (EFSA, 2012). Most of the women affected were inadvertently given
suppositories containing 2 mg ergotamine and 100 mg caffeine. In addition, one
case of fatal jejunal atresia was reported in an infant born preterm (35 weeks
gestation) to a mother who had taken ergotamine tartrate (6–8 mg/day) in tablet
form throughout her entire pregnancy (Graham, Marin-Padilla & Hoefnagel,
1983). A more recent case report describes a neonate delivered by caesarean

section at 32 weeks gestation, with unilateral renal agenesis, urethral atresia and
pulmonary hypoplasia, whose mother took oral ergotamine tartrate (0.75 mg
plus 80 mg caffeine tablets, 1.5 mg daily) during her first trimester of pregnancy
(Demirel et al., 2012). The infant died due to cardiopulmonary arrest after
13 hours.
2.4.3.2 Ergometrine
Adverse effects
Ergometrine maleate is used to induce uterine contractions and prevent
haemorrhage in the third stage of labour and for prevention and treatment of
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Ergot alkaloids
Table 12
Case reports of drug interactions with ergotamine
Navarro et al. (2017) 1 mg ergotamine plus 100 mg caffeine for 5 days in Acute leg ischaemia in a 33-year-old male patient with HIV
combination with antiretroviral therapy
Ferry et al. (2014) Two tablets each containing 1 mg ergotamine tartrate Ankle pain resulting in amputation in a 32-year-old female
plus 100 mg caffeine six times daily for 3 days in with HIV
combination with highly active antiretroviral therapy
(after discontinuing treatment 2 years previously)
Fröhlich, Kaplan & 1 mg ergotamine tartrate tablet per day for 2 weeks in Paraesthesiae and coldness of the left upper extremity
Amann-Vesti (2010) combination with antiretroviral therapy consistent with ergotism in a 29-year-old male positive for
HIV
Iardino et al. (2018) Ergotamine-containing medication (dose not A 34-year-old male positive for HIV presented with severe
reported) in combination with antiretroviral therapy vasospasm resulting in left foot pain and swelling (i.e. a cold,
purple, left foot, and absent pedal and tibial pulses, and no
arterial flow from the popliteal artery)
Reghukumar & Ergotamine twice daily (dose not reported) 4 days A 24-year-old female presented with severe, burning pain in
Benson (2020) before presentation in combination with antiretroviral the legs. Diffuse, symmetric narrowing of the arteries in both
therapy legs was observed. Perfusion improved 2 weeks after initial
presentation
Tseng et al. (2010) Three tablets each containing 1 mg ergotamine plus A 35-year-old woman presented with acute paraesthesia,
100 mg caffeine in combination with macrolide diffuse vasospasm of the femoral arteries and the right
antibiotic (erythromycin) brachial artery visible on angiogram
Demir et al. (2010) Ergotamine tartrate for 3 years (dose not reported) in Pain and mild cyanosis resulting from arterial vasospasm in
combination with erythromycin for 3 days an 18-year-old female
Ozpelit et al. (2016) Ergotamine tartrate (1.5 mg/day for the past 5 days) A 53-year-old female with Takotsubo cardiomyopathy
in combination with a 14-day course of clarithromycin
Adam et al. (2014) Ergotamine (long-term use, dose not reported) with Vasospastic ischaemia with acute arterial embolism
macrolide antibiotic for 4 days
postpartum haemorrhage. Although all EAs have uterotonic effects, ergometrine

(or methylergometrine) has been used clinically because it is more active as a
uterine-stimulating agent than ergotamine (Sanders-Bush & Mayer, 2006).
Uterotonic effects can be observed in postpartum women within 10 minutes
after oral administration of 0.2 mg of ergometrine; however, there may be a wide
variation in patient response (Sanders-Bush & Mayer, 2006).
Nausea and vomiting are common side-effects of ergometrine maleate
when used at normal therapeutic doses (Martindale, 2010). Less common
effects include abdominal pain, diarrhoea, headache, dizziness, tinnitus, chest
pain, palpitations, bradycardia and other cardiac arrhythmias, coronary artery
vasospasm, myocardial infarction, dyspnoea and pulmonary oedema (Martindale,
2010; EFSA, 2012). Intravenous administration has been associated with rapid
increases in blood pressure (Martindale, 2010; EFSA, 2012). Overdosages may
cause seizures and gangrene as well as gastrointestinal symptoms, dizziness,
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Table 13
Case reports of adverse effects associated with ergometrine use
Wang, Liu & Chen Oral ergometrine (0.2 mg), four doses Transient sick sinus syndrome with complete atrioventricular block
(2017) with junctional escape rhythm in 38-year-old woman treated with
oral ergometrine to induce uterine contractions
Birch & Lu (2019) Syntocinon followed by 250 IU ergometrine Atrial fibrillation in 36-year-old woman undergoing caesarean
administered intravenously section
Johnston & Hughes Oxytocin (5 IU) and 0.5 mg ergometrine maleate Bronchospasm in non-asthmatic patient
(2013) administered intramuscularly postpartum
increased blood pressure, loss of consciousness, numbness in the extremities,

chest pain and hypercoagulability (Martindale, 2010; EFSA, 2012).
Recent case reports are summarized in Table 13. Wang, Liu & Chen
(2017) reported a case of transient sick sinus syndrome following oral exposure
to ergometrine, whereas the other reports listed in Table 13 describe effects
following intravenous and intramuscular administration of ergometrine
(Johnston & Hughes, 2013; Birch & Lu, 2019).
2.4.3.3 Ergotoxine
Adverse effects
Ergotoxine is a mixture of ergocornine, ergocristine and ergocryptine. Ergotoxine
esilate and ergotoxine phosphate were formerly used as oxytocics and in the
treatment of migraine (Martindale, 2010). The effect of ergocornine (2 mg during
the post-ovulatory phase) on progesterone metabolism and its potential as an oral
inhibitor of implantation (at a dose of 2–20 mg) have been investigated, but the
results from the studies identified were inconclusive (Shelesnyak et al., 1963; Koi
et al., 1966; Lindner et al., 1967; Floss, Cassady & Robbers, 1973; EFSA, 2012).
No new studies of ergotoxine or its components (for example, ergocornine) were
identified.
2.4.4 Epidemiological studies

2.4.4.1 Ergotamine
As reported in a review by Tfelt-Hansen et al. (2000), ergotamine induces
bradycardia and can produce coronary vasoconstriction, ischaemia and angina in
coronary heart disease (Tfelt-Hansen et al., 2000). A more recent review identified
two observational studies (Velentgas et al., 2004; Wammes-van der Heijden et
al., 2006) that reported serious cardiovascular effects (i.e. myocardial infarction,
stroke, ventricular arrhythmia and transient ischaemic attack) in association with
intensity, but not recency, of ergotamine use. A pooled risk estimate of 2.28 (95%
132
Ergot alkaloids
confidence interval (CI): 1.18–4.41) for the association of ergotamine intensity

with serious cardiovascular effects was determined (Roberto et al., 2015).
Velentgas et al. (2004) defined current, recent and non-use categories for EAs.
The exposure intensity metric was defined as the total days that the medication
was dispensed within the previous 6 or 7–12 months. Roberto et al. (2015)
used the effect estimate from the study by Velentgas et al. (2004) for the highest
intensity category (≥61 days supplied) in the previous 6 months to derive the
pooled estimate (odds ratio (OR): CI: 1.47, 0.34–6.27). Velentgas et al. (2004) did
not observe a dose–response relationship across the intensity categories (i.e. the
OR for the association between EAs dispensed for 11–26 days in the previous 6
months was 4.54 (95% CI, 2.26–9.10) and no associations were observed in some
higher and lower categories). Wammes-van der Heijden et al. (2006) defined
intensity as the number of prescribed doses within the year before the index
event (i.e. hospitalization for ischaemic complications), including all prescribed
ergotamines. The highest intensity category (≥ 90 daily doses), indicating the
overuse of ergotamine, was associated with ischaemic complications in this study
(OR, 2.55; 95% CI, 1.22–5.36). Risk estimates for a specific agent or formulation
were not presented in either study (i.e. ergotamine, dihydroergotamine and
possibly methysergide, alone or in combination with other drugs were included
in the analyses).
Poisonings
Several retrospective studies of ergotamine poisoning were identified. Robblee
et al. (2020) analysed intentional overdoses of ergotamines as a single agent or
in combination with other drugs reported to the United States National Poison
Data System from 2014 to 2018. Of 48 reports of poisonings involving EAs, 16
involved oral exposure to ergotamines as a single agent and were included in the
authors’ main analysis. Major effects or death were not reported in association
with these EA overdoses. Common symptoms included abdominal pain,
vomiting, numbness, nausea, diarrhoea and vertigo. Although exposures were
characterized as intentional overdoses, no dose estimates were provided.
Srisuma, Lavonas & Wananukul (2014) conducted a retrospective study
of ergotism cases referred to the poison centre in Bangkok, Thailand (January
2006–2013). A total of 378 EA exposures were identified with 12 subjects showing
signs and symptoms of ergotism. All of the symptomatic cases and most (318
of 366) of the asymptomatic cases involved the ingestion of 1 mg ergotamine
(plus 100 mg caffeine). Other asymptomatic cases involved ingestion of tablets
containing 0.1 mg ergotamine/20 mg phenobarbital (n = 29) or 1 mg ergotamine
as a single agent (n = 17). Of the 12 exposures that resulted in symptoms, all
involved 1 mg ergotamine/100 mg caffeine tablets and most (n = 9) were the result
133
of interactions with CYP3A4 inhibitors. The remaining cases included suicide

attempts (n = 2) and unsupervised ingestion (unknown dose) by a 15-month-old
boy who presented in a continuous state of seizure.
Exposures of children to EAs reported to the California Poison Control
System from 1997 to 2008 were analysed to determine the risk of toxicity
due to oral exposure in children less than 7 years old (Armenian & Kearney,
2014). Median doses and ranges reported in this study were based on estimated
maximum possible doses that were determined using reported pill counts and
observer accounts. Only dose estimates coded as “certain” were included in
the analysis and doses were missing for 97 of 256 patients with symptoms. Of
56 reports of ergotamine exposure, seven (13%) resulted in gastrointestinal
symptoms (nausea, vomiting, abdominal pain and/or diarrhoea), whereas eight
(14%) resulted in other (CNS or respiratory) symptoms. None of the cases were
characterized as serious; the median dose among the children with clinical
symptoms was 1 mg (range: 0.2–11 mg). Although there were 61 reports of co-
exposure to caffeine, the authors noted that this information was incomplete.
There were also 15 reports of exposure to dihydroergotamine resulting in one
case involving gastrointestinal symptoms and two cases involving other CNS or
respiratory symptoms (median dose among the children with clinical symptoms:
1 mg (range: 0.25–3 mg)).
Pregnancy
As discussed previously, ergotamine is contraindicated in pregnancy. Amundsen
et al. (2015) reviewed studies of ergotamine exposure among pregnant and
lactating women and identified only one eligible study. In this case–control study,
Bánhidy et al. (2007) found an association of ergotamine with low birth weight
(OR, 2.8; 95% CI, 1.2–6.5) and preterm birth (OR, 1.9; 95% CI, 1.0–4.0), but the
authors did not control for maternal smoking, a known cause of low birth weight
(Bánhidy et al., 2007). The women in this study received ergotamine drops or
tablets. Mean daily dose of ergotamine ranged from 0.3 mg (tablets) to 1.5 mg
(drops) for 1 day to 7 months.
No increase in the overall incidence of congenital abnormalities was
found in a study of 924 children of women who had migraine headaches, 71%
of whom were said to have taken ergotamine at some time during pregnancy
(month and trimester not stated); the number of children with abnormalities
in the cohort was 31 (Wainscott, 1978). Two analyses of the Hungarian Case-
Control Surveillance of Congenital Abnormalities dataset reported associations
of ergotamine use during pregnancy with neural tube defects. In the first analysis
of 1202 infants with neural tube defects, there was an association with maternal
use of ergotamine during pregnancy (OR, 3.3; CI, 1.4–7.7), based on six cases
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Ergot alkaloids
(Medveczky & Czeizel, 2004). A later analysis, which was based on three cases,
found an association of ergotamine drops (mean dose 1.5 mg/day during the
second month of pregnancy) with neural tube defects (OR, 6.9; CI, 2.0–24.2)
(Ács et al., 2006).
2.4.4.2 Ergometrine
Liabsuetrakul et al. (2018) sought to compare the use of ergometrine (or
methylergometrine) by any route with no intervention to determine the
efficacy and side-effect profile. Of the eight studies identified one examined
oral ergometrine (De Groot et al., 1996). De Groot et al. (1996) conducted a
randomized trial to determine whether oral ergometrine reduced postpartum
blood loss by 30% and found that blood loss was reduced by a smaller amount.
The ergometrine group received 0.4 mg (two tablets of 0.2 mg). Among women
receiving oral ergometrine whose blood pressure was measured, no significant
elevation of blood pressure was observed (no other end-points were measured).
Another systematic review (Gallos et al., 2018) was designed to identify the
most effective drugs to prevent postpartum haemorrhage and examine their
side-effect profiles. The study reviewed 140 trials overall, including 21 studies
of ergometrine. The typical dosage was 500 µg ergometrine plus 5 IU oxytocin
administered intramuscularly. The side-effect profile for ergometrine was
relatively poor compared to other drugs. An association with hypertension was
reported (Gallos et al., 2018).
Poisoning
Reports of ergometrine maleate poisonings following accidental administration
to neonates were reviewed by the EFSA CONTAM Panel (EFSA, 2012). Effects
reported included peripheral vasoconstriction, encephalopathy, convulsions,
respiratory failure, acute renal failure and temporary lactose intolerance (EFSA,
2012). Over the long term most infants recovered, with the exception of one
infant who died after receiving an oral dose of 0.2 mg ergometrine maleate
(AHFS, 1995; EFSA, 2012). More recently, exposures of children to EAs reported
to the California Poison Control System from 1997 to 2008 were analysed to
determine the risk of toxicity due to oral exposure in children less than 7 years
old (Armenian & Kearney, 2014). The maximum possible dose was estimated
from information provided regarding pill counts and observer accounts. A
total of 353 ergot exposures were included in the analysis. Most (351 of 353)
exposures were due to ingestion by the oral route. As shown in Table 14, there
were reports involving exposure to ergometrine (n = 37), methylergometrine (n =
231) or methysergide (n = 3) totalling 271 reports. Forty-two (42) of the 271
reported exposures (15.5%) resulted in gastrointestinal symptoms, whereas 15
135
Table 14
Clinical effects and doses of EAs reported to the California Poison Control System (n = 271
children < 7 years old, 1997–2008)
EA (n) Gastrointestinal symptoms N Other symptomsa N Median dose (range) (mg)
Ergometrine (37) 4 1 0.4 (0.2–2)
Methylergometrine (231) 37 13 0.4 (0.1–3)
Methysergide (3) 1 1 2 (2)
a
Other symptoms include central nervous system and respiratory effects.
Source: adapted from Table 3 of Armenian & Kearney (2014).
(5.5%) resulted in CNS or respiratory symptoms. Two patients presented with

significant vascular and CNS symptoms that eventually resolved:
1) a 2-year-old child who ingested an unknown quantity of 0.2 mg
methylergonovine tablets was lethargic with cool, pale extremities
and prolonged capillary refill time when she or he was admitted to
the hospital (temperature 36.9 °C, pulse 90 beats per minute, blood
pressure 94/60 mmHg, respirations 20 per minute, oxygen saturation
82%); and
2) a 3.3-kg newborn who received 0.2 mg of intramuscular methergine
(methylergonovine maleate) experienced respiratory depression
with oxygen saturation of 80% (temperature 36.7 °C, pulse 120 to
140 beats per minute, blood pressure 73/43 mm Hg, and a normal
respiratory rate).
In another study, Davanzo et al. (2015) examined cases of inadvertent
oral administration of methylergometrine maleate to children up to 5 years old
which were reported to the national poison centre in Milan, Italy. A total of 642
cases of unintentional poisoning were reported during the study period. The
most common symptoms included vomiting and abdominal pain. Also reported
were hyperactivity, tachycardia and cyanosis. The severity of clinical symptoms
reported for most cases was minor (i.e. mild, transient, spontaneously resolved
(n = 43)). Infants aged 1 year or younger with minor symptoms included those
exposed to doses ranging from 0.003 to 0.050 mg/kg bw. Older children (1–5 years
old) with minor symptoms were exposed to doses ranging from 0.003–0.089 mg/
kg bw. Moderate symptoms (pronounced or prolonged) were reported in infants
(<1 year old) who were exposed to doses ranging from 0.11 to 0.86 mg/kg bw,
including three who experienced repeated exposures. Severe poisoning (i.e.
cyanosis, apnoea, coma and cardiac arrest) was reported in one child <1 week old
exposed to 0.100 mg/kg bw for 4 days. Sepsis-like symptoms and encephalopathy
136
Ergot alkaloids
were reported in a case-series study of 12 newborns exposed to intramuscular

methylergonovine in a hospital in Türkiye (Bas et al., 2011).
2.4.4.3 Ergotoxine
Poisoning
Exposures of children to EAs, reported to the California Poison Control System
from 1997 to 2008, were analysed to determine the risk of toxicity following oral
exposure in children less than 7 years old (Armenian & Kearney, 2014). There
was one report of dihydroergotoxine exposure (dose 1 mg), which did not result
in symptoms.
2.4.4.4 Sclerotia of Claviceps spp.

Two forms of ergotism can occur concurrently, namely, vasospastic gangrenous
and convulsive forms (EFSA, 2012). Lactation inhibition has also been reported
in association with outbreaks of ergotism. Preparations of sclerotia of C. purpurea
in tablet or liquid form have been used to accelerate labour. Single doses ranged
from 0.2–3 mg and daily doses from 6–7.5 mg (EFSA, 2012). The practice of oral
administration of ergot by midwives in Europe was discontinued because it was
associated with an increased risk of stillbirth.
Acute poisoning events are rare; the convulsive form of ergotism has not
been reported in Europe for more than a century (EFSA, 2012). The last reported
outbreak of gangrenous ergotism noted by EFSA occurred in Ethiopia in 2001.
EFSA (2012) and WHO-ICPS (1990) described poisonings associated with grain
contaminated with C. purpurea. Large outbreaks killing thousands of people
occurred in Europe between the ninth and eighteenth centuries. The most recent
outbreaks in Europe occurred in the United Kingdom and Russia (1926–1928).
Overall, ergot concentrations of 0.1% were thought to be harmless, whereas
concentrations of 1% were associated with the risk of toxicity, and concentrations
of 7–10% were associated with mortality (EFSA, 2012). The types of ergotism
associated with these outbreaks were the vasospastic gangrenous and convulsive
forms and some outbreaks were associated with high rates of infant mortality due
to the inhibition of lactation in mothers (Wirth & Gloxhuber, 1981; WHO-ICPS,
1990; EFSA, 2012).
The most recent outbreaks identified occurred in Ethiopia in 1978
and 2001 (EFSA, 2012; Belser-Ehrlich et al., 2013). The outbreak in 1978 was
associated with 2–3 months of exposure to grain with 0.75% ergot content (i.e.
ergometrine detected by thin layer chromotography) (Demeke, Kidane & Wuhib,
1979; King, 1979; Belser-Ehrlich, 2012; EFSA, 2012). The 2001 outbreak was
associated with concentrations ranging from 2.1–26.6 mg ergotamine/kg and
0.9–12.1 mg ergometrine/kg (Urga et al., 2002; EFSA, 2012; Belser-Ehrlich et al.,
137
2013). Finally, two case reports have been published. One described exposure of
a 13-year-old girl who ate muesli contaminated with 12% sclerotia of C. purpurea
for several months. The other described a farmer exposed via inhalation to
ergotamine-containing millet dust, resulting in high plasma concentrations of
ergotamine (Stange et al., 1996; EFSA, 2012).
Outbreaks associated with C. fusiformis in contaminated pearl millet
have also occurred in India, including outbreaks in the twentieth century. The
concentrations reported in unaffected villages ranged from 1–38 g ergot/kg (15–
26 mg/kg total EA content) whereas concentrations in affected villages ranged
from 15–175 g ergot/kg (15–199 mg/kg total EA content) (Krishnamachari &
Bhat, 1976; WHO-ICPS, 1990; EFSA, 2012).
3. Analytical methods
The following sections review analytical methods for EAs in cereals and cereal
products, with particular emphasis on the period since 2000. Databases searched
for publications since 1980 were Scopus and PubMed with the terms “ergot” and
“determination”, resulting in 3597 and 963 hits, respectively. The Scopus search
was further restricted by limiting it to chemistry and the term “alkaloids” (1131
hits). In addition, information has been drawn from the annual summaries of
advances in mycotoxin analytical methods previously published as general
referee reports of the Association of Official Analytical Chemists International
and subsequently by the World mycotoxin journal. Recent reviews have also
covered the topic (Komarova & Tolkachev, 2001b; Scott, 2007; Krska & Crews,
2008; EFSA, 2012; Crews, 2015).
3.1 Chemistry
The first EA to be identified was ergotamine in 1920 and since then about 50
more have been discovered (Lorentz, 1979; Flieger, Wurst & Shelby, 1997). They
are produced, among others, by species of the genus Claviceps, which infect a
range of cereal crops, including rye, wheat, barley, millet, oats and sorghum. The
EAs (also known as ergolines) are found in the fungal sclerotia in amounts that
range between 0.01 and 0.5% by weight. The alkaloid constituents and quantities
depend on factors such as fungal strain, geographical region and plant host
(Lorentz, 1979).
The EAs are characterized by the ergoline ring system consisting of
four fused rings (Flieger, Wurst & Shelby, 1997; Komarova & Tolkachev, 2001a).
Position N6 carries a methyl group, and there is a double bond at either C8,9
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Ergot alkaloids
or at C9,10. Substitution at C8 gives rise to the naturally occurring range of

alkaloids, the major ones being divided into four groups. Firstly, there are those
derived from precursors of lysergic acid (see Fig. 1), termed clavine alkaloids,
such as agroclavine (8,9-didehydro-6,8-dimethylergoline). The second group
are simple lysergic acid derivatives, such as ergometrine (D-lysergic acid-L-
propanolamide, also known as ergonovine). The third group are the peptide
alkaloids or ergopeptines (ergopeptides, cyclol EAs), in which the substituent at
C8 is a cyclized tripeptide containing a variable α-hydroxyl amino acid, a second
variable L-amino acid and L-proline. The fourth group are the ergopeptames
(noncyclol lactam EAs), which also contain a tripeptide substituent, but lack the
α-hydroxy group on the first amino acid and the second amino acid is cyclized with
D-proline. The EAs with a C9,10 double bond can readily undergo enolization to
form epimers with the opposite chirality at C8 (Komarova & Tolkachev, 2001a;
Smith & Shappell, 2002).
The 12 EAs most commonly associated with contaminated cereals
and hence with food safety are the lysergic acid derivative, ergometrine, and
the ergopeptides, ergocornine, ergocristine, ergotamine, ergocryptine and
ergosine, as well as their epimers, ergometrinine, ergocorninine, ergocristinine,
ergotaminine, ergocryptinine and ergosinine (EFSA, 2012). Also, ergocryptine
and ergocryptinine can occur as both an α- and a β-analogue, although these are
seldom distinguished in analysis.
The EAs are generally soluble in organic solvents (methanol, acetonitrile,
organic solvent/buffer mixtures) and are charged in acid pH and uncharged in
neutral or alkaline pH (Krska & Crews, 2008; Krska et al., 2008; Crews, 2015).
Those with a C9,10 double bond have natural fluorescence properties (Flieger,
Wurst & Shelby, 1997; Komarova & Tolkachev, 2001b; Scott, 2007). Two factors of
importance in their determination are their light sensitivity and the fact that the
epimerization reaction at C8 can proceed in both directions and occurs during
long storage and readily during chemical analysis, such that both epimers need to
be determined (Komarova & Tolkachev, 2001a; Lampen & Klaffke, 2006).
3.2 Description of analytical methods

3.2.1 Introduction
EAs may be extracted with non-polar organic solvents under alkaline conditions
or polar solvents under acidic conditions (Komarova & Tolkachev, 2001b; Scott,
2007; Krska & Crews, 2008; EFSA, 2012). Accelerated solvent extraction has also
been used (Kokkonen & Jestoi, 2009). Sample purification has been achieved by
either liquid/liquid partitioning exploiting the basic tertiary N6 amino group
(Scott & Lawrence, 1980), reversed-phase (C18) solid phase extraction (SPE)
139
(Fajardo et al., 1995), strong cation exchange SPE (Storm et al., 2008; Reinhold
& Reinhardt, 2011), mixed cation/reversed-phase SPE (Reinhard, Rupp & Zoller,
2008), Extrelut® columns (Baumann, Hunziker & Zimmerli, 1985), silica gel
columns (Rottinghaus et al., 1993) or immunoaffinity columns (IAC) (Kokkonen
& Jestoi, 2010). In addition, a molecularly imprinted polymer has been developed
for the alkaloids of interest and inserted as polymerized beads in an SPE column
(Lenain et al., 2012). An aptamer-functionalized silica gel has been trialled but
showed activity for only a limited number of the alkaloids (Rouah-Martin et al.,
2014).
The earliest tests for EAs were performed using simple colorimetric
reagents, resulting in the determination of total alkaloid content (Robbers
et al., 1975; Young, 1981). These tests were performed by adding aqueous
succinic acid to the dried extract, followed by modified van Urk’s reagent
(p-dimethylaminobenzaldehyde and aqueous ferric chloride in aqueous sulfuric
acid). The resultant blue colour was measured at 580 nm. The method was
improved with respect to reaction time and sensitivity by replacing the ferric
chloride with sodium nitrite (Michelon & Kelleher, 1963). Subsequent
development of chromatographic methods allowed the individual alkaloids to be
separated and individually quantified.
Official control of ergot is usually based on the presence by weight of
sclerotia in a grain sample as determined by visual inspection. The determination
of ricinoleic acid has been used as a surrogate marker for ergot content, as it is a
major component of the fatty lipids in Claviceps sclerotia (Franzmann et al., 2010).
Following lipid extraction and hydrolysis, ricinoleic acid in rye was determined as
the silyl derivative by gas chromatography (GC) with flame ionization detection.
Although capable of detecting ergot impurities at concentrations as low as 0.01%,
there is no correlation with total EAs owing to the variability of the levels of the
latter (Franzmann et al., 2010).
Although more a physical technique than analytical chemistry, near-
infrared spectroscopy (NIR) has been used to determine EA content in tall
fescue (Roberts et al., 2005, 2009). NIR hyperspectral imaging has been used to
identify sclerotia in cereal grains (Vermeulen et al., 2009) and has been applied
to scanning wheat on a conveyor belt (Vermeulen et al., 2012). The method was
further validated for wheat, rye, rapeseed, straw and barley straw (Vermeulen
et al., 2013). Extension of the technique to wheat flour showed potential but
was variable at low levels with over- and underestimation, possibly as a result of
sampling problems (Vermeulen et al., 2017).
All analytical operations need to be performed in subdued light and with
quantitation of both epimers. Stock analytical standards should be prepared in
aprotic solvents and stored in amber vials below 0 ºC. Individual calibrants should
be prepared immediately before use or stored at −18 ºC as a dried film. Several
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Ergot alkaloids
reviews of the available analytical methods have been published (Komarova &
Tolkachev, 2001b; Scott, 2007; Krska & Crews, 2008; EFSA, 2012; Crews, 2015).
3.2.2 Screening tests

3.2.2.1 Thin layer chromatography (TLC)
Of the chromatographic techniques applied to mycotoxins, TLC was the first,
although it has been largely superseded by instrumental HPLC. Separation of the
main EAs contaminating cereals was attempted with normal phase TLC using
silica- or alumina-coated plates. However, complete separation of all 12 main
alkaloids was not achieved, even with two-dimensional TLC (Scott, 2007). Some
reports in the older literature describe partial separations of most of the main
alkaloids (Reichelt & Kudrnac, 1973; Szepesi, Molnar & Nyiredy, 1979). EAs
with a C9,10 double bond exhibit fluorescence and can be determined on a TLC
plate by fluorodensitometry. Apart from this, several spray reagents have been
developed and were reviewed by Scott (2007).
An alternative TLC method was developed to measure total EAs in rye
as a single spot on a high-performance TLC (HPTLC) plate (Oellig & Melde,
2016). The alkaloids were extracted with ammonium acetate buffer and cleaned
up by liquid-liquid extraction. The amino HPTLC plates were developed with
methanol to separate the alkaloids from matrix components. Quantification was
by natural fluorescence.
3.2.2.2 Immunological methods

ELISAs are ideally suited for rapid screening of cereals for total EAs. They are
commercially available and can detect all six of the important EAs, as well as
their epimers. However, questions have been raised as to whether cross-reactivity
is the same for all 12 forms (Schnitzius et al., 2001; Crews, 2015). Besides the
commercial ELISAs, some laboratories have developed their own monoclonal
antibodies for determination of total EAs (Liesener et al., 2010; Gross, Curtui &
Usleber, 2018).
3.2.3 Quantitative methods

3.2.3.1 Gas chromatography (GC)
Although GC has been used to determine many mycotoxins, it is not the method
of choice for EAs other than the structurally simpler clavine alkaloids and lysergic
acid derivatives (Scott, 1993; Flieger, Wurst & Shelby, 1997). For the ergopeptines
with their amide linkage at C8, decomposition tends to occur in hot injector
ports and only fragments can be determined, usually by MS (Jegorov et al., 1997).
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3.2.3.2 HPLC with fluorescence detection (HPLC-FLD)

Quantitative determination of all 12 of the main EAs associated with
contaminated cereals is generally achieved by HPLC with either UV/fluorescence
detection or mass spectrometric detection (Scott, 2007; Krska & Crews, 2008;
EFSA, 2012; Crews, 2015). The alkaloids are separated on reversed-phase (for
example, C18) columns with isocratic or gradient elution programmes. Mobile
phases are typically acidified acetonitrile solutions or acetonitrile with basic
buffer. A drawback of acetonitrile with basic buffer is that the silica support of
the HPLC packing can degrade at an alkaline pH. Some of the earlier HPLC
methods only reported a limited number of the alkaloids. UV detection can be
employed at wavelengths of 225 to 254 nm (Blaney et al., 2009). However, better
sensitivity and selectivity can be achieved by utilizing the natural fluorescence
with excitation wavelengths between 235 and 250 nm, or 310 to 330 nm with
emission wavelengths 370 or 410 to 425 nm (Muller et al., 2009; Koppen et al.,
2013; Schummer et al., 2018). The detection limits reported are typically in the
low µg/kg range, although some studies have reported figures as low as 0.01 µg/
kg (Ware et al., 2000; Lombaert et al., 2003; Muller et al., 2006). Sensitivity of
the instrument clearly plays an important part in achieving low detection limits.
More recently, lysergic acid diethylamide (LSD) has been proposed as an internal
standard for determination of the 12 major alkaloids in rye flour and rye products
(Holderied, Rychlik & Elsinghorst, 2019).
3.2.3.3 HPLC coupled to mass spectrometry (HPLC-MS(/MS))

The coupling of HPLC with MS has resulted in a versatile analytical technique
that can incorporate both quantitative results and confirmatory mass spectra and
has become the instrumental technique of choice for most mycotoxin analyses.
Together with limited sample clean-up designed to remove impurities rather
than isolating the analytes (unlike the more traditional SPE or IAC), it provides a
platform for the development of multimycotoxin methods incorporating toxins
of very different chemistries.

HPLC is performed on reversed-phase columns, frequently with volatile
weak acids to provide efficient electrospray ionization in the positive electrospray
ionization mode (ESI(+)) at the MS interface. Many of the earliest methods
for analysing EAs by LC-MS determined only a limited number of alkaloids,
possibly due to the absence of standards, which have only become available
relatively recently (Shelby et al., 1997; Lehner et al., 2005; Burk, Hobel & Richt,
2006; Mohamed et al., 2006). Krska et al. (2008) developed an HPLC tandem MS
(HPLC-MS/MS) method for the six priority EAs and their epimers. Extracts were
purified with dispersive SPE (dSPE) and the 12 compounds were separated within
14 minutes by gradient reversed-phase HPLC. The method was validated for 10
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Ergot alkaloids
different cereal and food matrices and a similar method was applied to a range
of rye products (Crews et al., 2009). A variation of this method was developed
that omitted the dSPE extract purification and used dihydroergotamine as an
internal standard (Tittlemier et al., 2015). Di Mavungu et al. (2012) optimized
and validated an HPLC-MS/MS method for the six major EAs and their epimers
using alkaline extraction and simple liquid/liquid purification and applied it
successfully to a range of food commodities. Twenty-five EAs, including the main
six and their epimers, were included in an ultra-HPLC tandem mass spectrometry
(UHPLC-MS/MS) method developed by Guo et al. (2016).
A common feature of many analytes determined by HPLC-MS/MS is the
occurrence of matrix effects (signal enhancement or suppression). In the case of
the EAs, given the absence of labelled analogues such as those used as internal
standards for other mycotoxins, some analysts use matrix-matched standards
in which calibrants are prepared in an extract of toxin-free sample (Kokkonen
& Jestoi, 2009; Zachariasova et al., 2010). Malysheva et al. (2013) investigated
these effects in detail for wheat, barley, rye, triticale and oats. There was a wide
variation in the level of signal suppression between the different matrices and
the different alkaloids, even within different varieties of the same cereal (rye).
Signal suppression was strongly influenced by the extract purification technique
and was improved with the use of UHPLC. The use of atmospheric pressure
chemical ionization rather than ESI produced strong signal enhancement and an
overestimation of alkaloid levels.
The development of multi-mycotoxin analytical methods using HPLC-
MS/MS has led to the incorporation of EA into a suite of mycotoxins determined
in a single method. One of the earliest was that of Sulyok et al. (2007), which
included 12 major alkaloids in a total of 87 analytes in various food samples,
including bread. Similarly, five of the priority alkaloids (and their epimers)
were determined in a total of 32 toxins in beer using UHPLC coupled to high-
resolution MS (Orbitrap®) (Zachariasova et al., 2010). Arroyo-Manzanares et
al. (2018) developed a UHPLC-MS/MS method with positive ESI that included
the main EAs and their epimers together with 24 other mycotoxins. The aim of
their study was to provide a faster and simpler alternative to multi-mycotoxin
methods that use both positive and negative polarities and may require two
separate chromatographic runs per sample. Sample preparation and analysis time
was kept short to minimize epimerization and the method was validated under
European Commission guidelines.
The movement towards a more general multi-mycotoxin analytical
method using MS detection has resulted in the need to reconsider the traditional
extract purification methods. In general, two different approaches have been
adopted. The first is the complete removal of the extract purification step and the
use of the so-called “dilute-and-shoot” method, in which the extract is merely
143
diluted with an appropriate solvent (for example, HPLC mobile phase) and
directly injected into the HPLC (Sulyok et al., 2010; Zachariasova et al., 2010).
The second method is a more general easy-to-use clean-up designed not to
remove the mycotoxins from the extract, but to remove interfering substances
such as lipids and pigments. This latter method has been termed QuEChERS
(quick, easy, cheap, effective, rugged and safe) (Walker et al., 2015; Kowalczyk et
al., 2016; Leon et al., 2016; Arroyo-Manzanares et al., 2018). This is a relatively
fast method involving salting out with buffer salt such as magnesium sulfate or
citrate buffer, which may be followed by dispersive dSPE with magnesium sulfate,
primary secondary amine, octadecyl sorbent and neutral alumina.
4. Sampling protocols
The inherent non-homogeneous nature of contamination with EAs (and other
mycotoxins) in raw agricultural commodities is a major challenge in obtaining
representative samples (Brera, Miraglia & Colatosti, 1998; Kabak, Dobson & Var,
2006). This challenge has been addressed for other mycotoxins (for example,
aflatoxins, fumonisins and ochratoxin) by the adoption of suitable sampling
protocols. The distribution of EAs is more heterogeneous than that of other
mycotoxins (Karlovsky et al., 2016), because contamination does not occur on
single kernels but within large sclerotia which are prone to fragmenting. Although
this is a known problem, no sampling protocols have been developed specifically
for EAs. In some countries, the concentration of ergot sclerotia in unprocessed
grains is legally limited (EU 2015b, 0.05 g/kg of ergot sclerotia in grain) and
although there are publicly available sampling plans for visual inspection of
raw grain for sclerotia occurrence (USDA, 1995; Canadian Grain Commision
(CGC), 2015b), the value of these protocols when sampling prior to analytical
determination of EAs is very limited.
The problem of sampling for major mycotoxins has been addressed by

statistical means and the drawing up of sampling plans. Manuals on sampling
for inspection purposes were developed by the United States Department of
Agriculture, Agricultural Marketing Service, Federal Grain Inspection Service
(USDA, 1995), and the CGC (CGC, 2015a). In Europe the methods of sampling
and analysis for the official control of the levels of mycotoxins in foodstuffs
are detailed in the Commission Regulation (EC) No 401/2006 (EU, 2006). A
manual aimed at addressing sampling procedures and written for both food
analysts and regulatory officials, which explains some of the statistical issues,
was produced as part of the Joint FAO/IAEA Programme, Nuclear Techniques in
Food and Agriculture (Whitaker et al., 2010). Much of the statistical research was
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Ergot alkaloids
consolidated by FAO in a mycotoxin sampling tool, containing information on 26

mycotoxin–commodity combinations (FAO, 2013).1
To further assist in the practical application of sampling for mycotoxin
contamination, FAO, in collaboration with the Italian National Institute of
Health, produced two training videos (Brera, Miraglia & Pineiro, 2007; Istituto
Superiore di Sanità, 2015). Maximum levels for mycotoxins in various foods set
by the Codex Alimentarius Commission (CAC) are supported by sampling plans.
These plans for cereals and cereal products can be accessed online (FAO/WHO,
2016). At the time of writing, the Codex Committee on Methods of Analysis and
Sampling (CCMAS) was reworking the General Guidelines on Sampling (CAC/
GL 50-2004).
In the absence of specific sampling plans for EAs, aflatoxin sampling
plans are routinely used. Whenever possible, it is recommended that the
same sampling method is applied to the same product by buyer and seller. It
is further recognized that large-scale sampling plans may not be appropriate or
advantageous in smallholder settings, thus presenting an additional challenge.
5. Effects of processing
5.1 Sorting, cleaning and milling

Sorting, sieving, flotation, handpicking, washing and milling, can potentially
reduce EA levels. Of these processes, manual and automatic sorting, along with
sieving, are the most effective means of reducing fungal contamination (Grenier et
al., 2014; CGC, 2015a; Karlovsky et al., 2016). As a major portion of EAs has been
associated with small, shrivelled and discoloured material within grain samples
(Muthaiyan, 2009; Grenier et al., 2014), a reduction in EA levels of about 34–55%
is possible when mould-contaminated grains or seeds are physically removed by
sieving, handpicking and separation from intact raw samples (Muthaiyan, 2009).
In the rye industry, sclerotia are effectively removed by sorting, thus reducing EA
contamination (Young et al., 1983; Miedaner & Geiger, 2015). Therefore, these
procedures can be useful for reducing the level of human exposure to EAs (EFSA,
2012).
Conventional grain cleaning equipment (i.e. scalpers, shaker decks,
gravitational separators and electronic sensor-based sorters) that removes foreign
matter, dust, and broken or shrivelled grains can reduce EA contamination
(Belser-Ehrlich et al., 2012; CGC, 2015a). Methods using either high velocity
1
http://www.fstools.org/mycotoxins/
145
air cleaning or electronic sensor-based handling have been shown to reduce EA

levels by more than 80% (Miedaner & Geiger, 2015).
In common with other mycotoxins, EAs in cereals are not destroyed by
milling, which merely distributes them among the milling fractions. In general,
fractions intended for human food have reduced levels, whereas those intended
for animal feed (for example, bran, shorts and feeds) have elevated levels (Farjado
et al., 1995; Franzmann et al., 2011; Tittlemier et al., 2019).
5.2 Thermal and chemical food processing

Heat treatment can reduce EA levels in processed products; the level of reduction
is strongly dependent on temperature and treatment duration. Temperatures
above 100 °C used for frying, roasting, toasting and extrusion cooking have been
reported to reduce EA levels. Studies on the fate of EAs in different flours and
during different food processing operations suggested a reduction during baking
(Wolff et al., 1988; Fajardo et al., 1995; Scott, 2009).
Merkel et al. reported that EAs are degraded and epimerized during
bread baking; thus, the ratio shifts from the -ine form to the -inine form (Merkel
et al., 2012). Similar observations on the instability and shift in the ratio of
epimeric forms were also reported by others (Baumann et al., 1985; Franzmann
et al., 2010). Meleard reported a reduction of up to 60% of EAs during a French
baking test at 250 °C of a commercial wheat flour spiked with a sclerotia grind
(Meleard, 2016).
Bryla et al. investigated the stability of six R-configuration EAs
(ergometrine, ergocornine, ergocristine, ergocryptine, ergosine and ergotamine)
and their respective S-enantiomers (ergometrinine, ergocorninine, ergocristinine,
ergocryptinine, ergosinine and ergotaminine) during the process of baking rye
bread at 190 °C (Bryla et al., 2019). Although the authors did not observe a
reduction of EAs during dough formation, they reported a 22% loss of total EAs
during baking; specific degradation products were not identified. Interestingly,

the authors also noted that the concentration of R-enantiomers decreased from
dough formation to baking by 46%, but the concentration of S-enantiomers
increased by 21%. During the baking process, the ratio of R-enantiomers to
S-enantiomers shifted from 65:35 to 45:55.
Fajardo et al. reported losses of EAs during preparation and cooking
of different Asian noodles and spaghetti prepared from highly contaminated
wheat flour. However, the analytical method used in this study only reported
the R-enantiomers (Fajardo et al., 1995). Epimerization to S-enantiomers during
cooking would not have been detected.
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Ergot alkaloids
In a well-designed study, Tittlemier et al. prepared spaghetti from

durum contaminated with different amounts of ergot sclerotia (0.01–0.1%)
monitoring the levels of 10 different EAs during the processing steps (Tittlemier
et al., 2019). An analytical method (LC-MS/MS) was used that included four
R/S enantiomeric pairs (ergosine, ergosinine, ergocristine, ergocristinine,
ergocornine, ergocorninine, ergocryptine and ergocryptinine) and two further
R-enantiomers (ergometrine and ergotamine) as analytes. The authors noted no
significant epimerization during spaghetti extrusion at 45 °C, but the cooking
process led to a marked shift in enantiomers from R- to S- forms. Notably, the
authors did not detect a reduction in the total EA concentration during the
production or cooking processes; EAs were also not detected in the cooking
water.
5.3 Fermentation
Reduction of levels of major R-enantiomer forms of EAs during malting and
brewing has been documented (Schwarz et al., 2007). Steeping appears to
solubilize a small amount of EAs, with kilning resulting in a greater decrease
(≈30%) and an overall reduction in beer of about 46% of the total EAs, as a result
of thermal degradation (Schwarz et al., 2007).
6. Prevention and control
6.1 Preharvest control

Factors influencing the concentration of EAs in plants are poorly documented;
however, limited data are available on cereals.
Among the cereals, rye and triticale are the most susceptible to infection
by Claviceps spp., followed by wheat, barley and oats (Platford & Bernier, 1976;
Menzies & Turkington, 2015). Rye and triticale are more susceptible mainly
because their flowers are cross-pollinated, whereas wheat, barley and oats are self-
pollinated (Gaudet et al., 2000). A French survey of 1919 fields sampled between
2012 and 2014 found that rye was 16 times more contaminated than wheat, and
that triticale was twice as contaminated as wheat (Orlando et al., 2017).
Differences between cultivars also play a role in susceptibility, but this
has not been thoroughly investigated. A few studies have been conducted on
rye (Sosulski & Bernier, 1975; Mainka et al., 2007), wheat (Watkins & Littlefield,
147
1976; Pageau et al., 1994; Menzies, 2004) and barley (Pageau & Lajeunesse, 2006;
Oxley et al., 2009) to identify resistant cultivars.
Wild grasses within or outside fields are the primary source of ergot
inoculum due to the wide host range of C. purpurea. The above-mentioned
French survey showed that fields with grasses, both in the main part of the field
and in the field margins, were 50% more contaminated than those without grasses
(Orlando et al., 2017).
Because ergot sclerotia usually do not survive longer than a year in the
soil (Mitchell & Cooke, 1968; Maunas & Leclère, 2013), crop rotation can be used
to control EA contamination. However, this strategy is only effective if grasses are
simultaneously controlled. For example, difficulties controlling grasses in oilseed
rape fields lead to a 20 to 60% increase in EA contamination in subsequent cereal
crops (Orlando et al., 2017).
Similarly, the tillage system can help to control plant contamination, as
deep ploughing buries sclerotia in the soil. As a result, ascospores are not formed
or they cannot be released into the air in spring. Maumené et al. (2016) described
the depth distribution of sclerotia artificially dispersed on the soil surface, under
different tillage systems (ploughing, shallow cultivation and a combination of
these two techniques) over a 2-year crop sequence. The authors recommended
limiting tillage the subsequent year to shallow cultivation, to avoid bringing the
buried sclerotia back up to the soil surface. The French field study indicated that
fields were slightly less contaminated after deep tillage than after other tillage
practices, but there was no statistically significant difference (Orlando et al.,
2017).
Lastly, the use of ergot-free or certified seed could significantly improve
control, by preventing the introduction of primary inoculum into fields. The
treatment of seeds with fungicide has also been identified as a way of decreasing
the germination of fungal spores (Shaw, 1988; Puhl et al., 2007; Maunas et al.,
2016).
6.2 Postharvest control

There are very few studies on postharvest control of EAs. Cleaning grain during
and after harvest by removing sclerotia will reduce EA contamination (Miedaner
& Geiger, 2015).
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Ergot alkaloids
6.3 Decontamination
Effective decontamination measures should be irreversible, products should be
non-toxic and grain should keep its nutritional value while maintaining storablity
and palatability.
An effective physical decontamination procedure is removal of sclerotia
by sieving (Seaman, 1980; Muthaiyan, 2009). Opto-electric sorting is another
highly effective way of sorting sclerotia from cereal (Young et al., 1983; Miedaner
& Geiger, 2015). Berg et al. (1995) reported winnowing prior to sorting as an
effective decontamination procedure. Studies by Franzmann et al. showed that
milling, even after carefully applied cleaning practices, only redistributed sclerotia
fragments and dust in the grinding stock (Franzmann et al., 2011). Peeling was
also reported as a successful EA reducing procedure, but cannot be universally
applied (Franzmann et al., 2011).
Young et al. reported that EAs in wheat ergot sclerotia could be reduced
by up to 90% by applying chlorine at either 150 °C or 200 °C. Treatment with
sulfur dioxide and hydrogen chloride at the same temperatures reduced the
amounts of EAs by only 20%. The authors further observed that treatment of
wheat sclerotia in an autoclave for 30 minutes at 121 °C prior to feeding to
growing chickens reduced toxic effects (Young et al., 1983). Ergot separation
from rye grains by flotation in sodium chloride solution also showed promising
effects, but the treatment resulted in damp and salty grain (Plante & Sutherland,
1944; Seaman, 1980).
Although EAs are reported to be photosensitive (Stoll & Schlientz, 1955),
Young et al. observed that irradiation of ground sclerotia with UV light for
54 hours did not change EA levels (Young et al., 1983). Other techniques including
enzymatic or microbial decontamination are reported to have been successfully
applied for other mycotoxins (Petruzzi et al., 2014; Karlovsky, 2016), but not for
sclerotia or EAs.
A challenge of applying chemicals, irradiation or other procedures is that
the processing agents, procedures, or microorganisms used for decontamination
must be authorized for use in food (Codex Alimentarius, 2015). Procedures
authorized in national food legislation were not found. National context may be
nuanced; for example, European Commission Regulation EC 2015/786 defines
acceptability criteria for detoxification processes applied to products intended for
animal feed (EU, 2015a), but these criteria do not apply to food.
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7. Levels and patterns of contamination in food

commodities
7.1 Surveillance data

The GEMS/food contaminants database was queried for records relating to the
analytical results for ergot alkaloids referring either to the sum of EAs or to
the different ergot alkaloids: ergometrine (ergonovine), ergosine, ergocornine,
ergotamine, ergocristine, α-ergocryptine, β-ergocryptine and the corresponding
-inine (S)-epimers (ergometrinine (ergonovinine), ergosinine, ergocorninine,
ergotaminine, ergocristinine, α-ergocryptinine and β-ergocryptinine).
Data extracted (n = 203 453 results) were submitted between 2004 and
2019 and originated from four WHO regions: African Region (Benin, Cameroon,
Mali and Nigeria), European Region, Region of the Americas (Canada) and the
Western Pacific Region (Hong Kong SAR, New Zealand and Singapore). No
concentration data for EAs have been submitted to the GEMS/Food contaminants
database from countries in the WHO South-East Asia and Eastern Mediterranean
regions. Overall, data extracted originated from 30 countries or country groups,
representing 13 of the 17 GEMS/Food cluster diets.
The extracted database was cleaned by:

1) removing individual results related to animal feed;
2) removing aggregated data, keeping only results from individual
samples;
3) converting all results and the analytical limits: LOD and LOQ to
the same units (µg/kg) and ensuring that the naming of foods was
consistent;
4) removing results expressed as a summation when analytical results
for the individual EAs were available for the same food sample
identifier;
5) reclassifying food descriptors for some data to establish consistency
within the database (for example, some records of EAs in pasta and
cookies were reported in “composite foods” and/or in “snacks and
dessert” and these records were reclassified as “bread and other
cooked cereal products”, and since spelt is a wheat species (Triticum
spelta) records relating to spelt were reclassified as “wheat”;
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Ergot alkaloids
6) excluding some quantified samples classified in the “herbs and

spices” food category, which referred to baking ingredients that are
not ready-to-eat;
7) excluding records of grain as crops that referred to samples of
unprocessed grains of unknown end-use, as such samples are usually
not considered when estimating human dietary exposure;
8) substituting for left-censored data, as recommended in Chapter 6
Dietary exposure assessment of chemicals in food (2020) of the EHC
240 “principles and methods for the risk assessment of chemicals in
foods” (WHO/IPCS, 2009). At the lower bound (LB), results below
the LOQ and LOD were replaced by zero and at the upper bound
(UB), results below the LOD were replaced by the LOD and those
below the LOQ were replaced by the LOQ.
After cleaning, as indicated in Table 15 the database contained 178 184
data items originating from the WHO African Region (1.4%), Region of the
Americas (13.6%), European Region (83.8%) and the Western Pacific Region
(1.2%).
For 11 of the EAs considered, the highest levels were observed in food
samples from the European Region: ergocristine (9279 µg/kg), ergocristinine
(3538 µg/kg), ergocornine (619 µg/kg), ergocorninine (396 µg/kg), ergocryptine
(661 µg/kg), ergocryptinine (1007 µg/kg), ergosine (1287 µg/kg), ergosinine
(1066 µg/kg), ergometrine (760 µg/kg), ergometrinine (234 µg/kg) and
ergotaminine (339 µg/kg). For ergotamine, the highest level (3343 µg/kg) was
observed in food samples from the Region of the Americas (Table 16). Table 17
summarizes data on EA concentrations by region and country from the GEMS/
Food contaminants database.
No quantified concentration data were submitted for alcoholic beverages
(n = 4198); eggs and egg products (n = 48); fats and oils (n = 1315); fish and
others (n = 72); fruits and fruit products (n = 11 471); fruit and vegetable juices
(n = 1822); herbs and spices (n = 865); meat and meat products (n = 136); milk
and milk products (n = 156); non-alcoholic beverages (n = 81); nuts and oilseeds
(n = 21 945); products for special nutritional use (n = 192); snacks and desserts
(n = 496); starchy roots and tubers (n = 594); stimulant beverages (n = 72); sugar
and confectionery (n = 1572), or vegetables and vegetable products (n = 1368).
Quantified concentrations of EAs were available for some food
commodities: cereals and cereal-based products (n = 19 635/124 252); legumes
and pulses (n = 33/2513, mainly in dry soya beans), and foods for infants and
young children (n = 4/4780, mainly cereal-based infant foods).
As the relative potency of individual EAs is uncertain, the Committee
calculated the total EA concentration as the simple sum, for all food samples
151
Table 15
Summary of EA concentrations from the GEMS/Food contaminants database
EA No. of records % positive Mean positive (µg/kg) Maximum (µg/kg)
Ergometrine 13 469 14.4 34.7 760
Ergometrinine 11 940 5.0 16.0 234
Ergotamine 13 176 18.9 62.1 3 343
Ergotaminine 11 549 9.6 26.1 339
Ergosine 18 432 9.8 33.2 1 287
Ergosinine 11 485 6.1 22.4 1 606
Ergocristine 20 484 17.3 64.9 9 279
Ergocristinine 13 544 11.2 39.2 3 538
Ergocryptine 13 519 14.6 27.8 661
Ergocryptinine 22 245 5.7 20.0 1 007
Ergocornine 14 516 12.2 34.2 617
Ergocorninine 13 435 6.8 21.8 396
Total EAs (not specified)a 390 20.0 46.5 1 200
Overall 178 184 11.1 39.9 9 279
a
Analytical records with no specification of the EA measured were from Hong Kong SAR and Singapore.
Table 16
Summary of maximum EA concentrations in the different WHO regions, from the GEMS/
Food contaminants database
WHO region
African Region European Region Western Pacific Region Region of the Americas
EA maximum (µg/kg) maximum (µg/kg) maximum (µg/kg) maximum (µg/kg)
Ergometrine 27 760 22 153
Ergometrinine 1 234 2 33
Ergotamine 31 2 131 23 3 343
Ergotaminine 2 339 16 91
Ergosine 29 1 287 10 296
Ergosinine 2 1 066 6 38
Ergocristine 37 9 279 27 951
Ergocristinine 11 3 538 22 414
Ergocryptine 14 661 25 414
Ergocryptinine 6 1 007 5 88
Ergocornine 5 619 8 172
Ergocorninine 3 396 12 58
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Ergot alkaloids
Table 17
Summary of EA concentrations by region and country from the GEMS/Food contaminants
database
Region/country No. of records % positive Mean positive (µg/kg) Maximum (µg/kg)
African 2520 2.1 7.2 37
Americas 24 236 19.7 38.7 3 343
Canada 24 236 19.7 38.7 3 343
European 149 358 9.6 39.9 9 279
European Union 149 358 9.6 39.9 9 279
Western Pacific 2 070 28.1 7.8 1 200
Hong Kong SAR 351 22.2 46.5 1 200
New Zealand 1 680 29.9 1.8 27
Singapore 39 0.0 0.0 0.00
Overall 178 184 11.1 39.9 9 279
for which concentration data were available for individual EAs (Tables 18–21).
The upper bound (UB) estimate of concentration for the sum of EAs was only
calculated for food commodities for which at least one quantified sample was
available. The UB estimates per food sample were derived by adding quantified
results to the mean of the LODs or LOQs of the individual EAs reported as not
detected or not quantified. For samples for which EAs were reported only as not
detected or not quantified, UB estimates were derived by averaging the LODs
or LOQs. For example, of the 84 samples of barley reported from the WHO
European Region, only one sample was quantified. The mean UB was calculated
by adding the value of the quantifed sample to the sum of analytical limits of each
individual EA analysed in the 83 other samples, and dividing by the total number
of individual EAs analysed in each sample. This approach was used rather than
summing the LOD/LOQ of each unquantified individual alkaloid to avoid overly
conservative estimates of UB concentrations (Tables 18–21). The Committee did
not consider food commodities for which results were reported only as “none
detected” or “none quantified”.
Owing to the paucity of data for regions other than Europe, a
literature search was conducted using Web of Science with the search term
“TS=((mycotoxin)+(ergot alkaloids) and (surve* or occurr* or contam*) and
(food or feed) NOT(Europe*))”. This search recovered only 82 publications (as
of January 2021), which were screened for relevance by title, abstract and then
the full paper. The results from the GEMS/Food contaminants database and the
literature search are summarized below by WHO region.
153
7.1.1 African Region

A total of 2520 analytical results from 210 food samples from African countries
were present in the GEMS/Food contaminants database. The samples were
from the African Total Diet Study performed by Ingenbleek et al. across four
sub-Saharan countries (Benin, Cameroon, Mali and Nigeria). All samples
were analysed for 12 EAs (ergometrine, ergotamine, ergosine, ergocristine,
ergocryptine, ergocornine and the corresponding -inine epimers); 97.9% of the
analytical results were below the LOD (0.03 to 1.1 µg/kg depending on the EA).
Food consumption data were estimated as the daily amount of food
consumed (in g per adult male equivalent per day) derived from previously
existing household budget surveys generated by national statistics authorities
from 72 979 households. In total, 4020 food samples, representing at least 90% of
each national total diet by weight, were obtained from a core list of 84 subgroups.
A food composite approach was taken, pooling 12 samples of the same core
food to form 335 pooled samples. Samples were collected from each of the eight
study centres. They were purchased during the rainy season (October 2017) and
again during the dry season (February 2018) (Ingenbleek et al., 2020). The study
centres included three located in coastal areas (Duala, the Littoral of Benin and
Lagos) and five in non-coastal areas (Bamako, the Borgou region of Benin, Kano,
North Cameroon and Sikasso) (Ingenbleek et al., 2019). Of the 335 composite
samples, 194 composite samples of cereals, tubers, legumes, vegetables, nuts and
seeds, dairy products, oils, beverages and miscellaneous foods were considered
to be potentially contaminated by mycotoxins and were analysed by LC-MS/MS.
Only bread and other cooked cereal products were found to be
contaminated (Table 18). The most contamination was observed in a “bread and
other cooked cereal products” sample contaminated with 36.91 µg/kg ergocristine
and having a total EA contamination of 165.7 µg/kg.
Except the total diet study by Ingenbleek et al., the results of which
are already included in the GEMS/Food contaminants database, the literature
search identified no other study that investigated the occurrence of the 12 EAs
(ergometrine, ergotamine, ergosine, ergocristine, ergocryptine, ergocornine and
the corresponding -inine epimers) in African food.
One study investigated the variation of fungal metabolites in sorghum
malts used to prepare the traditional Namibian fermented beverages omalodu and
otombo. Six clavine EAs (agroclavine, chanoclavin, elymoclavine, festuclavine,
fumigaclavine A and fumigaclavine B) were quantified with a prevalence of 50–
100% in malt samples used to prepare both beverages, except for elymoclavine,
which was only found in 3% and 38% of otombo and omalodu malt samples,
respectively (Nafuka et al., 2019).
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Ergot alkaloids
Table 18
Summary of mean and maximal EA concentrations in food from the WHO African Region
from the GEMS/Food contaminants databasea
Food commodities No. of samples % <LOD or LOQ mean LB, µg/kg mean UB, µg/kg Max. µg/kg
Cereals and cereal-based products 59 89.8 6.2 7.0 166
Bread and other cooked cereal 9 33 40.9 43.0 166
products
Maize 16 100 0.0 0.5
Millet 8 100 0.0 0.5
Rice 16 100 0.0 0.5
Sorghum 10 100 0.0 0.5
LB, lower bound; LOD, limit of detection; LOQ, limit of quantitation; UB, upper bound.
a
The mean LB estimates were derived by substituting zero for analytical results below the LOD or LOQ. The mean UB estimates were derived by averaging the sum of
individual EAs reported as not detected or not quantified.
7.1.2 Region of the Americas

A total of 24 236 analytical results from 6240 food samples from the Region of the
Americas were present in the GEMS/Food contaminants database. All these data
came from Canada and were collected between 2009 and 2018. Most samples
(5394) were analysed for three EAs (ergocristine, ergocryptine and ergosine), 286
were analysed for six EAs (ergocornine, ergometrine, ergocristine, ergocryptine,
ergosine and ergotamine), 191 for 10 EAs (ergometrine, ergocristine, ergocryptine,
ergosine, ergotamine and the corresponding -inine epimers), and 369 for 12 EAs
the corresponding -inine epimers). More than 80% of the analytical results were
below the LOD (1.7 to 9 µg/kg depending on the EA).
Only cereals and cereal-based products were found to be contaminated
(Table 19). The maximum level was observed in a barley sample contaminated
with 3343 µg/kg ergotamine and a total EA contamination of 3549 µg/kg.
As for other parts of the world, the literature survey did not identify any
additional data.
7.1.3 Eastern Mediterranean and South-East Asia regions

There were no EA analyses submitted to the GEMS/Food contaminants database
from the Eastern Mediterranean and South-East Asia regions. The literature
survey did not provide any additional data.
7.1.4 European Region

The European Region was the most represented in the GEMS/Food contaminants
database, with a total of 149 538 analyses (83.8% of total analyses) from 14 972
155
Table 19
Summary of mean and maximal EA concentrations in food from Canada, from the GEMS/
Food contaminants databasea
Cereals and cereal-based products 5 593 72.2 33.0 40.4 3549
Barley 264 66.3 45.9 54.8 3 549
Bran, unprocessed of cereal grain 186 90.3 3.2 11.9 97
(except buckwheat, canihua, quinoa)
Bread and other cooked cereal products 1 425 69.1 16.9 23.9 1 060
Buckwheat 140 95 2.3 7.8 217
Cereals and cereal-based products NES 612 98 0.8 6.0 234
Maize 449 8.4 0.4 5.7 53
Oats 434 90.8 7.2 14.9 481
Rye 132 41.7 195.0 200.5 1 917
Wheat 1 404 38.5 83.9 93.5 1 066
a
samples having reported results for EAs. All samples were from the European
Union and had been collected between 2004 and 2019. Most of them were
analysed for the 12 EAs (ergometrine, ergotamine, ergosine, ergocristine,
ergocryptine, ergocornine and the corresponding -inine epimers). Overall, 9.6%
of all analytical results from the European Region were positive for EAs.
Only two food commodities were found to be contaminated: cereals and
cereal-based products, and legumes and pulses (Table 20). The maximum levels
were observed in rye grain (13 783 µg/kg) and undefined cereals and cereal-
based products (5649 µg/kg). Within the cereals and cereal-based products, four
categories had a relatively high proportion of contaminated test samples, i.e.
bread and other cooked cereal products (37.1% of the samples in this category
were contaminated; mean LB 29.8 µg/kg and mean UB 61.0 µg/kg); cereal and
cereal-based products unidentified (38.6% samples contaminated; mean LB
99.4 µg/kg and mean UB 126.1 µg/kg); rye (45.9% samples contaminated; mean
LB 93.4 µg/kg and mean UB 124.6 µg/kg) and wholemeal bread (60.5% samples
contaminated; mean LB 68.5 µg/kg and mean UB 114.3 µg/kg). Among the
samples of legumes and pulses, 6 of the 67 samples of beans, shelled (immature
seeds) were contaminated (mean LB 6.5 µg/kg and mean UB 14.2 µg/kg) as well
as 11 of the 109 dried soya bean samples (mean LB 2.3 µg/kg and mean UB
9.1 µg/kg).
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Ergot alkaloids
Table 20
Summary of mean and maximal EA concentrations in food from the WHO European Region,
from the GEMS/Food contaminants databasea
Cereals and cereal-based products 9 381 66.9 63.5 89.8 13 783
Barley 84 99 1.6 11.6 133
Bran, unprocessed of cereal grain 1 100 0.0 2.0
(except buckwheat, canihua, quinoa)
Bread and other cooked cereal products 2 334 62.9 29.8 61.0 923
Buckwheat 110 89.1 3.7 11.5 80
Cereal grains NES 519 95 9.5 28.7 1 280
Cereals and cereal-based products NES 4261 61.4 99.4 126.1 5645
Maize 123 100 0.0 16.2
Millet 41 100 0.0 5.2
Oats 183 90.7 7.8 20.0 359
Rice 155 100 0.0 15.4
Rye 910 54.1 93.4 124.6 13 783
Sorghum 1 100 0.0 2.0
Wheat 549 83.6 15.0 33.7 1101
White bread 72 87.5 4.1 23.5 199
Wholemeal bread 38 39.5 68.5 114.3 552
Legumes and pulses 197 91.4 3.5 11.7 318
Beans except broad bean and soya bean 18 100 0.0 16.7
(green pods and immature seeds)
Beans, shelled (immature seeds) 67 91 6.5 14.2 318
Lentil (dry) 2 100 0.0 20.0
Peas (dry) 1 100 0.0 20.0
Soya bean (dry) 109 89.9 2.3 9.1 36
a
7.1.5 Western Pacific Region

Three countries from the Western Pacific Region supplied data on EAs to the
GEMS/Food contaminants database: New Zealand (1650 analytical results from
140 food samples), Hong Kong SAR (351 analytical results from 351 food samples)
and Singapore (39 analytical results from 39 food samples). These samples were
collected between 2016 and 2019.
The samples from Singapore and Hong Kong SAR were analysed for total
EAs with no indication of the specific EAs included, whereas the samples from
New Zealand were analysed separately for the 12 EAs (ergometrine, ergotamine,
ergosine, ergocristine, ergocryptine, ergocornine and the corresponding ‑inine
epimers). All samples from Singapore were below the LOD (10 µg/kg).
157
Table 21
Summary of mean and maximal EA concentrations in food from the WHO Western Pacific
Region, from the GEMS/Food contaminants databasea
Cereals and cereal-based products 496 67.3 9.1 9.8 1200
Barley 11 91 49.1 49.4 540
Bread and other cooked cereal products 149 45 7.7 8.2 130
Buckwheat 5 100 0.0 0.5
Cereals and cereal-based products NES 176 81.3 3.7 4.6 180
Maize 9 100 0.0 0.5
Millet 3 100 0.0 0.5
Oats 26 92.3 1.7 3.3 42
Rice 23 95.7 0.4 1.9 10
Rye 29 34.5 15.8 16.1 169
Sorghum 3 100 0.0 0.5
Spelt 9 66.7 7.0 7.4 31
Wheat 9 100 0.0 0.5
Wheat flour 17 29.4 4.6 5.2 21
Wheatgerm 3 0 464 464 1200
White bread 6 16.7 15.0 15.1 44
Wholemeal bread 3 66.7 14.0 14.3 42
Legumes and pulses 10 90 0.9 2.8 9
Lentils (dry) 3 100 0.0 1.7
Peas (dry) 7 85.7 1.3 3.3 9
a
Only two food commodities were found to be contaminated: cereals

and cereal-based products and legumes and pulses (Table 21). The maximum
level was observed in a wheatgerm sample from Hong Kong SAR contaminated
with 1200 µg/kg total EA. More than half of the bread and other cooked cereal
products had a detectable level of EAs (mean LB 7.7 µg/kg and mean UB 8.2 µg/
kg). Of the samples of legumes and pulses, one out of the 10 had detectable levels
of EAs.
No report on the occurrence of EAs in the Western Pacific Region was
identified in the literature search.
7.2 Conclusions
Based on data from the GEMS/Food contaminants database, comparisons of
analyses for EA across global regions have indicated that, despite large differences
158
Ergot alkaloids
in the number of analyses reported, the only foodstuffs testing positive for EAs
were cereals and cereal-based products and, to a lesser extent, legumes and pulses.
Most of the analytical records were supplied from the European Region. Results
were also obtained from the African Region (Benin, Cameroon, Mali and Nigeria),
the Region of the Americas (Canada) and the Western Pacific Region (Hong
Kong SAR, New Zealand and Singapore). In all regions, contamination (10.2 to
32.7% positive samples) was observed in cereals and cereal-based products, but
the level of contamination was higher in Europe (mean UB level 89.8 µg/kg) and
in the Region of Americas (mean UB level 40.4 µg/kg) than in the Western Pacific
Region (mean UB level 9.8 µg/kg) or in the African Region (mean UB level 7.2 µg/
kg). In the European Region and the Western Pacific Region, contamination (8.6
to 10% positive samples) was also observed in legumes and pulses at lower levels
than in cereals and cereal-based products (mean UB level 11.7 µg/kg and 2.8 µg/
kg, respectively). The highest levels of total EAs were observed in rye (13 783 µg/
kg) and wheat-based products (5649 µg/kg) from the European Region.
8. Food consumption and dietary exposure estimates
8.1 Concentrations in food used in the dietary exposure estimates

The GEMS/food contaminants database was used as the source of information.
The query as well as the cleaning steps are described in section 7.1. Estimates of
dietary exposure derived by the Committee only considered food commodities
for which quantified levels of any individual EAs were reported.
8.2 Food consumption data used in the dietary exposure estimates

In addition to national and regional estimates of dietary exposure published
in the literature, the Committee derived international or regional estimates of
dietary exposure to EAs using the GEMS/Food cluster diets. Considering that
cereals and cereal-based products are foods consumed on a regular basis and
considering that wheat and wheat-based products represent the main foods
contributing to chronic dietary exposure to EAs across national estimates, the
Committee considered that the use of GEMS/Food cluster diets rather than the
use of summarized individual food consumption data (Chronic individual food
consumption database – Summary statistics (CIFOCOss)) data was sufficient to
capture international estimates of dietary exposure to EAs.
159
The GEMS/Food cluster diets provide mean per capita food consumption
values based on FAO food balance sheet data for raw commodities and some
semi-processed commodities for 17 clusters of countries (Sy et al., 2013). Relevant
food consumption data for each of the 17 GEMS/Food cluster diets categorized
by WHO regions are presented in Table 22. Although some clusters defined in
2013 do not map exactly to a single WHO region, the Committee considered that
the differences noted in clustering were limited to a small number of countries
and were not likely to affect the chronic dietary estimates.
8.3 Assessments of dietary exposure

Estimates of national and international chronic and acute dietary exposure to EAs
reported from the scientific or grey literature, or derived by the Committee were
all based on total EA concentration calculated as the simple sum of individual
EAs. Table 23 summarizes national and international estimates of chronic dietary
exposure to EAs from the literature or derived by the Committee.
8.3.1 National or regional estimates of chronic dietary exposure from the

published literature
Few publications or reports on dietary exposure to EAs have appeared in the
scientific or grey literature. The Committee evaluated studies on dietary exposure
to EAs in sub-Saharan African countries, European countries and New Zealand.
(a) WHO African Region

Twelve EAs were detected in foods processed from wheat (five of six bread
samples), with a mean concentration of 62.4 µg/kg, ranging from not detected
to 165.7 µg/kg, for the sum of 12 EAs. The treatment of left-censored data for
estimation of dietary exposure (LB–UB) was only applied for products in which
alkaloids were detected. Assessment of dietary exposure to the sum of EAs was
not carried out in the original study (only assessment of dietary exposure to each
individual EA was performed for all food commodities including those where
EAs were not quantified) and was subsequently estimated by the Committee,
based on information available from FAO. Dietary exposure estimates for the
adult population are summarized for mean and high percentile (P95) for the
eight study centres (Table 23).
(b) WHO European Region

Both chronic and acute human dietary exposure to EAs were assessed for European
countries (EFSA Panel on Contaminants in the Food Chain (CONTAM) 2012).
Dietary exposure to EAs was estimated using occurrence data submitted by
160
Table 22
Summary of consumption data for 17 GEMS/Food cluster diets grouped by WHO region
Food consumption for WHO GEMS/Food cluster diet in g/person per day, grouped by WHO region
Eastern Mediterranean European African Americas Western Pacific South-East Asia
Food commodities G01 G04 G06 G02 G07 G08 G10 G11 G15 G03 G13 G16 G05 G12 G14 G17 G09
Grains and grain-based products
Barley, including flour and 12.5 0.1 0.6 7.6 0.6 2.6 2.0 1.0 1.9 0.1 5.5 0.0 1.3 0.1 0.0 0.0 0.4
grits
Buckwheat, including flour 0.0 0.0 0.1 0.3 0.0 0.6 0.3 0.0 0.0 0.0 0.0 0.0 0.1 0.0 2.8 0.0 0.1
Cereals preparations NES 0.9 9.1 0.7 0.9 3.3 1.6 8.2 1.5 4.2 0.7 0.7 0.1 2.0 5.0 0.6 1.9 0.7
(and flour)
Maize, including maize flour 23.4 36.3 61.6 36.1 17.8 14.7 17.1 5.8 29.6 87.8 94.4 56.0 48.0 53.1 8.1 28.1 21.1
and sweet corn
Oats, including rolled oats 0.0 1.1 0.0 3.9 4.1 3.4 2.7 1.7 1.5 0.1 0.2 0.1 0.5 1.7 0.0 0.0 0.1
Rice, including milled, 35.4 87.7 72.6 11.9 16.8 12.8 58.3 13.1 14.9 72.8 43.8 15.2 150.7 67.9 222.0 60.2 263.2
husked, broken and flour
Rye, including rye flour 0.1 0.1 1.7 15.5 2.6 28.3 5.2 1.2 11.2 0.1 0.0 0.0 0.0 0.0 0.0 0.7 0.2
Wheat, including flour, 301.8 226.1 343.5 271.3 199.1 193.7 185.8 171.7 216.3 30.5 57.8 20.5 137.0 134.4 87.8 105.6 106.4
macaroni and bread
Wheatgerm 0.0 0.0 0.0 0.0 1.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.4 0.0 0.0 0.0 0.0
Pulses, nuts and oilseeds
Beans (green pods and 1.6 1.2 7.8 0.6 2.2 5.2 1.6 16.9 4.1 0.5 0.3 0.0 0.9 0.2 3.1 0.0 4.2
immature seeds)
Peas (dry) 1.6 1.5 0.2 3.2 3.8 1.3 2.3 2.7 3.5 0.9 1.5 3.6 2.9 3.8 2.5 0.7 0.9
Soya beans (dry) 0.6 0.0 0.3 0.0 0.0 0.3 3.9 0.0 0.3 0.4 2.8 3.2 1.6 5.8 0.1 0.0 6.6
NES, not otherwise specified.
Ergot alkaloids
161
Table 23
Summary statistics of chronic dietary exposure to the sum of EAs (ng/kg bw per day) across
eight studies in sub-Saharan African countries
Dietary exposure Cameroon Benin Mali Nigeria
(ng/kg bw per day) Duala Garoua Cotonou Borgou Bamako Sikasso Lagos Kano
Mean LB sum 12 EAs 37 10 0.3 0.1 28 5 92 34
Mean UB sum 12 EAs 42 11 0.3 0.1 31 6 94 35
P95 LB sum 12 EAsa 52 24 1 1 54 12 177 91
P95 UB sum 12 EAsa 73 25 1 1 57 13 179 94
LB, lower bound; P95, 95th percentile; UB, upper bound.
a
Highest P95 individual EAs + sum of the mean of the 11 other EAs.
Member States, combined with food consumption information from the EFSA
Comprehensive European Food Consumption Database (EFSA, 2017). The main
results reported by EFSA are summarized below.
The initial data set with 12 675 samples representing 123 367 analytical
results on EAs was collected between 2004 and 2016 in 22 European countries:
Austria, Belgium, Bulgaria, Croatia, the Czech Republic, Denmark, Estonia,
Finland, France, Germany, Hungary, Ireland, Italy, Lithuania, Luxembourg, Malta,
Poland, Slovenia, Sweden, Switzerland, the Netherlands and the United Kingdom.
The analytical results for EAs referred to the 12 main Claviceps purpurea EAs:
ergometrine, ergosine, ergocornine, ergotamine, ergocristine, ergocryptine, and
the corresponding -inine (S)-epimers (ergometrinine, ergosinine, ergocorninine,
ergotaminine, ergocristinine and ergocryptinine). Additional analytical data on
the two isomers (α- and β-) of ergocryptine/ergocryptinine were reported either
as individual results or as the sum of both isomers.
The left-censored data were treated by the substitution method as
recommended in the Principles and methods for the risk assessment of chemicals
in food (FAO/WHO, 2009). At the LB, results below the LOQ and LOD were
replaced by zero; at the UB, the results below the LOD were replaced by the
LOD and those below the LOQ were replaced by the value reported as the LOQ.
Additionally, as a point estimate between the two extremes, a middle bound
(MB) scenario was calculated by assigning a value of LOD/2 or LOQ/2 to the
left-censored data.
The highest levels of EAs were reported in rye and rye-containing
commodities, particularly in raw agricultural or minimally processed
commodities (for example, “rye milling products”, 198–239 μg/kg, LB–UB).
Among processed foods, the highest levels of EAs were found in “mixed wheat
and rye bread and rolls” (33–82 μg/kg), “rye bread and rolls” (29–67 μg/kg) and
162
Ergot alkaloids
“rye flakes” (35–83 μg/kg), with the range representing the LB–UB scenarios in
all cases.
For chronic dietary exposure assessment, the Committee considered
food consumption data and body weight information from 35 recent dietary
surveys carried out in 19 European countries present in the latest version of
the comprehensive database. Occurrence and consumption data were linked
at the lowest food level possible. The mean and the high (P95) chronic dietary
exposures were calculated by combining mean occurrence values for EAs with
the average daily consumption for each food at individual level in each dietary
survey. Dietary exposure estimates were calculated for each dietary survey and
age class. In addition, the different food commodities were grouped within each
food category to better assess their contribution to the total dietary exposure to
EAs.
Considering both the LB and the UB scenarios, the estimates for mean
dietary exposure ranged from 10 ng/kg bw per day (minimum LB) in diverse age
classes to 470 ng/kg bw per day (maximum UB) for “toddlers”. Estimates for P95
dietary exposure ranged from 20 ng/kg bw per day (minimum LB) in diverse
age classes to 860 ng/kg bw per day (maximum UB) for “toddlers”. Estimates of
chronic dietary exposure are summarized in Table 24.
The main contributors to dietary exposure to EAs were different types
of bread and rolls, particularly those containing or made exclusively of rye. In
some countries such as Austria, the contribution of “mixed wheat and rye bread
and rolls” to the exposure to EAs represented up to 84% (MB) of the total dietary
exposure. The consumption of rye-derived processed foods was also important
in Nordic countries, where the consumption of “rye bread and rolls” contributed
61% of the total dietary exposure and up to 65% for “mixed wheat and rye
bread and rolls” in some dietary surveys. “Wheat bread and rolls” were also a
major contributor to the dietary exposure to EAs (up to 80% of the total, MB)
in some countries. However, in general, these countries were not among those
with the highest dietary exposure estimates for EAs. Other food commodities
that contributed to the exposure to EAs were “breakfast cereals” in general, and
“cereal flakes” in particular, with contributions up to 43% at the MB.
(c) WHO Pacific Region

New Zealand Food Safety runs an ongoing mycotoxin surveillance programme,
which in 2017–2018 included survey and assessment of dietary exposure to EAs
in the New Zealand food supply (NZFS, 2020).
The analytical results for the dietary exposure assessment were taken
from a survey of cereal-based foods, with a particular focus on rye-based foods.
The study analysed each sample for the 12 EAs: ergometrine, ergometrinine,
163
Table 24
Summary statistics of chronic dietary exposure to ergot alkaloids (ng/kg bw per day) across
European dietary surveys and different age classes
Mean dietary exposure (ng/kg bw per day)
Lower bound Upper bound
Age class N Min. Median Max. Min. Median Max.
Infants (<12 months) 6 10 20 80 30 150 340
Toddlers (12 to <36 months) 10 30 60 120 180 250 470
Other children (36 months to <10 years) 18 20 50 170 140 200 460
Adolescents (10 to <18 years) 17 10 30 150 70 120 290
Adults (18 to <65 years) 17 10 20 50 60 90 180
Elderly (65 to <75 years) 14 10 20 50 50 90 140
Very elderly (over 75 years) 12 10 20 60 50 90 160
95th percentile dietary exposurea (ng/kg bw per day)
Infants (<12 months) 5 50 –b 190 290 –b 760
Toddlers (12 to <36 months) 7 70 180 300 380 590 860
Other children (36 months to <10 years) 18 50 120 390 290 420 790
Adolescents (10 to <18 years) 17 30 70 330 140 270 560
Adults (18 to <65 years) 17 20 40 120 120 180 360
Elderly (65 to <75 years) 14 20 40 100 110 170 280
Very elderly (over 75 years) 9 20 30 90 120 150 260
BW, body weight; Max., maximum; Min., minimum; N, number of surveys.
a
The 95th percentile estimates obtained on dietary surveys/age classes with fewer than 60 observations may not be statistically robust. Those estimates were not
included in this table.
b
A minimum number of six dietary surveys are required to estimate a statistically robust median.
ergotamine, ergotaminine, ergosine, ergosinine, ergocornine, ergocorninine,

ergocryptine, ergocryptinine, ergocristine and ergocristinine. Total EA concen-
trations were calculated as the sum of the 12 individual EA results for each food
sample. For left-censored data (<LOQ), the UB was assigned a value of the LOQ
to any EA detected at >LOD and <LOQ and a value of the LOD to any EA detected
at <LOD, whereas the LB was assigned a concentration value of zero to any EAs
below the LOQ. The LB–UB concentration estimates for each sample were used
to derive the arithmetic means of LB–UB for each food type. For LB, mean
total EA concentrations of all flour samples followed the order rye > spelt > oats >
wheat. The order of this data set was comparable to the results reported by EFSA
(2017).
Over half of the samples (60%) contained quantifiable levels of EAs
(LOQs were in the range 0.5–1.25 μg/kg for the individual EAs). Just over one-
third of samples (35%) contained quantifiable concentrations of total EAs
≤10 μg/kg. Concentrations of total EAs in the range 11–50 μg/kg were found in
20% of samples and the remaining 5% of samples had concentrations of total EAs
164
Ergot alkaloids
>50 μg/kg. The highest concentration was observed in a sample of rye flour. In the
foods studied, ergotamine and ergotaminine were by far the most prevalent EAs.
Individual food consumption data were derived from the 2009 Adult
Nutrition Survey (4721 respondents aged 15 years and over) and the 2002
National Children’s Nutrition Survey (3098 respondents aged 5–15 years). Both
surveys used a single-pass 24-hour dietary recall. Use of a single day of dietary
recall tends to inflate the upper percentiles of the food consumption distribution,
compared to habitual long-term consumption levels. Mean UB estimates of
dietary EA exposure were 9.4 ng/kg bw per day for adults and 25 ng/kg bw per
day for children. The main contributors to dietary EA exposure were wheat bread,
wheat flour and multigrain bread.
NZFS concluded that the dietary exposure assessment for EAs in New
Zealand is approximately 10-fold lower than in similar European studies. This
is primarily because of the lower EA concentrations measured in cereal and rye-
based foods.
8.3.2 National or regional estimates of chronic dietary exposure derived by the

Committee
Additional national estimates of dietary exposure may be derived by the
Committee. Considering that estimates of chronic dietary exposure for EAs
are available for European countries, sub-Saharan African countries and New
Zealand, and considering that those national estimates were of the same order
of magnitude as those performed by the Committee with corresponding GEMS/
Food cluster diets, the Committee decided not to derive additional national
estimates of dietary exposure to EAs (for example, for countries in the WHO
Region of the Americas).
8.3.3 International estimates of chronic dietary exposure derived by the

Committee
International estimates of dietary exposure to EAs were derived by the Committee
using food consumption information from the WHO GEMS/Food cluster diets.
The concentration data for EAs in foods used for these international estimates
of dietary exposure are shown in Tables 17–20. Concentration data on EAs in
foods from the GEMS/Food contaminants database were pooled for countries
within the same WHO region. Mean (LB and UB) concentrations for food
commodities were then combined with food consumption information for the
clusters associated with that WHO region (see Table 22) to derive international
estimates of dietary exposure. Based on the assumption of equipotency for each
individual EA, the Committee derived estimates of dietary exposure for the sum
of EAs. The Committee decided not to derive estimates of dietary exposure for
165
clusters within WHO regions where no data had been submitted to the GEMS/
Food contaminants database. Therefore, no dietary exposure was estimated for
clusters G01, G04 and G06 associated with the WHO Eastern Mediterranean
Region and cluster G09 associated with the WHO South-East Asia Region.
The Committee derived international estimates of chronic dietary
exposure using the 13 GEMS/Food cluster diets for the WHO African, Americas,
European and Western Pacific regions (Table 22) with the concentration data for
EAs in foods from the same WHO regions and food commodities as displayed
in Tables 17–20.
Table 25 provides the estimated total and per commodity LB–UB dietary
exposure to EAs for the GEMS/Food cluster diets in ng/kg bw per day. The
GEMS/Food cluster diets are mean per capita estimates of food consumption
and do not allow estimation of high percentiles of dietary exposure. However,
the Committee used a previously established approximation in which the 90th
percentile is approximately twice the mean dietary exposure (FAO/WHO, 2009).
A standard body weight of 60 kg was used for all data reported in all GEMS/Food
clusters to assess exposure per kg of body weight.
LB–UB mean estimates of dietary exposure to EAs ranged from 12.2 ng/
kg bw per day (G14, Western Pacific) to 184.2 ng/kg bw per day (G02, Europe).
LB–UB mean estimates of dietary exposure to EAs ranged from 24.4 ng/kg bw
per day (G14, Western Pacific) to 368.5 ng/kg bw per day (G02, Europe). Wheat
and wheat products are the main foods contributing to dietary exposure to EAs
in all clusters (ranging from 67 to 100%). Rye and rye products also contributed
to dietary exposure in all clusters, but to a lesser extent (ranging from 2 to 33%).
8.3.4 Dietary chronic exposures summary

National and international estimates of dietary exposure to EAs described in the
previous sections are summarized in Table 26.
Across national and international estimates, LB–UB estimates of mean
dietary exposure were in the range 10–470 ng/kg bw per day for children and
0.1–180 ng/kg bw per day for adults. High percentile LB–UB estimates of dietary
exposure (P90 or P95) were in the range 28–860 ng/kg bw per day for children and
1–369 ng/kg bw per day for adults. For all national and international estimates
the main foods contributing to dietary exposure were wheat and wheat-based-
products.
The Committee noted that the dietary exposure estimates for GEMS/
Food clusters within WHO regions for which national estimates were available
(African, European and Western Pacific regions, Table 25) were of approximately
the same order of magnitude as the corresponding national estimates. The
166
Table 25
Estimated LB–UB dietary exposure to the sum of EAs in the GEMS/Food cluster diets
Estimated LB–UB dietary exposure to EAs in the GEMS/Food cluster diets (grouped by WHO region) in ng/kg bw per day
Western
European African Americas Pacific
Food commodities G02 G07 G08 G10 G11 G15 G03 G13 G16 G05 G12 G14
All foods LB UB LB UB LB UB LB UB LB UB LB UB LB UB LB UB LB UB LB UB LB UB LB UB
Meana 89.3 184.2 60.8 127.9 81.4 153.2 65.9 138.0 50.6 106.4 74.5 151.6 20.8 23.2 39.4 42.5 14.0 15.3 116.0 152.3 113.0 142.7 12.2 18.6
High (90th percentile)b 178.6 368.5 121.6 255.9 162.8 306.4 131.8 276.1 101.3 212.9 149.0 303.2 41.6 46.4 78.7 85.0 28.0 30.6 232.0 304.6 226.0 285.5 24.4 37.3
Grains and grain-based products
Barley, including flour 0.2 1.5 0.0 0.1 0.1 0.5 0.1 0.4 0.0 0.2 0.1 0.4 0.0 0.0 0.0 0.0 0.0 0.0 1.0 1.2 0.0 0.0 0.0 0.0
and grits
% 0.2 0.8 0.0 0.1 0.1 0.3 0.1 0.3 0.1 0.2 0.1 0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.9 0.8 0.0 0.0 0.1 0.1
Buckwheat, including 0.0 0.1 0.0 0.0 0.0 0.1 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
flour
% 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1
Cereals preparations, 1.1 1.4 4.2 5.4 2.0 2.6 10.5 13.5 2.0 2.5 5.3 6.8 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.2 0.1 0.6 0.0 0.0
NES (and flour)
% 1.2 0.8 6.9 4.2 2.5 1.7 15.9 9.8 3.9 2.4 7.1 4.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.2 0.1 0.4 0.3 0.3
Maize, including maize 0.0 7.3 0.0 3.6 0.0 3.0 0.0 3.5 0.0 1.2 0.0 6.0 0.0 0.7 0.0 0.8 0.0 0.5 0.3 4.6 0.4 5.1 0.0 0.1
flour and sweetcorn
% 0.0 4.0 0.0 2.8 0.0 2.0 0.0 2.5 0.0 1.1 0.0 4.0 0.0 3.1 0.0 1.8 0.0 3.0 0.3 3.0 0.3 3.6 0.0 0.4
Oats, including rolled 0.3 0.9 0.4 0.9 0.3 0.8 0.2 0.6 0.2 0.4 0.1 0.3 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.1 0.2 0.4 0.0 0.0
oats
% 0.4 0.5 0.6 0.7 0.4 0.5 0.4 0.4 0.3 0.4 0.2 0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.1 0.2 0.3 0.0 0.0
Rice, including milled, 0.0 1.8 0.0 2.6 0.0 2.0 0.0 9.0 0.0 2.0 0.0 2.3 0.0 0.6 0.0 0.4 0.0 0.1 0.0 12.6 0.0 5.7 1.5 7.1
husked, broken and
flour
% 0.0 1.0 0.0 2.0 0.0 1.3 0.0 6.5 0.0 1.9 0.0 1.5 0.0 2.5 0.0 0.8 0.0 0.8 0.0 8.2 0.0 4.0 12.6 38.1
Ergot alkaloids
167
168
Table 25 (continued)
Estimated LB–UB dietary exposure to EAs in the GEMS/Food cluster diets (grouped by WHO region) in ng/kg bw per day
Western
European African Americas Pacific
Food commodities G02 G07 G08 G10 G11 G15 G03 G13 G16 G05 G12 G14
Grains and grain- LB UB LB UB LB UB LB UB LB UB LB UB LB UB LB UB LB UB LB UB LB UB LB UB
based products
(continued)
Rye, including flour 14.5 19.3 2.4 3.2 26.5 35.3 4.9 6.5 1.1 1.5 10.4 13.9 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.1 0.0 0.0 0.0 0.0
% 16.2 10.5 4.0 2.5 32.5 23.0 7.4 4.7 2.2 1.4 14.0 9.2 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.1 0.0 0.0 0.0 0.0
Wheat, including flour, 73.1 151.2 53.7 111.0 52.2 107.9 50.1 103.5 46.3 95.7 58.3 120.5 20.8 21.9 39.4 41.4 14.0 14.7 114.5 133.4 112.3 130.9 10.6 11.2
macaroni and bread
% 81.9 82.1 88.3 86.8 64.1 70.5 76.0 75.0 91.3 89.9 78.2 79.5 100.0 94.4 100.0 97.3 100.0 96.1 98.7 87.6 99.4 91.7 86.4 60.3
Wheatgerm 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
% 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.1
Pulses, nuts and oilseeds

Beans (green pods and 0.0 0.1 0.1 0.3 0.3 0.7 0.1 0.2 1.1 2.4 0.3 0.6 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
immature seeds)
% 0.0 0.0 0.2 0.2 0.4 0.5 0.2 0.2 2.2 2.3 0.4 0.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Peas (dry) 0.0 0.6 0.0 0.8 0.0 0.3 0.0 0.5 0.0 0.5 0.0 0.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Ninety-first JECFA
% 0.0 0.3 0.0 0.6 0.0 0.2 0.0 0.3 0.0 0.5 0.0 0.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Soya beans (dry) 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.1
% 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.2 0.0 0.0 0.0 0.0 0.4 0.7
%: percentage of dietary exposure to EAs that is due to consumption of the associated food commodity to the total dietary exposure from all foods, based on LB estimates of dietary exposure to EAs; bw: body weight; GEMS/Food: Global Environment
Monitoring System – Food Contamination Monitoring and Assessment Programme; LB: lower bound; UB: upper bound.
a
The range of dietary exposure estimates refers to LB and UB estimates of mean dietary exposure. The LB mean estimates were derived by substituting zero for analytical results below the LOD or LOQ. The UB mean estimates were derived by averaging the
sum of individual EAs reported as not detected or not quantified. All exposure estimates are based on a 60-kg body weight.
b
High percentiles are an approximation of the 90th percentile dietary exposure, calculated as twice the mean dietary exposure (FAO/WHO, 2009).
Table 26
Summary of national and international LB–UB estimates of dietary exposure to EAs from the literature and derived by the Committee
Estimated dietary
exposure, mean (P90
Country/ Food concentration or P95) in ng/kg bw
WHO region data used Consumption data used Population groups (age) per day b Major contributors Reference
WHO GEMS/Food contaminants Per capita consumption Adult 113–152 (226–305) Wheat and wheat-based products Derived by this Committee
Americasc
Benin Survey mean Household budget survey Adult (18 to <65 years) 0.1–0.3 (1) Wheat and wheat-based products Derived by this Committee
(Ingenbleek et al., 2019)
Cameroon Survey mean Household budget survey Adult (18 to <65 years) 10–42 (24–73) Wheat and wheat-based products Derived by this Committee
Mali Survey mean Household budget survey Adult (18 to <65 years) 5–31 (12–57) Wheat and wheat-based products Derived by this Committee
Nigeria Survey mean Household budget survey Adult (18 to <65 years) 34–94 (91–179) Wheat and wheat-based products Derived by this Committee
WHO Africanc GEMS/Food contaminants
Per capita consumption Adult 14–42 (28–85) Wheat and wheat-based products Derived by this Committee
New Zealand Survey mean National food consumption Children (5–15 years) 12–25 (28–59) Wheat breads (NZFS, 2020)
surveys (24-hour dietary Adults (>15 years) 4.6–9.4 (12–22) Wheat flour-containing foods
recall) Multigrain breads
WHO Western GEMS/Food contaminants Per capita consumption Adult 12–19 (24–37) Wheat and wheat-based products Derived by this Committee
Pacificc Rice and flour
Europe (22 EFSA database National food consumption Infants (<12 months) 10–34 (50–760) Wheat breads and rolls (EFSA, 2017)
countries)a surveys (at least two survey Toddlers (12 to <36 months) 30–470 (70–860) Breads and rolls containing or made
days) Other children (36 months to <10 years) 20–460 (50–790) exclusively of rye
Adolescents (10 to <18 years) 10–290 (30–560) Breakfast cereals and cereal flakes in
Adults (18 to <65 years) 10–180 (20–360) particular
Elderly (65 to <75 years) 10–140 (20–280)
Very elderly (over 75 years) 10–160 (20–280)
Ergot alkaloids
169
170
Estimated dietary
exposure, mean (P90
Country/ Food concentration or P95) in ng/kg bw
WHO region data used Consumption data used Population groups (age in years) per day b Major contributors Reference
WHO GEMS/Food contaminants Per capita consumption Adult 51–184 (101–369) Wheat and wheat-based products Derived by this Committee
Europeanc Rye and flour
a
Austria, Belgium, Bulgaria, Croatia, the Czech Republic, Denmark, Estonia, Finland, France, Germany, Hungary, Ireland, Italy, Lithuania, Luxembourg, Malta, Poland, Slovenia, Sweden, Switzerland, the Netherlands and the United Kingdom.
b
The range of dietary exposure estimates refers to LB and UB estimates of mean dietary exposure. National and international dietary exposure to EAs were reported in the scientific or grey literature or derived by the Committee. The LB mean estimates
were derived by substituting zero for analytical results below the LOD or LOQ. The UB mean estimates were derived by averaging the sum of individual EAs reported as not detected or not quantified. All international exposure estimates are rounded and
based on a 60-kg body weight.
c
Ninety-first JECFA
Ergot alkaloids
concentration data used to derive international estimates are exactly the same
as those used to derive national estimates for the African and European regions.
8.4 Assessments of acute dietary exposure

8.4.1 National estimates of acute dietary exposure from the published literature
Except for European countries, no acute dietary exposure estimates for EAs have
been reported in the scientific literature.
Acute dietary exposure to EAs was estimated by EFSA using a
probabilistic approach. Single-day food consumption information from 41 recent
dietary surveys carried out in 23 European countries was used. Acute exposure
was assessed for each reporting day by multiplying the total daily consumption
for each food by one occurrence level randomly drawn among the individual
results available for that type of food. EFSA considered EA content in the food
samples as the sum of up to 12 individual EAs. Most of the available data were
left-censored (only 11% of the analytical results were quantified). Acute dietary
exposure was estimated using the MB scenario for sample EA concentrations,
as the UB scenario would represent an overly conservative exposure. The MB
scenario was calculated by assigning a value of LOD/2 or LOQ/2 to the left-
censored data. Respective exposures from the different foods consumed that
day were then summed and finally divided by the individual’s body weight. This
process was iterated 1000 times for each reporting day. For each of these end-
points, the 95% confidence interval was defined as the 2.5th and 97.5th percentiles
obtained from the 1000 iterations.
The range of acute exposure estimates (average and P95, MB scenario)
to EAs across European dietary surveys with their corresponding confidence
intervals (2.5th and 97.5th percentiles) are presented in Table 27.
Mean acute exposure ranged from 20 ng/kg bw per day in “infants” up to
320 ng/kg bw per day in “other children”. The highest estimate of MB P95 acute
dietary exposure was for a dietary survey within the age class “other children”
(980 ng/kg bw per day).
The food types making the greatest contribution to acute dietary exposure
to EAs were the same as those identified for chronic dietary exposure: “mixed
wheat and rye bread and rolls” and “rye bread and rolls”. For high consumers, a
single consumption of “mixed wheat and rye bread and rolls” can lead to acute
exposure estimates up to 740 (95% CI = 590–930) ng/kg bw per day while for “rye
bread and rolls”, the exposure can reach values up to 640 (95% CI = 600–690) ng/
kg bw per day.
171
Table 27
Summary statistics of acute dietary exposure to EAs (ng/kg bw per day) from European
dietary surveys and different age classes
Mean acute dietary exposure (ng/kg bw per day)
Age class Number of dietary surveys Minimum (95% CI) Median Maximum (95% CI)
Infants (<12 months) 6 20 (10–40) 80 210 (200–210)
Toddlers (12 to <36 months) 11 110 (110–110) 150 300 (290–300)
Other children (36 months to <10 years) 20 80 (80–80) 130 320 (300–330)
Adolescents (10 to <18 years) 20 40 (40–50) 70 230 (210–240)
Adults (18 to <65 years) 22 30 (30–30) 60 170 (160–170)
Elderly (65 to <75 years) 16 30 (30–30) 50 130 (110–140)
Very elderly (over 75 years) 14 30 (30–40) 50 120 (100–150)
95th percentile acute dietary exposure (ng/kg bw per day)
Infants (<12 months) 5a 260 (250–270) – 650 (620–680)
Toddlers (12 to <36 months) 10 350 (310–380) 500 790 (760–830)
Other children (36 months to <10 years) 20 270 (250–300) 400 980 (900–1060)
Adolescents (10 to <18 years) 20 130 (110–160) 260 770 (670–890)
Adults (18 to <65 years) 22 100 (100–110) 210 490 (460–530)
Elderly (65 to <75 years) 16 100 (90–110) 160 370 (290–480)
Very elderly (over 75 years) 14 100 (90–110) 160 390 (270–510)
95% CI: 95th percentile confidence interval.
a
A minimum number of six dietary surveys are required to estimate a statistically robust median.
8.4.2 National estimates of acute dietary exposure derived by the Committee

Given that there is no match between the countries in the FAO/WHO global
individual food consumption database (GIFT) and the countries that have
submitted data on concentrations of EAs in foods, the Committee decided not to
derive additional national estimates of acute dietary exposure.
8.5 Summary of global dietary exposure estimates

Across national studies and international estimates, LB–UB estimates of mean
dietary exposure were in the range 0.01–0.47 µg/kg bw per day for children and
<0.01–0.18 µg/kg bw per day for adults. High percentile LB–UB estimates of
dietary exposure (P90 or P95) were in the range 0.03–0.86 µg/kg bw per day for
children and <0.01–0.37 µg/kg bw per day for adults. The main foods contributing
to dietary exposure for all national and international estimates were wheat and
wheat-based products.
The Committee noted that for Europe no major differences were observed
between estimates of chronic and acute dietary exposure to EAs, indicating that
the main contributors to the exposure are foods consumed on a regular basis
172
Ergot alkaloids
within particular populations, together with a relatively symmetrical distribution

of their occurrence values. Considering that wheat and wheat-based products
have been identified as the main foods contributing to both chronic and acute
dietary exposure to EAs in European estimates, it is likely that, for other regions
in the world where wheat-based products are staple foods, the chronic exposure
estimates will be comparable to acute exposure estimates.
9. Dose–response analysis and estimation of toxicity/

carcinogenic risk
9.1 Identification of key data for risk assessment

Data from studies in experimental animals described in section 2.2.2 and from
clinical and epidemiological studies in humans described in section 2.4 were
identified and evaluated for their relevance in assessing the human health risk
and suitability for dose–response modelling and in the derivation of the 95%
lower confidence limits of the benchmark dose (BMDL) as point of departure.
9.1.1 Pivotal data from biochemical and toxicological studies

Limited toxicological data were available on naturally occurring EAs. The longest
exposure period was in a 13-week toxicity study conducted in rats treated with
ergotamine tartrate (Speijers et al., 1993). No long-term toxicity study of specific
naturally occurring EAs was available.
Seven key studies in experimental animals were identified by the
Committee as having data that might be applicable for dose–response modelling.
In addition to the 13-week toxicity study conducted in rats with ergotamine
tartrate (Speijers et al., 1993), three short-term toxicity (4 weeks) studies in rats
treated with ergotamine tartrate (Speijers et al., 1992), ergometrine maleate
(Peters-Volleberg, Beems & Speijers, 1996) and α-ergocryptine (Janssen et al.,
1998, 2000a,b) and two short-term toxicity studies (4 weeks) in pigs given feed
contaminated with ergot sclerotia (Oresanya et al., 2003, Maruo et al., 2018) were
also considered. A further study conducted in pigs exposed for 50 days to feed
contaminated with ergot sclerotia was also considered (Dignean, Schiefer & Blair,
1986).
(a) Rats
Ergotamine tartrate administered at concentrations of 0, 4, 20, 100 or 500 mg/kg
diet showed concentration-dependent effects on body and relative organ weight,
173
feed intake, and histopathological and biochemical parameters in female and

male rats (Speijers et al., 1992). The concentration-dependent incidence of tail
abnormality (redness of the tail tip, which in some cases progresses to necrosis
and degenerative changes in the longitudinal skeletal muscle) assessed in this
study at the two highest dietary concentrations was identified as the most relevant
adverse health effect from this study for dose–response modelling.
Ergotamine maleate administered at concentrations of 0, 2, 10, 50 or
250 mg/kg diet caused significant effects, mainly at the highest concentration
(relative organ weights, histopathological changes and effects on T4 levels (Peters-
Volleberg, Beems & Speijers, 1996). Prolactin levels were decreased in animals
given the two highest concentrations. Tail abnormalities were not observed and
this study was not considered further for dose–response modelling.
The study by Janssen et al. (1998, 2000a,b) using concentrations of 0, 4, 20,
100 or 500 mg α-ergocryptine/kg diet showed concentration-dependent effects
on body weight, body weight gain, feed intake and feed efficiency as well as on
relative organ weights, which exhibited some non-monotonicity. Concentration-
dependent effects were also observed in several parameters of blood and urine
chemistry and after microscopical examination of tissues at autopsy, including
a detailed examination of the tail. Focal muscle degeneration in the tail was
considered relevant for dose–response modelling. Concentration-dependent
changes also occurred in carbohydrate metabolism and thyroid and pituitary
functions. Significant decreases in serum prolactin were observed, in particular,
in females where the levels were statistically significantly decreased in animals
that received concentrations of 20 and 500 mg/kg feed. The concentration-
response of prolactin was, however, non-monotonic in animals that received
100 mg/kg feed. In these animals, the mean was about twice that of the next lower
concentration of 20 mg/kg feed. Only a few of the suite of dose–response models
could be fitted satisfactorily in a tentative benchmark dose (BMD) analysis of the
data on serum prolactin in females. The highly variable prolactin levels seen in
this study were anticipated in females, probably because of their unknown estrus
cycle status at the time of blood collection. Serum prolactin levels in female rats
typically increase during the early stages of estrus. It was also noted that the
prolactin levels in this study were mainly within the normal range. The prolactin
levels in males were much lower and the fitted curves were flatter than those of
the females. The Committee concluded that the data available on the suppression
of prolactin were not suitable for determining a BMDL using the BMD approach
and considered the data on tail muscle abnormalities observed in this study for
dose–response modelling.
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Ergot alkaloids
(b) Pigs
The Committee noted that a dose–response evaluation of data on pigs exposed
to ergot sclerotia at different concentrations is restricted to a concentration–
response analysis. This is because the calculations of doses of specific EAs
would not be possible from the limited information available in the published
reports. Results of concentration–response modelling in pigs might, however,
be informative when these results are compared with concentration–response
modelling from other animals.
In the 50-day study by Dignean, Schiefer & Blair (1986), pigs were fed an
experimental diet that included ergot sclerotia at concentrations of 0, 0.225, 0.45,
0.9 or 1.8 g sclerotia/kg diet. Although the extent and severity of haematological,
hepatic, urinary and histological alterations were associated with increasing
concentrations of sclerotia in the diet, the Committee could not identify relevant
end-points suitable for concentration–response modelling, particularly because
only two animals (one female and one male) were assigned to each concentration.
Ergot sclerotia added to the basal diet of groups of pigs in small but
increasing amounts (0, 0.05, 0.10, 0.25, 0.50 and 1.00%) (n = 32 per group) was
associated with a concentration-dependent decrease of body weight, feed intake
and feed efficiency as well as with the suppression of prolactin and urea nitrogen
(Oresanya et al., 2003). The Committee considered prolactin reduction as relevant
but also noted the absence of a clear concentration–response relationship.
A recently published study of ergot sclerotia in pigs (n = 24 per group)
compared a control group with two groups receiving concentrations of 1.2 or
2.5 g sclerotia/kg diet with mean EA concentrations of 2.36 and 5.05 mg/kg diet,
respectively (Maruo et al., 2018). The Committee identified a clear concentration–
response relationship between the occurrence and severity of histopathological
lesions in the intestine and liver. Lesion scores for animals given concentrations
of 2.36 and 5.05 mg/kg diet were statistically significantly different from those
of the control animals in the three subsamples of n = 6 pigs per group. The
Committee obtained from the authors individual data for the pigs (body weights,
feed intake and the frequency and severity of jejunal lesions). A dimensionless
individual jejunal lesion score ((1, 1, 1, 2, 4, 5) for controls and (3, 4, 5, 7, 7, 9)
and (4, 4, 9, 9, 10, 12) for 1.2 or 2.5 g sclerotia/kg diet, respectively) based on
different histopathological observations and representing frequency and severity
of lesions showed a clear concentration–response relationship. The mean scores
(SD) were 2.33 (1.75), 5.83 (2.23) and 8 (3.29) for the control group and the two
concentration groups, respectively. The Committee considered using a BMD
approach to calculate a reference concentration from these data. Application
of Benchmark Dose Software version 3.2 of the United States Environmental
Protection Agency (BMDS 3.2) to both the individual and the summary data
175
resulted in wide benchmark concentration (BMC) intervals for benchmark

response (BMR) values of 10% or larger. Most of the BMCL values (the LB of the
BMC interval calculated as a BMDL) were orders of magnitude lower than the
low sclerotia concentration of 1.2 mg/kg feed. Model averaging was not possible
given the sparsity of the data (n = 6 per group and only two concentrations).
The Committee noted that the test material in this study was feed contaminated
with ergot sclerotia in which mixtures of EAs were present and the proportions
differed depending on whether the feed was for the low- or high-concentration
group. The relevance of this mixture with regard to human dietary exposure was
difficult to assess, taking into account the variability in food contamination data
according to the region of the world.
Considering the difficulties, the Committee refrained from dose–
response modelling of this dataset using a BMD approach and decided that the
dose–response assessment should revert to the LOAEL of 100 µg EAq/kg bw
calculated by the authors (see section 2.2.2).
In the 13-week study by Speijers et al. (1993), female and male rats were
exposed to ergotamine tartrate administered at concentrations of 0, 5, 20 or
80 mg/kg diet. Concentration-dependent effects were observed for body weight
gain and feed intake, relative organ weight, haematological, biochemical and
histopathological effects. The Committee identified the concentration-dependent
incidence of muscular atrophy in the caudal longitudinal muscles of the tail of
animals as the most relevant adverse health effect for dose–response modelling.
9.1.2 Pivotal data from human clinical/epidemiological studies

The Committee considered the human data on the clinical use of EAs to be
relevant for establishing a health-based guidance value (HBGV). As certain effects
induced by EAs in humans can be due to acute or short-term exposures, the
Committee decided it would be appropriate to establish an acute oral reference
dose from the available data. Ergotamine and ergometrine have been used in
human medicine for the treatment of migraine headache and for management
of the third stage of labour and postpartum blood loss. Ergometrine is the most
toxic of the naturally occurring EAs based on LD50 values. Humans appear to
be more sensitive to some effects of ergometrine, including on uterine smooth
muscle contraction, than experimental animals, and ergometrine maleate is more
active than ergotamine as a uterine-stimulating agent.
The initial oral dosage of ergometrine maleate to induce smooth muscle
contractions of the uterus is typically 0.2 mg and can be increased to 0.4 mg,
2–4 times daily for up to 7 days. Side-effects, including vomiting and nausea, are
well-characterized in the normal therapeutic dose range. Overdosages may cause
176
Ergot alkaloids
seizures and gangrene as well as more severe gastrointestinal symptoms, vascular

effects, dizziness or loss of consciousness, and cardiovascular effects.
Relatively higher doses of ergotamine tartrate are used to treat migraine
(vasoconstriction) compared with the therapeutic doses of ergometrine maleate.
The usual oral dose is 2 mg, which can be repeated until maximums of 6 mg per
day, 8 mg per attack, 12 mg per week and two courses per month are reached.
Common side-effects, which typically occur as a result of prolonged use, include
nausea and vomiting, and effects consistent with ergotism (i.e. cardiovascular
effects, gangrene, confusion and convulsions). In rare cases, pleural and peritoneal
fibrosis and fibrosis of the cardiac valves have been reported.
9.2 General modelling considerations

The Committee identified the tail muscular atrophy effect in experimental
animals as the critical effect suitable for dose–reponse modelling. This tail
muscular atrophy can be explained by vasoconstriction. The most consistent
toxicity findings for ergotamine tartrate and α-ergocryptine were tail muscular
atrophy in female rats in 4-week and 13-week studies. Data from clinical uses
of ergotamine and ergometrine in humans were not considered suitable for
quantitative dose–reponse modelling.
9.2.1 Selection of data

For acute toxicity, data from short-term studies were considered by the Committee
for dose–response modelling, based on tail muscular atrophy or degeneration in
rats (Speijers et al., 1992; Janssen et al., 2000a,b).
For long-term toxicity, data from the subchronic study in rats were
considered by the Committee for dose–response modelling, based on tail
muscular atrophy (Speijers et al., 1993).
9.2.2 Measure of exposure

EA doses were derived from EA concentrations using available information on
initial and final body weights and food intake data separately for each sex and
each dose group and the control group. In some cases, initial body weights were
calculated by subtracting from the reported final body weight an estimated body
weight gain calculated by the authors (for example, when based on modelling
of all measured weights over the duration of the experiment). An average
weekly food intake was calculated when mean weekly food intake was available.
The Committee calculated doses using the respective molecular weights when
exposure was reported in terms of the concentrations of EA salt (for ergotamine
tartrate, see Table 28).
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Table 28
Dose–response data for the two 4-week studies and the 13-week study identified for dose–
response modelling using the benchmark dose approach for EAsa
Speijers et al., 1992
ergotamine tartrate in diet for 4 weeks
0, 100, 500 mg/kg diet
Approximately six males and six females per group
Ergotamine (mg/kg bw per day) Incidence of tail abnormality
females 0, 7.3, 41 0/4, 0/6, 3/6
males 0, 7.5, 37 0/3, 2/6, 4/5
Janssen et al., 1992

α-ergocryptine in diet for 4 weeks
0, 4, 20, 100, 500 mg/kg diet
six males and six females per group
α-Ergocryptine (mg/kg bw per day) Incidence of muscular degeneration
females 0, 0.37, 1.7, 8.9, 60 0/6, 0/6, 0/6, 0/5, 4/6
males 0, 0.34, 1.4, 6.6, 44 0/6, 0/6, 0/6, 2/6, 6/6
Speijers et al., 1993

Ergotamine tartrate in diet for 13 weeks
0, 5, 20, 80 mg/kg diet
10 males and 10 females per group
Ergotamine (mg/kg bw per day) Incidence of tail abnormality
females 0, 0.48,1.99, 7.63 0/10, 0/10, 2/10, 7/10
males 0, 0.42, 1.65, 6.18 1/10,1/10,1/10,7/10
a
The Committee calculated the doses of ergotamine from the concentrations of ergotamine tartrate in the diet using the reports of the authors. Doses of α-ergocryptine
were calculated by the authors.
9.2.3 Measure of response

In all three studies, statistically significant dose-dependent effects were noted.
The Committee concluded that the data on these three studies were relevant for
BMD modelling and a BMR of 10% extra risk was used for these quantal dose–
response data.
9.2.4 Selection of mathematical model

The BMD approach was used for dose–response modelling (DRM) on the data
from the experimental animals selected for the risk assessment of EAs using the
guidance of FAO/WHO (2020), Chapter 5.
BMDS 3.2 was applied for DRM (https://www.epa.gov/bmds) on the
data considered suitable for this purpose. The models available in BMDS 3.2
for dichotomous (quantal) response were used as the default set of models
and Bayesian model averaging (Bayesian MA) was used for the calculation of
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Ergot alkaloids
BMD confidence intervals (BMD-CI). The BMR default value of 10% extra risk
was selected for the dichotomous critical end-point of incidence of tail muscle
degeneration or muscular atrophy in Sprague-Dawley rats investigated over 4
and 13 weeks. For the quantitative end-points of prolactin levels in Sprague-
Dawley rats and the jejunal lesion score in pigs, the relative deviation from the
level at dose zero of 10% or higher was used as the BMR.
The benchmark dose at 10% extra risk (BMD10) estimates and the BMD10-
CI of Bayes MA are reported below in detail for the two 4-week studies (Speijers
et al., 1992; Janssen et al., 2000a,b) and the 13-week study by Speijers et al. (1993),
separately for females and males. The results of Bayesian MA are summarized
in Table 29 and details are given in Tables 31 and 32 when using the BMDS 3.2
default of non-informative priors (11% prior probability assigned to each of the
nine models used to calculate the BMD and the BMD-CI. Figure 3 shows the
overall curve fitting of the suite of models for Bayesian MA by BMDS 3.2 for the
13-week study by Speijers et al. (1993) for females and males. Figure 4 shows the
fitted curve of the model with the largest posterior probability, i.e. the highest
weight, among the nine single Bayesian models used for Bayesain MA with its
individual BMD10 and BMD10-CI since no comprehensive graphics on the overall
outcome results of BMD model averaging BMD-CI were available.
From the result of Bayesian MA, the lower confidence bound (BMDL10)
corresponding to the BMR of 10% extra risk was selected as the reference point
for hazard characterization.
9.3 BMD analysis

Table 29 shows the BMD10 and the BMD10-CI with upper and lower 95% con-
fidence limits (BMDL10 and BMDU10) using Bayesian MA. The data from the two
4-week studies on ergotamine tartrate and α-ergocryptine provided the lowest
reference point (BMDL10) of 1.3 mg/kg bw from males in the Janssen et al. (2000a)
study, based on muscular degeneration in the tail, secondary to vasoconstriction.
The Committee considered the dose–response data from the 13-
week study by Speijers et al. (1993) on muscular atrophy in the tail as suitable
for calculating a reference point for chronic exposure in humans. Female and
male Sprague-Dawley rats were exposed to ergotamine tartrate at three doses
equivalent to a range from 0.4 to 7.6 mg ergotamine/kg bw per day (Table 28).
Bayesian MA performed with BMDS 3.2 provided a BMD10-CI of 0.61–3.20 mg
ergotamine/kg bw per day for females and 0.63–3.53 mg ergotamine/kg bw per
day for males. The Committee established an overall BMDL10 of 0.6 mg/kg bw per
day for ergotamine for the subchronic exposure of rats.
179
Table 29
Dose–response analysis using the benchmark dose (BMD) approach (benchmark
response = 10%) in rats (BMDS 3.2 Bayesian Model Averaging)
Ergot alkaloid/ BMD BMD-confidence
concentration in the Dose (mg/kg bw interval
diet Sex (mg/kg bw per day) per day) (mg/kg bw per day) Reference
4-week studies
Ergotamine tartrate Females 0, 7.3, 41 16.6 5.25–64.7 Speijers et al. (1992)
0, 100, 500 mg/kg diet Males 0, 7.5, 37 6.37 1.68–28.4
α-Ergocryptine 0, 4, 20, Females 0, 0.37, 1.7, 8.9, 60 18.1 5.36–37 Janssen et al. (2000a)
100, 500 mg/kg diet Males 0, 0.34, 1.4, 6.6, 44 3.88 1.29–9.59
13-week study
Ergotamine tartrate Females 0, 0.48,1.99, 7.63 1.54 0.61–3.20 Speijers et al. (1993)
0, 5, 20, 80 g/kg diet Males 0, 0.42, 1.65, 6.18 1.68 0.63–3.53
BMDS 3.2, Benchmark Dose Software from the United States Environmental Protection Agency, version 3.2.
Table 30
Benchmark dose (BMD) analysis of the tail abnormality observed in the 4-week study in
female and male rats (Speijers et al., 1992) using BMDS 3.2a
Females Males
BMDS 3.2 Bayesian MA MA weights BMDS 3.2 Bayesian MA MA weights
BMD-CI probability BMD-CI probability
Model BMD posterior (%) rounded BMD posterior (%) rounded
Probit 17.5 9.08–61.0 17 6.93 3.57–30.2 12
Logit 22.8 9.43–inf 17 9.03 3.74–66.5 15
LogProbit 19.4 7.67–33.9 10 11.3 2.01–27.1 7
LogLogistic 13.6 3.81–26.9 12 6.01 0.713–19.8 9
Weibull 17.3 6.41–33.9 17 10.3 1.99–28.4 8
Gamma 15.6 5.12–76.3 6 6.78 1.34–87.7 5
Multistage 2 6.92 3.91–11.9 2 4.41 2.27–8.33 11
Multistage 1 6.71 3.72–12.9 not used 4.08 2.11–8.81 not used
Quantal linear 8.36 3.77–24.8 12 3.54 1.78–13.1 28

Hill dichotomous 15.0 3.68–68.4 10 4.07 0.435–42.3 4
Model average 16.6 5.25–64.7 6.37 1.68–28.4
BMDS 3.2, Benchmark Dose Software from the United States Environmental Protection Agency, version 3.2; MA, model average.
a
BMR=10% for extra risk, all results rounded to two significant digits after decimal for doses in mg/kg bw per day.
Detailed results are shown in Tables 30–32 indicating acceptable fits for
all nine models incorporated in the Bayesian model averaging. The posterior
probabilities used for model averaging by the BMDS 3.2 software ranged between
6 and 15%. A cross-check with results obtained by independent software available
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Ergot alkaloids
Table 31
Benchmark dose (BMD) analysis of the tail muscular degeneration in the 4-week study in
female and male rats (Janssen et al., 2000a) using BMDS 3.2a
Females Males
Probit 23.4 13.0–39.1 26 6.54 3.01–12.0 14
Logit 21.9 12.6–44.4 8 6.89 3.29–12.7 5
LogProbit 22.5 7.96–41.5 12 4.26 1.58–13.0 3
LogLogistic 15.9 4.57–39.6 12 3.71 1.01–11.6 2
Weibull 19.9 7.06–37.2 11 4.60 1.37–14.5 7
Gamma 14.6 5.11–35.6 5 3.40 1.14–8.58 5
Multistage 4 9.71 5.32.16.5 not used 4.46 2.20–8.63 not used
Multistage 3 9.39 5.11–15–9 not used 4.25 2.10–8.18 not used
Multistage 2 8.71 4.83–15.1 5 4.00 1.98–7.35 28
Quantal linear 8.27 4.00–20.4 12 2.19 1.07–4.74 33
Model average 18.1 5.36–37.4 3.88 1.29–9.59
a
Table 32
Benchmark dose (BMD) analysis of the tail muscular atrophy observed in the 13-week study
in female and male rats (Speijers et al., 1993) using BMDS 3.2a
Females Males
Probit 2.20 1.43–3.35 15 1.60 1.05–2.67 15
Logit 2.23 1.44–3.43 8 1.71 1.09–3.26 13
LogProbit 1.85 0.85–4.24 7 2.42 1.08–4.18 14
LogLogistic 1.55 0.58–3.27 13 1.80 0.62–3.45 14
Weibull 1.86 0.75–3.75 11 2.07 0.82–3.67 14
Gamma 1.42 0.59–2.86 7 1.47 0.58–3.25 6
Multistage 2 1.06 0.63–1.74 12 0.94 0.55–1.55 6
Quantal linear 0.90 0.52–1.74 21 0.85 0.47–1.90 12
Model average 1.55 0.61–3.20 1.70 0.63–3.53
a
181
Fig. 3
Fit of the suite of models available in BMDS 3.2 for Bayesian modelling used for model
averaging of the dose–response assessment of ergotamine in the 13-week study by
Speijers et al. (1993)a
182
Ergot alkaloids
Fig. 4
Fit of the model with highest weight (largest posterior probability) included in the Bayesian
model averaging of BMDS 3.2 of US EPA of the dose–response assessment of ergotamine in
the 13-week study by Speijers et al. (1993) shown in Figure 3 and Table 32
from EFSA (https://shiny-efsa.openanalytics,eu/bmd) based on a slightly different

set of models was also applied to the male and female data, both separately and
combined. This resulted in similar reference points for the response of muscular
atrophy in rats as an end-point for vasoconstriction in tails.
The Committee noted that applying a default uncertainty factor (UF) of
100 for intra- and interspecies differences together, a UF of 2 for the use of the
13-week study for chronic toxicity in the absence of long-term data on chronic
toxicity, and an additional UF of 3 (to take into account the limitations of the
available toxicity data) converts the overall BMDL10 of 0.6 mg/kg bw per day to a
tolerable daily intake (TDI) of 1 µg/kg bw per day.
183
10. Comments

No reports of kinetic studies are available for most of the naturally occurring EAs
(for example, ergosine, ergocristine, ergocryptine and ergocornine). However,
for ergotamine and ergometrine some human and limited animal data were
available.
Absorption following oral administration of radiolabelled ergotamine in
rats and rhesus monkeys ranged from 40% to 10%, respectively (Nimmerfall &
Rosenthaler, 1976; Eckert et al., 1978). Following absorption in rats and rhesus
monkeys, ergotamine was excreted mostly in bile (33% and 24%, respectively)
whereas only a small amount (9% and 7%, respectively) was present in urine
(Nimerfall & Rosenthaler, 1976; Eckert et al., 1978). In humans, excretion of
various radiolabelled EAs including some dihydro-derivatives in urine, was low
(range 1 to 5%) (Schmidt & Fanchamps, 1974; Meier & Schreier, 1976; Little et al.,
1982; Maurer & Frick, 1984; Ronca et al., 1996). No EAs were detected in cow’s
milk following consumption of ergot-contaminated feed (equal to 4.1–16.3 μg
total alkaloids/kg bw per day) (Schumann et al., 2009).
Apart from their likely presence in liver and kidneys, little is known
about the distribution of EAs in other tissues and organs. No information is
available on EAs administered by the oral route in laboratory animals. In mice,
after intraperitoneal administration, ergotamine is found mainly in the kidneys
with relatively low levels detected in liver and brainstem. Following intravenous
administration in pregnant rats, radiolabelled ergotamine could be detected in
the uterus, placenta and yolk-sac with minor amounts in amniotic fluid and fetal
tissues. The transplacental passage of ergotamine was estimated to be around
2.8% (Leist & Grauwiler, 1973; Reddy et al., 2020).
In humans, most data on the pharmacokinetics come from studies involving
a range of radiolabelled synthetic or semi-synthetic EAs administered to healthy

volunteers. Wide variability in the pharmacokinetic parameters was observed
for different alkaloids and between individuals. Following oral administration of
0.2 mg of ergometrine maleate, the elimination half-life from plasma was calculated
to be 1.9 hours (de Groot et al., 1994). Peak plasma ergotamine levels following
oral administration (1 mg/kg bw) are generally achieved 2 hours later but have
been reported to occur as early as 30 minutes after administration in some studies.
Absorption of ergotamine is up to 62% but bioavailability of parent compound is
approximately 1% or less due to extensive metabolism during its passage through
the intestinal wall and liver (Meier & Schreier, 1976; Aellig & Nüesch, 1977;
Ala-Hurula et al., 1979a; Little et al., 1982).
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Ergot alkaloids
There is no information on the likely presence of ergometrine in the milk

of lactating women (EMEA, 1999). However, for the analogue methylergometrine,
up to 1.3 µg/L was present in the milk of lactating women after oral administration
of 0.25 mg/day (Erkkola et al., 1978; Vogel et al., 2004).
Only limited data are available on the metabolic pathway of EAs either in
humans or in laboratory animals. Ergotamine metabolism occurs largely through
undefined pathways but is likely to involve cytochrome P450 3A4 (CYP3A4), an
important phase I drug-metabolizing enzyme in humans. The evidence comes
from co-administration of therapeutic compounds known to be potent CYP3A4
inhibitors, such as clarithromycin (Horowitz, Dart & Gomez, 1996) and ritonavir
(Liaudet et al., 1999). Both are reported to be associated with ergotism following
co-administration with ergotamine. The most likely biotransformation of the
ergopeptine alkaloids involves opening the tricyclic amino acid ring structure
at the proline moiety (Eckert et al., 1978). Maurer & Frick (1984) have proposed
that in humans dihydroergotamine undergoes oxidation of the peptide moiety.
After a single oral administration of dihydroergotamine to healthy volunteers,
the plasma levels of 8'-OH-dihydroergotamine were several times greater than
those of the parent compound (Chen et al., 2002; Bicalho et al., 2005). This
metabolite, 8'-OH-dihydroergotamine, has been shown to have approximately
the same potency as dihydroergotamine for vasoconstrictive activity in human
volunteers (Aellig, 1984).

Oral median lethal dose (LD50) values are available for some of the naturally
occurring EAs, namely ergometrine, ergotamine, ergocornine, ergocryptine,
ergostine and ergonine (Griffith et al., 1978). Oral LD50 values range from 150–
3200 mg/kg bw for mice, rats and rabbits, with the exception of ergometrine in
rabbits (27.8 mg/kg bw). Based on available oral LD50 values, ergometrine is the
most toxic of the naturally occurring EAs. The clinical signs of acute sublethal
poisoning relate to neurotoxicity, including restlessness, miosis or mydriasis,
muscular weakness, tremors and rigidity. Tail gangrene was observed in rats 5–7
days after a single intraperitoneal exposure to a mixture of EAs (ergocornine,
α- and β-ergocryptine and ergocristine) at 25 mg/kg bw. More recent studies of
the effects of a single intraperitoneal administration to rats and mice of naturally
occurring alkaloids (Thorat et al., 2019; Reddy et al., 2020) also reported signs of
neurotoxicity (head and whole-body shakes, reciprocal forepaw treading, lateral
head weaving, flat body posture and hind limb abduction) and cardiotoxicity
(bradycardia and elevated systolic and diastolic blood pressure).
185
Short-term (4-week) toxicity studies have been conducted in rats treated

with ergotamine tartrate (Speijers et al., 1992), ergometrine maleate (Peters-
Volleberg, Beems & Speijers, 1996) and α-ergocryptine (Janssen et al., 1998,
2000a, b) and in pigs given feed contaminated with ergot sclerotia (Oresanya
et al., 2003; Maruo et al., 2018). A further study was conducted in pigs exposed
for 50 days to feed contaminated with ergot sclerotia (Dignean, Schiefer & Blair,
1986).
Ergotamine tartrate at concentrations of 0, 4, 20, 100 or 500 mg/kg diet
was given to five groups of six rats per group per sex for 4 weeks (Speijers et al.,
1992). At the highest concentration (500 mg/kg diet), redness of the tail tip was
seen in all animals tested, which in some cases progressed to necrosis of the tail
tip (two of the six males and three of the six females). A significant decrease in
body weight and feed intake was observed in both sexes at 100 and 500 mg/kg.
Slight changes in some haematological parameters were seen in the groups that
received the 100 and 500 mg/kg concentrations. Increases in relative weights of
some organs (heart, brain and liver) were observed in the females fed ergotamine
tartrate at 20, 100 and 500 mg/kg.
Ergometrine maleate at concentrations of 0, 2, 10, 50 or 250 mg/kg diet
was administered to six groups of six rats per group per sex for 4 weeks (Peters-
Volleberg, Beems & Speijers, 1996). Two control groups were included: one
received the control diet ad libitum and the other was pair-fed with the highest
concentration group, to determine any effects secondary to a decreased feed
intake. No treatment-related clinical signs were observed during the experiment.
Tail tips were not affected. Body weight was not influenced by ergometrine maleate
treatment, except in females fed 10 mg/kg diet after 4 weeks of exposure, which
showed a significant weight increase. Plasma glucose levels were significantly
decreased in females fed 50 and 250 mg/kg diet, but not in males. T4 levels were
significantly decreased in males fed 250 mg/kg diet. Prolactin was determined
in serum samples taken from a limited number of animals and showed a wide
interindividual variation in all groups. The authors reported that the levels were
markedly decreased in the 50 and 250 mg/kg diet groups for both sexes (without
statistical analysis). In females given the highest concentration, relative organ
weights of heart, liver and ovaries were increased. Microscopic examination
showed moderate reactive hyperplasia in enlarged mediastinal lymph nodes.
Treatment-related histopathological changes were observed in the liver of males
and females fed 250 mg/kg diet, with significant evidence of increased glycogen
storage (Peters-Volleberg, Beems & Speijers, 1996).
α-Ergocryptine at concentrations of 0, 4, 20, 100 or 500 mg/kg diet was
given to six groups of six rats per group per sex for 28–32 days (Janssen et al.,
1998, 2000a, b). Two control groups were included: one received the control diet
ad libitum and the other was pair-fed with the highest concentration group. Mean
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Ergot alkaloids
body weight, body weight gain, feed intake and feed efficiency decreased in both
sexes in a non-monotonic manner. In animals receiving concentrations higher
than 4 mg/kg diet, significant changes were observed in some haematological
parameters (decreased mean corpuscular volume (MCV) and mean corpuscular
haemoglobin (MCH)), serum enzyme activities (slightly increased or decreased
alanine aminotransferase (ALAT), serum aspartate aminotransferase (ASAT)
and gamma-glutamyltransferase (GGT)), serum urea concentrations (increased),
glomerular filtration (decreased creatinine and urea clearances). Prolactin was
decreased in animals of both sexes in the 20, 100 and 500 mg/kg diet groups.
U-shaped changes were observed for some parameters, which might be caused
by the U-shaped concentration–response relationship for feed intake, owing
to the dopaminergic properties of α-ergocryptine. This could be related to
inhibition of feed intake at an intermediate concentration due to activation of
satiety mechanisms in the lateral hypothalamic area and/or the activation in
the forebrain of behaviours incompatible with feeding (Janssen et al., 1998).
Microscopic examination at autopsy revealed treatment-related findings in the
kidneys (nephrosis), liver (atrophy, glycogen storage), thymus (atrophy), tail
(muscular degeneration), ovaries (atrophy) and uterus (atrophy). The muscular
degeneration in the tail was assumed to be due to the vasoconstrictive properties
of ergocryptine. Ergocryptine influenced carbohydrate metabolism and affected
thyroid and pituitary function (Janssen et al., 2000a).
From the available studies in rats, the Committee concluded that
there is no major potency difference in the subacute toxicity of ergotamine,
ergometrine and α-ergocryptine (Speijers et al., 1992; Peters-Volleberg, Beems &
Speijers, 1996; Janssen et al., 2000a, b). The most prominent effect of ergotamine
and ergocryptine is vasoconstriction, whereas the most prominent effect of
ergometrine is smooth muscle contraction.
Feed contaminated with 0, 1.2 or 2.5 g of sclerotia/kg was given to three
groups of 24 weaned piglets for 28 days (Maruo et al., 2018). Based on the mean
feed intake and body weight, the doses of total EAs were 159 and 83 μg/kg bw per
day after 2 and 4 weeks, respectively for the animals given the low dose. The most
abundant alkaloid was ergotamine, followed by ergosine, ergocristine and their
corresponding -inine epimers. During the experimental period, the daily feed
intake of animals exposed to the higher dose of ergot was reduced by about 18%
in comparison with that in the control group. This reduction in feed ingestion was
associated with a decrease in animal weight gain. Exposure to ergot led to mild
to moderate lesions of the liver and the jejunum of the pigs. In the liver, tissue
disorganization of hepatic cords, inflammation and vacuolation of hepatocytes,
megalocytosis and necrosis were the main morphological alterations contributing
to a significant increase in the liver lesion score. The main histological changes
observed in the jejunum were villi atrophy, oedema of lamina propria and
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cytoplasmic vacuolation of enterocytes. Animals exposed to the higher dose of

ergot displayed a significant increase of the lesion score in the jejunum compared
to control animals.
Wheat ergot sclerotia were added to a basal diet at concentrations of 0,
1.04, 2.07, 5.21, 10.41 and 20.82 mg/kg for total alkaloids and fed to 32 weaned
pigs per concentration for 28 days. The most abundant alkaloid was ergocristine,
followed by ergotamine, ergosine, ergocryptine and ergocornine. Pigs fed the
highest concentration gained 82% and 38% less weight than the control animals
in weeks 1 and 2 respectively, and body weight on day 28 was significantly
reduced. EAs decreased average daily feed intake and feed efficiency over the
entire period, but average daily feed intake was not affected during the initial
14 days. EAs significantly decreased serum prolactin in all treated groups as
measured in samples collected on day 28 (Oresanya et al., 2003). The Committee
noted that there was no dose–response relationship in the prolactin reduction
(Oresanya et al., 2003).
Barley ergot sclerotia were added to a basal diet (0.0, 0.225, 0.45, 0.9 or
1.8 g ergot sclerotia/kg diet) and fed for 50 days to 10 weaned pigs, paired one
male to one female (Dignean, Schiefer & Blair, 1986). Ergot sclerotia contained
2.27 g alkaloid/kg, mainly ergocristine, followed by ergotamine, ergocristinine,
ergocryptine, ergometrine, ergosine and ergocornine. Pigs fed the highest
concentration of ergot were less efficient at conversion of feed to weight gain,
and animals receiving the higher levels of ergot were observed to be much more
excitable and difficult to restrain than control animals after 3 weeks of feeding with
ergot sclerotia. No other clinical signs were observed. Histopathological changes
(for example, cellular vacuolation and cytoplasmic disruption) were observed in
the liver, kidney and spleen. The severity of the changes was associated with the
concentration of ergot in the diet.
Four groups of 10 rats per group per sex fed ergotamine tartrate at
concentrations of 0, 5, 20 or 80 mg/kg diet were observed for 13 weeks (Speijers et
al., 1993). Both body weight gain and feed intake were significantly decreased in
females (but not in males) fed the high concentration (80 mg/kg diet) compared to
the controls. The only treatment-related histopathological finding was muscular
atrophy in the caudal longitudinal muscles of the tail of animals in groups fed
the higher concentration (see Table 37). The atrophy consisted of partial or
total disappearance of fibres, tinctorial changes and fibrosis. The low incidence
in the control and lower concentration groups was considered by the authors
to be a background level. In addition to the atrophy in the high-concentration
group, degenerative changes of nerve fibres in that region were also apparent.
No vascular abnormalities could be detected that might be responsible for these
putative ischaemic changes.
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No long-term toxicity studies of specific naturally occurring EAs (i.e.

ergometrine, ergotamine, ergosine, ergocristine, ergocryptine or ergocornine)
were available. In an early study (Fitzhugh, Nelson & Calvery, 1944), three series of
experiments were conducted with rats (3 weeks of age) fed powdered crude ergot
(composition not known) in a high protein diet. In a first experiment, groups of
20 females received a diet containing 0, 1, 2 or 5% crude ergot for 6 months. In a
second experiment, groups of nine males and nine females received a low protein
diet with 0, 1, 2 or 5% crude ergot for 6 months. In a third experiment, groups of
eight males and eight females received a low protein diet with 0 or 5% crude ergot,
5% defatted ergot, 5% ergot oil, or ergotoxine ethanesulfonate at a concentration
equivalent to 5% ergot for 1 to 2 years. The body weight gain was significantly
reduced at week 15 in the 5% groups compared to the controls. The pathological
changes observed only in the treated animals included: neurofibromas of the
ears, necrosis and calcification of the lower ends of the renal pyramids, and
enlargement of the ovaries from marked corpus luteum hyperplasia. These lesions
were noted in 46, 45 and 41 of the 218 treated animals, respectively. The earliest
tumour was noted after 9 months of exposure. The tumours regressed when the
feeding of ergot was stopped and resumed growth when it was begun again.
In vitro and in vivo genotoxicity studies are available only for a limited
number of naturally occurring EAs or their salts. Ergometrine tartrate showed
no mutagenic potential in vitro in Salmonella Typhimurium strains (Zeiger
et al., 1987). α-Ergocryptine did not induce sister chromatid exchange in
Chinese hamster ovary cells (Dighe & Vaidya, 1988). Agroclavine (a precursor
compound) showed a weak mutagenic response when activated with rat liver S9
in S. Typhimurium strains (Glatt et al., 1987). However, ergometrine maleate and
ergotamine tartrate induced chromosomal damage in human leukocytes in vitro
(Jarvik & Kato, 1968; Dighe & Vaidya, 1988; Roberts & Rand, 1977a). Ergotamine
tartrate and ergometrine maleate induced sister chromatid exchange in Chinese
hamster ovary cells (Dighe & Vaidya, 1988). Semi-synthetic dihydrogenated
derivatives (dihydroergocristine and α-dihydroergocryptine) also gave negative
results in tests with S. Typhimurium strains (Dubini et al., 1990; Adams et al.,
1993).
In vivo, ergotamine tartrate showed no genotoxic potential in
the micronucleus test after intraperitoneal injection to mice and Chinese
hamster (Matter, 1976) and gave negative results in the dominant lethal
test after intraperitoneal injection in mice (Roberts & Rand, 1978; Matter,
1982). Semi-synthetic dihydrogenated derivatives (dihydroergocristine and
α-dihydroergocryptine) also gave negative results in the in vivo mouse
micronucleus assay after oral administration (Dubini et al., 1990; Adams et al.,
1993). However, ergotamine tartrate was reported to induce a significant number
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of chromosomal aberrations in bone marrow cells after intraperitoneal injection

in mice (Roberts & Rand, 1977b).
Taking all the available information into account, the Committee
concluded that naturally occurring EAs do not raise concerns for genotoxicity.
In animals, EAs induce effects on ovulation, implantation, early
pregnancy, and on embryonic and fetal development, resulting in abortion,
high neonatal mortality, fetal malformations and growth retardation (Carlsen
Zeilmaker & Shelesnyak, 1961; Deanesly, 1968; Carpent & Desclin, 1969; Mantle,
1969; Grauwiler & Schön, 1973; Floss, Cassady & Robbers, 1973; Schön, Leist &
Grauwiler, 1975; Griffith et al., 1978; Holstege & Traven 2014). The effects vary
according to the EA (Griffith et al., 1978). The Committee noted that the effects
on implantation and pregnancy maintenance in rodents are due to reduced
secretion of prolactin and this is not relevant for humans at that stage of pregnancy
(Ben-Jonathan, LaPensee & LaPensee, 2008). EAs inhibit prolactin secretion and
impair lactation in rodents and humans (Mantle, 1968; Shaar & Clemens, 1972;
Griffith et al., 1978; Flint & Ensor, 1979; Kopinski et al., 2007, 2008).
The pharmacological mechanisms associated with ergot toxicity are
complex and have not been fully delineated (Holstege & Traven 2014). They
include peripheral vasoconstriction, peripheral adrenergic blockade, reduced
secretion of prolactin and stimulation of uterine smooth muscle (Peters-
Volleberg, Beems & Speijers 1996).
EAs are structurally related to biogenic amines such as norepinephrine,
dopamine and serotonin. This structural similarity allows EAs to interact with
G-protein coupled receptors (GPCR) (for example, dopaminergic, noradrenergic
and serotoninergic ones), as agonists and/or antagonists. The receptor affinity
and selectivity, as well as the intrinsic activity (efficacy), of these compounds
are highly dependent upon the substituents present at positions 1, 6, 8 and
10 of the lysergic acid moiety. In addition, the specific interaction between
EAs and monoaminergic receptors appears to be organ-specific (Zajdel et al.,
2015). Ergotamine displayed high affinity for adrenergic (α1, α2), dopaminergic
(D1, D2) and serotoninergic ((5-hydroxytryptamine) 5-HT1A, 5-HT1B,

5-HT1D, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT5A, 5-HT5B and 5-HT6) receptors.
Ergotamine behaves as an antagonist at adrenergic receptors, partially inhibits
the 5-HT1 receptor, modulates neurotransmitter release presynaptically, and
excites the 5-HT2, 5-HT3, 5-HT4, 5-HT6 and 5-HT7 receptors. Because of their
structural differences from the physiological monoamine neurotransmitters, EAs
are generally characterized by a low specificity and selectivity with respect to the
above-mentioned neuroreceptors and, depending on the individual structure,
they can display a complex behaviour as receptor agonists, partial agonists or
antagonists (Mantegani, Brambilla & Varasi, 1999).
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Ergot alkaloids
The vascular effects of EAs have been known for centuries. The occurrence
of gangrene in various animal species correlates closely with the vasoconstrictor
potential of the EAs (Griffith et al., 1978). Repeated-dose toxicity experiments
with ergotamine in rats showed necrosis and fibrosis in the tail tips of animals in
the high-concentration groups, explained by the vasoconstrictive properties of
ergotamine (Speijers et al., 1992, 1993).
Cherewyk et al. (2020) showed in vitro that four (S)-epimers
(ergocryptinine, ergocristinine, ergocorninine and ergotaminine) were vaso-
active and produced a concentration-dependent arterial contractile response
similar to that reported for the (R)-epimers. The arterial contractile response to
ergotaminine was the strongest, followed by ergocorninine, ergocristinine and
ergocryptinine.
Several hydroxylated metabolites were found to retain the biochemical
activity and receptor-binding potential of the parent compound in vitro (Müller-
Schweinitzer, 1984).
EAs have an agonist effect on the α-adrenergic receptors. Activation of
α1-adrenergic receptors produces anorexia. Reduced appetite was observed in rat
studies (Speijers et al., 1992, 1993; Janssen et al., 2000a, b) and in rabbit studies
(Canty et al., 2014; Solano-Baez et al., 2018) and, consequently, decreased body
weight.
In the uterus, EAs can play an agonist role on α-adrenergic receptors
leading to oxytocic effects (promotion of uterine contractions). The activation
of receptors is characterized by an increase in the three parameters of uterine
contraction: frequency, amplitude and basic tone.
10.3 Observations in domestic animals/veterinary toxicology

EAs have negative impacts on growth (decreased feed intake and weight gain) and
reproductive performance (decreased prolactin levels, lower conception rates and
birth weights (and, in males, reduced fertilization potential) of domestic animals,
such as cattle, horses, pigs and sheep (reviewed by Klotz (2015)). Gangrenous
ergotism (i.e. fescue foot or fescue lameness in livestock) is one of the most
acute and obvious visible effects of exposure to EAs. EAs induce vasoconstrictive
responses in arteries and veins (Poole et al., 2018; Britt et al., 2019; Cowan et
al., 2018, 2019; Klotz et al., 2019). The effects of EAs on lactation vary with
the livestock species. Consumption of EAs reduces milk yield in cattle, horses
and sheep (Poole & Poole, 2019). Poole & Poole (2019) reviewed the effects of
EAs on female reproduction in grazing livestock species. The effects reported
included altered cyclicity, suppressed hormone secretion, reduced pregnancy
rates, early embryonic loss, agalactia and reduced offspring birth weights. In
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cattle, reproductive failure following exposure to EAs can be attributed to altered

ovarian follicle development, luteal dysfunction and reduced concentrations of
circulating steroid hormones, leading to reduced pregnancy rates.

10.4.1 Biomarkers
A small number of studies of EA exposure biomarkers were identified. One study
measured EAs in serum and urine and collected 24-hour dietary recall data that
were used to estimate dietary exposure for 600 men and women participating in
the European Food Consumption Validation Project (DeRuyck et al., 2020). The
six EAs relevant to this report and their ‑inine epimers were included in the study.
Overall, EAs were detected in 116 of 268 serum samples and 106 of 188 urine
samples. Across all mycotoxins evaluated, only slight agreement was observed
between exposure estimates and concentrations in serum or urine, suggesting
that biological measurements were generally not sufficient for describing chronic
dietary exposure, although they might provide useful information following
single exposures. No studies of biomarkers of effect were identified.
10.4.2 Clinical observations

Ergotamine tartrate is used to treat migraine and cluster headaches (Silberstein
& McCrory, 2003; Tfelt-Hansen & Diener, 2014). The usual oral dose is 2 mg
(1–2 mg, and can be repeated at 30-minute intervals up to a maximum of 6 mg per
day, 8 mg per attack, 12 mg per week and two courses per month) (Martindale,
2010).
The adverse effects of ergotamine are related to its vasoconstrictive
properties and its effects on the CNS. Common side-effects at therapeutic doses,
including nausea and vomiting, are well characterized. Side-effects typically
occur as a result of prolonged use for migraine headaches rather than after acute
single doses. In cases of prolonged use, effects consistent with ergotism have
been observed (i.e. cardiovascular effects, gangrene, confusion and convulsions)
(Martindale, 2010). In rare cases, pleural and peritoneal fibrosis and fibrosis of the
cardiac valves have been reported (Martindale, 2010). Recent case studies report
severe cardiovascular effects (i.e. acute coronary syndrome, electrocardiogram
changes compatible with myocardial infarction, valvulopathy and decompensated
heart failure) and drug interactions in HIV-infected individuals on antiretroviral
therapy and individuals taking macrolide antibiotics. These case reports involved
the oral administration of ergotamine tartrate at therapeutic doses for several
days in the case of drug interactions (for example, 1 mg ergotamine plus 100 mg
caffeine daily for 5 days) (Navarro et al., 2017), and up to several decades in
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Ergot alkaloids
patients treated for migraine (for example, 1–3 mg ergotamine plus caffeine for
30 years (Maréchaux et al., 2015).
Ergometrine maleate is used to induce uterine contractions and prevent
postpartum haemorrhage in the third stage of labour and to treat excessive
haemorrhage postpartum. Although all EAs have uterotonic effects, ergometrine
(or methylergometrine) has been used clinically because it is more active as a
uterine-stimulating agent than ergotamine (Sanders-Bush & Mayer, 2006). The
usual oral dose is 0.2 mg. This dosage can be increased to 0.4 mg, 2–4 times daily
for up to 2–7 days. Uterotonic effects can be observed in women postpartum
within 10 minutes after oral administration of 0.2 mg of ergometrine; however,
a larger initial dose may be required given the wide variation in patient response
(Sanders-Bush & Mayer, 2006). Ergometrine is contraindicated in patients with
cardiovascular disease, sepsis, hepatic or renal impairment, during the first stage
of labour, in women with pre-eclampsia or eclampsia, and those who are at risk
of preterm birth. Side-effects of ergometrine maleate, including vomiting and
nausea, are well characterized at the normal therapeutic doses (Martindale,
2010). Overdosages may cause seizures and gangrene as well as more severe
gastrointestinal symptoms, vascular effects, dizziness or loss of consciousness
and cardiovascular effects (Martindale, 2010).
10.4.3 Epidemiology
Epidemiological studies report associations of overuse of ergotamine or
dihydroergotamine (i.e. daily doses for 60–90 days in a period of 6 months to
1 year) with serious cardiovascular effects (i.e. ischaemic complications including
angina, myocardial infarction, stroke, cerebral ischaemia and peripheral vascular
disease) (Velentgas et al., 2004; Wammes-van der Heijden et al., 2006). Analyses
of poison control data indicate that most overdoses involving oral ergotamine
(including 1 mg ergotamine/100 mg caffeine tablets) have not resulted in major
effects or deaths (Robblee et al., 2020), and that most of the symptoms reported
after overdose in another study were due to interactions with CYP3A4 inhibitors
(Srisuma, Lavonas & Wananukul, 2014). Similarly, of 56 accidental exposures to
ergotamine in children less than 7 years of age reported to the California Poison
Control System, none were characterized as serious (median dose in children
with mild clinical symptoms: 1 mg (range: 0.2–11 mg)) (Armenian & Kearney,
2014).
One epidemiological study found an association of ergotamine (0.3 mg
(tablets) to 1.5 mg (drops) for 1 day to 7 months) with low birth weight and
preterm birth but maternal smoking, a known cause of low birth weight, was not
controlled for in the analysis (Bánhidy et al., 2007). No increase in the overall
incidence of congenital abnormalities was found in a study of 924 children (n=31
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abnormalities) of women who had migraine headaches, 71% of whom were said
to have taken ergotamine at some time during pregnancy (month/trimester not
stated) (Wainscott, Volans & Wilkinson, 1974). Two analyses, which were based
on either three or six cases, from the Hungarian Case-Control Surveillance of
Congenital Abnormalities dataset reported associations of ergotamine use during
pregnancy with neural tube deficits (Medveczky, Puhó & Czeizel, 2004; Ács et al.,
2006).
Ergometrine may be administered orally or via the intravenous or
intramuscular routes to control postpartum haemorrhage. One study was
identified that examined the effects of oral ergometrine administered for this
purpose (De Groot et al., 1996). Women in the ergometrine group received two
0.2 mg tablets or 0.4 mg total; no significant elevation of blood pressure was
observed in women for whom these data were available. Poisonings in neonates
accidentally administered ergometrine are described, including a fatality
involving a 0.2 mg oral dose of ergometrine maleate (AHFS, 1995; EFSA, 2012).
Of 37 cases reported to the California Poison Control System of oral ergometrine
exposure in children less than 7 years of age, five resulted in symptoms, all of which
were characterized as minor (median dose: 0.4 mg (range: 0.2–2) (Armenian &
Kearney, 2014).
Oral preparations of sclerotia of C. purpurea were previously used to
accelerate labour (single dose indications ranged from 0.2–3 mg with daily doses
from 6–7.5 mg) (EFSA, 2012). This practice has been discontinued due to an
increased risk of stillbirth.
The two most recent outbreaks of gangrenous ergotism occurred in
Ethiopia. The first of these involved 2–3 months of exposure to grain with a
0.75% ergot content (King, 1979; EFSA, 2012) and the second was associated
with exposure to concentrations ranging from 2.1–26.6 mg ergotamine/kg and
0.9–12.1 mg ergometrine/kg (Urga et al., 2002; EFSA, 2012; Belser-Ehrlich et al.,
2013). Outbreaks associated with C. fusiformis from contaminated pearl millet
have occurred in India. The concentrations reported in unaffected villages ranged
from 1–38 g ergot/kg (15–26 mg total ergot alkaloids/kg), whereas concentrations

in affected villages ranged from 15–175 g ergot/kg (15–199 mg total ergot
alkaloids/kg) (Krishnamachari & Bhat, 1976; WHO-ICPS, 1990; EFSA, 2012).
10.5 Analytical methods

The Committee reviewed the analytical methods for the determination of the 12
EAs most commonly associated with contaminated cereals, namely the lysergic
acid derivative, ergometrine, and the ergopeptines, ergocornine, ergocristine,
ergotamine, ergocryptine and ergosine, as well as their -inine epimers. Although
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Ergot alkaloids
ergocryptine and ergocryptinine can occur as both α- and β-analogues, these are
seldom determined separately.
EAs are generally soluble in organic solvents, charged at acid pH and
uncharged at neutral or alkaline pH. Those with a C9,10 double bond have natural
fluorescence properties. They are also light-sensitive, and prone to epimerization
at C8 during long storage and chemical analysis, requiring determination of
epimers.
EAs may be extracted with non-polar organic solvents under alkaline
conditions or with polar solvents under acidic conditions. They are isolated by
either liquid/liquid partitioning reversed-phase solid phase extraction (SPE),
strong cation exchange SPE, mixed cation/reversed-phase SPE, silica gel columns
or immunoaffinity columns.
Separation of individual EAs by one-dimensional or two-dimensional
thin-layer chromatography (TLC) has been only partially successful, whereas
total EA content can be resolved as a single spot by high-performance TLC.
Quantification is either by fluorodensitometry or by using selected spray reagents.
Commercial ELISAs are available for rapid screening of cereals for total EAs;
however, questions have been raised as to whether cross-reactivity is the same
for all 12 forms.
Gas chromatography may be used to determine structurally simpler
clavine alkaloids and lysergic acid derivatives, but ergopeptines tend to
decompose in hot injector ports and only fragments can be determined, usually
by mass spectrometry (MS).
Quantitative determination of the main EAs associated with contaminated
cereals is generally achieved by high-performance liquid chromatography
(HPLC) with either UV/fluorescence detection or MS detection. Earlier HPLC
methods were only able to identify a limited number of alkaloids, possibly owing
to the lack of reference standards. Detection by natural fluorescence provides
better sensitivity and selectivity with reported detection limits typically in the
low µg/kg range. Recently, lysergic acid diethylamide (LSD) has been used as an
internal standard for determination of the 12 EAs.
HPLC-MS has been used to obtain both quantitative results and
confirmatory mass spectra. It is the instrumental technique of choice for most
mycotoxin analyses and provides a platform for the development of multi-
mycotoxin methods incorporating toxins of very different chemistries. For
HPLC-MS/MS of EAs, reversed-phase HPLC is frequently performed with mobile
phases containing volatile weak acids to provide efficient positive electrospray
ionization (ESI) at the MS interface.
Matrix effects (signal enhancement or suppression) are common in
HPLC-MS/MS analysis. The level of signal suppression can vary widely between
matrices and the individual EAs, even among varieties of the same cereal. Signal
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suppression is strongly influenced by extract purification technique and can

be improved with the use of ultra-high-performance liquid chromatography
(UHPLC). The use of atmospheric pressure chemical ionization rather than ESI
produced strong signal enhancement and an over-estimation of EA levels. Given
the lack of available labelled standards, some analysts use EA calibrants prepared
in an extract of toxin-free sample to account for these effects.
EAs have been included in multimycotoxin HPLC-MS/MS methods.
The development of multimycotoxin analytical methods has resulted in the use
of so-called “dilute-and-shoot” techniques. Another popular method is termed
QuEChERS (quick, easy, cheap, effective, rugged, and safe), which removes
interfering substances such as lipids and pigments and is potentially followed by
dispersive SPE.
10.6 Sampling protocols

The Committee reviewed the available information regarding sampling
protocols. It noted that, in general, designing statistically-based sampling plans
for mycotoxins is a complex task because of the heterogeneity of contamination.
Sampling was discussed at the fifty-sixth meeting of the Committee (Annex 1,
reference 152). In the absence of sampling protocols designed specifically for EAs,
sampling protocols for aflatoxin are used for EA analysis. Further investigation
of their distribution in different foods is needed to develop specific sampling
protocols for EAs.
10.7 Effects of processing

Information on the effects of various processing procedures on the levels of EAs
in food comes largely from Canada, Europe and the USA where EAs frequently
occur in cereal crops, particularly in rye and wheat. Contamination of cereal grains
with EAs is mostly associated with fungal sclerotia and shrivelled, discoloured
grains. Conventional grain-cleaning equipment (for example, scalpers, shaker
decks, gravitational separators and electronic sensor-based sorters) can reduce
EA contamination. Methods using either high-velocity air cleaning of grains or
electronic sensor-based handling have been shown to reduce EA levels.
In common with other mycotoxins, the milling of cereals does not
destroy EAs, but merely distributes them among the milling fractions. In general,
fractions intended for human food have lower levels of EAs than those intended
for animal feed.
Heat treatment can reduce EA levels in final processed products; this
reduction is strongly dependent on processing temperature and duration.
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Ergot alkaloids
Temperatures above 100 °C used for frying, roasting, toasting and extrusion
cooking reduce EA levels; however, thermal treatment leads to epimerization and
a shift in the ratio of the -ine to the -inine forms (Tittlemier et al., 2019).
In beer brewing, the steeping and kilning steps lead to a reduction of the
final EA content.
10.8 Prevention and control

Factors that affect the concentration of EAs in plants are poorly documented.
Among the cereals, rye and triticale are the most susceptible to infection by
Claviceps species, followed by wheat, barley and oats. Cultivar differences also
play a role in susceptibility. Wild grasses growing within or outside fields are the
primary source of ergot inoculum due to the wide range of hosts for C. purpurea.
Crop rotation can be used to control EA contamination; however, this strategy
is only effective if grasses are simultaneously controlled. Tillage practices can
also contribute to the control of plant contamination, as deep ploughing buries
sclerotia in the soil. Lastly, the use of ergot-free or certified seed improves control
by preventing the introduction of a primary inoculum into fields.
There are very few studies on post-harvest control of EAs. Cleaning grain
during and after harvest by removing sclerotia will reduce EA contamination.
Effective decontamination measures must be irreversible, products must
be non-toxic and grain should keep its nutritional value while maintaining
storability and palatability (Codex Alimentarius, 2015). Removal of sclerotia by
sieving, opto-electric sorting or winnowing prior to sorting are effective physical
procedures. While treatment with chemicals such as chlorine, sulfur dioxide and
hydrogen chloride, or flotation in saline solution has some effect, these measures
would need to be allowed for use in foods. The Committee did not find any
authorized procedures in national food legislation.
10.9 Levels and patterns of contamination in food commodities

The GEMS/Food contaminants database contains 178 184 records for EAs
submitted between 2004 and 2019 originating from four WHO regions: African
Region (Benin, Cameroon, Mali and Nigeria), European Region, Region of the
Americas (Canada) and Western Pacific Region (China, Hong Kong SAR, New
Zealand and Singapore). No concentration data for EAs have been submitted
from countries in the WHO South-East Asian and Eastern Mediterranean
regions. Most of the analytical records were supplied by the European Region
(83.8%), followed by the Region of the Americas (13.6%), African Region (1.4%)
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and Western Pacific Region (1.2%). These data represented 13 of the 17 GEMS/
Food cluster diets.
All samples from Benin, Cameroon, Mali, Nigeria and New Zealand,
as well as most samples from the European Region were analysed for 12 EAs
the corresponding -inine epimers). Most samples from Canada were analysed for
three EAs (ergocristine, ergocryptine and ergosine), whereas the samples from
Hong Kong SAR (China), and Singapore were analysed for total EAs with no
indication of the specific EAs included.
For 11 EAs, the highest levels were observed in food samples from
the European Region: ergocristine (9279 µg/kg), ergocristinine (3538 µg/kg),
ergocornine (619 µg/kg), ergocorninine (396 µg/kg), ergocryptine (661 µg/kg),
ergocryptinine (1007 µg/kg), ergosine (1287 µg/kg), ergosinine (1066 µg/kg),
ergometrine (760 µg/kg), ergometrinine (234 µg/kg) and ergotaminine (339 µg/
kg). For ergotamine (3343 µg/kg), the highest level was observed in food samples
from the Region of the Americas.
As the relative potency of individual EAs is uncertain, the Committee
calculated the total EA concentration as the simple sum for all food samples for
which concentration data were available for individual EAs. The UB estimate of
concentration for the sum of EAs was only calculated for food commodities for
which at least one quantified sample was available. The UB estimates per food
sample were derived by adding quantified results to the mean of the LODs or
LOQs of the individual EAs reported as not detected or not quantified. For
samples with only EAs reported as not detected or not quantified, UB estimates
were derived by averaging the LODs or LOQs.
The Committee did not consider food commodities for which results were
reported only as “none detected” or “none quantified”. Despite large differences
in the number of analyses reported, the only foodstuffs that tested positive for
EAs were cereals and cereal-based products and, to a lesser extent, legumes
and pulses. In all regions, contamination (10.2 to 32.7% positive samples) was
observed in cereals and cereal-based products, but the level of contamination

was higher in the European Region (mean UB level 89.8 µg/kg) and in the Region
of the Americas (mean UB level 40.4 µg/kg) than in the Western Pacific Region
(mean UB level 9.8 µg/kg) or in the African Region (mean UB level 7.2 µg/kg).
In the European Region and the Western Pacific Region, contamination (8.6 to
10% positive samples) was also observed in legumes and pulses at lower levels
than in cereals and cereal-based products (mean UB level 11.7 µg/kg and 2.8 µg/
kg, respectively). Table 33 provides a summary of data from the GEMS/Food
contaminants database on the sum of concentrations of EAs by WHO region.
The highest levels of total EAs were observed in rye (13 783 µg/kg) and
wheat-based-products (5649 µg/kg) from the European Region.
198
Table 33
Summary of data from the GEMS/Food contaminants database on concentrations of ergot alkaloids (EAs) by WHO region according to the
food commodities used in the GEMS/Food cluster dietsa,b
WHO African Region WHO European Region WHO Western Pacific Region WHO/PAHO Region of the Americas
Food No. of % <LOD Mean LB Mean UB No. of % <LOD Mean LB Mean UB No. of % <LOD Mean LB Mean UB No. of % <LOD Mean LB Mean UB
commodities samples or LOQ (µg/kg) (µg/kg) samples or LOQ (µg/kg) (µg/kg) samples or LOQ (µg/kg) (µg/kg) samples or LOQ (µg/kg) (µg/kg)
Cereals and 59 89.8 6.2 7.0 9381 66.9 63.5 89.8 496 67.3 9.1 9.8 5593 72.2 33.0 40.4
cereal-based
products
Barley NA 84 99 1.6 11.6 11 91 49.1 49.4 264 66.3 45.9 54.8
Bran, unprocessed NA 1 100 0.0 2.0 NA 186 90.3 3.2 11.9
of cereal
grain (except
buckwheat,
canihua, quinoa)
Bread and other 9 33 40.9 43.0 2334 62.9 29.8 61.0 149 45 7.7 8.2 1425 69.1 16.9 23.9
cooked cereal
products
Buckwheat NA 110 89.1 3.7 11.5 5 100 0.0 0.5 140 95 2.3 7.8
Cereal grains NES NA 519 95 9.5 28.7 15 100 0.0 0.5 NA
Cereals and NA 4261 61.4 99.4 126.1 176 81.3 3.7 4.6 612 98 0.8 6.0
cereal-based
products NES
Maize 16 100 0.0 0.5 123 100 0.0 16.2 9 100 0.0 0.5 449 8.4 0.4 5.7
Oats NA 183 90.7 7.8 20.0 26 92.3 1.7 3.3 434 90.8 7.2 14.9
Rice 16 100 0.0 0.5 155 100 0.0 15.4 23 95.7 0.4 1.9 420 100 0.0 6.8
Rye NA 910 54.1 93.4 124.6 29 34.5 15.8 16.1 132 41.7 195.0 200.5
Wheat NA 549 83.6 15.0 33.7 18 83 3.5 3.9 1404 38.5 83.9 93.5
Wheat flour NA NA 17 29.4 4.6 5.2 NA
Wheatgerm NA NA 3 0 464.0 464.0 NA
White bread NA 72 87.5 4.1 23.5 6 16.7 15.0 15.1 NA
Ergot alkaloids
199
200
WHO African Region WHO European Region WHO Western Pacific Region WHO/PAHO Region of the Americas
Food No. of % <LOD Mean LB Mean UB No. of % <LOD Mean LB Mean UB No. of % <LOD Mean LB Mean UB No. of % <LOD Mean LB Mean UB
commodities samples or LOQ (µg/kg) (µg/kg) samples or LOQ (µg/kg) (µg/kg) samples or LOQ (µg/kg) (µg/kg) samples or LOQ (µg/kg) (µg/kg)
Wholemeal bread NA 38 39.5 68.5 114.3 3 66.7 14.0 14.3 NA
Legumes and 18 100 0.0 0.5 197 91.4 3.5 11.7 10 90 0.9 2.8 4 100 0.0 2.9
pulses
Beans, shelled NA 67 91 6.5 14.2 NA NA
(immature seeds)
Peas (dry) 2 100 0.0 0.5 1 100 0.0 20.0 7 85.7 1.3 3.3 NA
Soya bean (dry) NA 109 89.9 2.3 9.1 NA 4.0 100 0.0 2.9
LB: lower bound; LOD: limit of detection; LOQ: limit of quantification; NA: not analysed; UB: upper bound.
a
The total content of EAs in each sample was estimated by summing the reported concentrations for each of the individual alkaloids. Numbers of individual alkaloids tested were: 12 for the African Region (Benin, Cameroon, Mali and Nigeria: ergometrine,
ergocornine, ergocristine, ergocryptine, ergosine, ergotamine and their corresponding -inine (S)-epimers); between 3 (ergosine, ergocristine, ergocryptine) and 12 (ergometrine, ergosine, ergocornine, ergotamine, ergocristine and their corresponding
-inine (S)-epimers) for the Region of the Americas and the European Region depending on the sampling year and the food tested; either 12 for the Western Pacific Region (New Zealand; ergometrine, ergocornine, ergocristine, ergocryptine, ergosine,
ergotamine and their corresponding -inine (S)-epimers) or expressed as EAs for China, Hong Kong Special Administrative Region and Singapore).
b
The mean LB estimates were derived by substituting zero for analytical results below the LOD or LOQ. The mean UB estimates were derived by averaging the sum of individual EAs reported as not detected or not quantified.
Ninety-first JECFA
Ergot alkaloids
10.10 Food consumption and dietary exposure estimates

National and international assessments of chronic and acute dietary exposure to
EAs reported from the scientific or grey literature or derived by the Committee
were all based on total EA concentration calculated as the simple sum of individual
EAs. Table 34 provides a summary of the national and international estimates of
chronic dietary exposure to EAs from the literature or derived by the Committee.
The Committee evaluated published studies on chronic dietary exposure
to EAs in sub-Saharan African countries (Benin, Cameroon, Mali and Nigeria),
European countries and New Zealand.
Across national estimates of dietary exposure, LB–UB estimates of mean
dietary exposure to the sum of EAs were in the range of 0.010–0.47 µg/kg bw
per day for children and <0.01–0.18 µg/kg bw per day for adults. High percentile
LB–UB estimates of dietary exposure (P95) were in the range of 0.03–0.86 µg/
kg bw per day for children and <0.01–0.37 µg/kg bw per day for adults. For all
national estimates, the main foods contributing to dietary exposure were wheat
and wheat-based products.
In addition to national estimates of chronic dietary exposure, the Committee
derived international estimates of chronic dietary exposure to EAs using the 13
GEMS/Food cluster diets for the African Region, the Region of the Americas, the
European Region and the Western Pacific Region, with the concentration data
for EAs in foods from the same WHO regions and food commodities described
in Table 33. Even though some clusters defined in 2013 do not map exactly to a
single WHO region, the Committee considered that differences noted in clustering
were limited to a small number of countries and are not likely to affect the chronic
dietary exposure estimates. No international estimates were derived for clusters
within WHO regions from which no data had been submitted to the GEMS/Food
contaminants database. Therefore, no dietary exposure was estimated for clusters
G01, G04 and G06 associated with the Eastern Mediterranean Region and cluster
G09 associated with the South-East Asia Region.
LB–UB mean international estimates of chronic dietary exposure
to EAs ranged from 0.01 (G14, Western Pacific Region) to 0.18 µg/kg bw per
day (G02, European Region). LB–UB high estimates of dietary exposure (P90)
to EAs ranged from 0.02 (G14, Western Pacific Region) to 0.37 µg/kg bw per
day (G02, European Region). Wheat and wheat-based products were the main
foods contributing to dietary exposure to EAs in these clusters (ranging from
67 to 100%). Rye and rye products also contributed to dietary exposure in these
clusters, but to a lesser extent (ranging from 2 to 33%).
Except for European countries, no acute dietary exposure estimates for EAs
were reported in the scientific literature. Mean acute exposure ranged from 0.02 µg/
kg bw per day for “infants” up to 0.32 µg/kg bw per day for children aged 3–9 years.
201
High estimates of acute dietary exposure (P95) to EAs ranged from 0.1 to 0.49 µg/
kg bw per day for adults and from 0.13 to 0.98 µg/kg bw per day for children aged
3–9 years. The food types contributing most to the acute dietary exposure to EAs
were “mixed wheat and rye bread and rolls” and “rye bread and rolls”.
Since there was no match between the countries in the FAO/WHO global
individual food consumption database (GIFT) and the countries that submitted
data on concentrations of EAs in foods, the Committee did not derive additional
national estimates of acute dietary exposure.
The Committee noted that for Europe no major differences were observed
between estimates of chronic and acute dietary exposure to EAs, indicating that
the main contributors to the exposure are foods consumed on a regular basis
within particular populations. Considering that wheat and wheat-based products
have been identified as the main foods contributing to both chronic and acute
dietary exposure to EAs in European estimates, it is likely that, for other regions
in the world where wheat-based products are staple foods, the chronic exposure
estimates will be comparable to acute exposure estimates.
10.10.1 Transfer from feed to food

The very limited data on tissue distribution and residual concentrations in edible
tissues, milk and eggs provide no evidence of accumulation of EAs in edible
tissues.
10.11 Dose–response analysis

10.11.1 Dose–response data in humans
Data on the use of drugs containing EAs in humans provide relevant
information for assessing the acute effects of EAs, both with respect to doses
with a pharmacological (therapeutic) effect and those that may have a toxic effect
(Table 35). The therapeutic use of oral ergometrine maleate in management
of the third stage of labour or to prevent or treat postpartum haemorrhage

has largely been superseded by other drugs, sometimes in combination with
ergometrine, administered by the intramuscular or intravenous routes. But
from its use in the past, it is known that an oral dose of 0.2 mg ergometrine
maleate has pharmacological activity. This is the lowest single dose of an EA used
therapeutically; oral doses of ergotamine tartrate used for treatment of migraine
are 1–2 mg at onset (up to 4–6 mg per day).
10.11.2 Dose–response data in animals

The key studies in experimental animals identified by the Committee are
summarized in Table 36.
202
Ergot alkaloids
Table 34
Summary of national and international LB–UB estimates of chronic dietary exposure to
ergot alkaloids (EAs) from the literature or derived by the Committee
Estimated dietary
exposure, mean (P90
or P95) in µg/kg bw
WHO region Population group per dayb Reference
National estimates
African Region (Benin, Cameroon, Mali and Adult (18 to <65 years) <0.01–0.09 (<0.01–0.18) Derived by this
Nigeria) Committee
European Region (22 countries)a Infants (<12 months) 0.01–0.03 (0.05–0.76) EFSA, 2017
Toddlers (12 to <36 months) 0.03–0.47 (0.07–0.86)
Other children (36 months to <10 years) 0.02–0.46 (0.05–0.79)
Adolescents (10 to <18 years) 0.01–0.29 (0.03–0.56)
Adults (18 to <65 years) 0.01–0.18 (0.02–0.37)
Elderly (65 to <75 years) 0.01–0.14 (0.02–0.28)
Very elderly (over 75 years) 0.01–0.16 (0.02–0.28)
Western Pacific Region (New Zealand) Children (5–15 years) 0.01–0.03 (0.03–0.06) NZFS, 2020
Adults (>15 years) <0.01–0.01 (0.01–0.02)
International estimatesc
African Region (clusters GO3, G13, G16) Adult (>15 years) 0.01–0.04 (0.03–0.09) Derived by this
Committee
Region of the Americas (clusters G05, G12) Adult (>15 years) 0.11–0.15 (0.23–0.31) Derived by this
Committee
European Region (clusters G02, GO7, GO8, G10, Adult (>15 years) 0.05–0.18 (0.1–0.37) Derived by this
G11, G15) Committee
Western Pacific Region (clusters G14, G17) Adult (>15 years) 0.01–0.02 (0.02–0.04) Derived by this
Committee
a
Austria, Belgium, Bulgaria, Croatia, the Czech Republic, Denmark, Estonia, Finland, France, Germany, Hungary, Ireland, Italy, Lithuania, Luxembourg, Malta, Poland,
Slovenia, Sweden, Switzerland, the Netherlands and the United Kingdom. Range from minimum LB to maximum UB estimates across studies.
b
The range of dietary exposure estimates refers to LB and UB estimates of mean dietary exposure. National and international dietary exposure to EAs were reported
from scientific or grey literature or derived by the Committee. The LB mean estimates were derived by substituting zero for analytical results below the LOD or LOQ.
The UB mean estimates were derived by averaging the sum of individual EAs reported as not detected or not quantified. All international exposure estimates are
rounded and based on a 60 kg body weight.
c
Table 35
Daily oral doses of ergot alkaloids used therapeutically and doses causing adverse effects
in humans
Ergot alkaloid (EA) Dose (as mg EA Dose (as mg Dose (as mg EA/
(Effect) salt/person) EA/person) kg bw) Reference
Ergometrine maleate
Therapeutic dose, adult 0.2 0.15 0.0025a Martindale, 2010
(Uterine contraction, vasoconstriction)
Adverse effects, neonate 0.2 0.15 0.04–0.05b EFSA, 2012
(Vasoconstriction, CNS, respiratory, renal)c
Adverse effects, children <7 years old 0.4 (median dose) 0.3 0.015–0.02d Armenian &
(Gastrointestinal symptoms)e 0.2–2 (range Kearney, 2014
203
Ergot alkaloid (EA) Dose (as mg EA Dose (as mg Dose (as mg EA/
(Effect) salt/person) EA/person) kg bw) Reference
Ergotamine tartrate
Therapeutic dose, adultf 1–2 0.9–1.8 0.015–0.03a Martindale, 2010
(Vasoconstriction cerebral arteries)
Adverse effects, children <7 years old 1 (median dose) 0.9 0.045–0.06d Armenian &
(Gastrointestinal, CNS, respiratory symptoms)g 0.2–11 (range) Kearney, 2014
CNS, central nervous system.
a
Calculated for a 60 kg adult.
b
Calculated on the basis of a body weight range at birth of 3.0–3.5 kg.
c
Accidental administration of adult dose of ergometrine instead of vitamin K, including one death.
d
Calculated on the basis of a body weight range at 3–7 years of age of 15–20 kg.
e
Four cases of gastrointestinal symptoms, none serious, in 37 reports of exposure to ergometrine.
f
Up to 6 mg/person per day.
g
Fifteen cases of symptoms, none serious, in 56 reports of exposure.
Table 36
Summary of all critical studies of ergot alkaloids in experimental animals and their NOAELs/
LOAELs
Effect at the
Substances Species NOAELs LOAELs LOAEL Duration Reference
Ergotamine tartrate Rat 4 mg/kg diet 20 mg/kg diet Increased relative 4 weeks Speijers et al.,
Ergotaminea 0.34 mg/kg bw per day 1.7 (♀), 1.6 (♂) mg/kg organ weight 1992
(♀ and ♂) bw per day in ♀
Ergometrine maleate Rat 10 mg/kg diet 50 mg/kg diet Decreased plasma 4 weeks Peters-Volleberg,
Ergometrineb 0.72 (♀), 0.70 (♂) mg/ 3.3 (♀), 3.4 (♂) mg/kg glucose level in ♀ Beems &
kg bw per day bw per day Speijers, 1996
α-ergocryptine Rat 4 mg/kg diet 20 mg/kg diet Increased relative 28–32 Janssen et al.,
0.37 (♀), 0.34 (♂) mg/ 1.7 (♀), 1.4 (♂) mg/kg organ weight days 1998, 2000a,b
kg bw per day bw per day in ♀
Ergot sclerotia Pig Not determined 0.1 mg EAs/kg bw Jejunum lesions 4 weeks Maruo et al.,
per dayc 2018
Ergotamine tartrate Rat 5 mg/kg diet 20 mg/kg diet Tail muscular 13 weeks Speijers et al.,
Ergotaminea 0.48 (♀), 0.42 (♂) mg/ 1.99 (♀), 1.65 (♂) mg/ atrophy in ♀ 1993
kg bw per day kg bw per day
EAs, ergot alkaloids; LOAEL, lowest-observed-adverse-effect level; NOAEL, no-observed-adverse-effect level.
a
For the conversion of concentrations in diet to doses in mg/kg bw, the Committee used the molecular weights of ergotamine tartrate (1313.4 g/mol) and ergotamine
(1163.4 g/mol for two molecules) and the data on body weights and feed intake available in the report.
b
For the conversion of concentrations in diet to doses in mg/kg bw, the Committee used the molecular weights of ergometrine maleate (441.5 g/mol) and ergometrine
(325.4 g/mol) and the data on body weights and feed intake available in the report.
c
LOAEL of 0.1 mg/kg bw identified by the authors based on body weights and feed intake. The exposure was 0.159 and 0.083 mg/kg bw per day after 2 and 4 weeks
of exposure, respectively.
The Committee identified the tail muscular atrophy effect as the critical
effect suitable for the hazard characterization. This tail muscular atrophy can be
explained by vasoconstriction. Dose–response analyses were performed on the
204
Ergot alkaloids
Table 37
Dose–response analysis using the BMD approach (BMR = 10%) in rats (BMDS 3.2 Bayesian
Model Averaging)
BMD mg/ BMD confidence
Concentration of ergot Dose mg/kg bw kg bw per interval mg/kg Tail effect
alkaloids in the diet Sex per day day bw per day and incidence Reference
4-week studies
Ergotamine tartrate Ergotamine Abnormality Speijers et al.,
0, 100, 500 mg/kg diet Females 0, 7.3, 41 16.6 5.25–64.7 0/4, 0/6, 3/6 1992
Males 0, 7.5, 37 6.37 1.68–28.4 0/3, 2/6, 4/5
α-Ergocryptine α-Ergocryptine Muscle degeneration Janssen et al.,
concentration Females 0, 0.37, 1.7, 8.9, 60 18.1 5.36–37.4 0/6, 0/6, 0/6, 0/5, 4/6 2000a
0, 4, 20, 100, 500 mg/kg diet Males 0, 0.34, 1.4, 6.6, 44 3.88 1.29–9.59 0/6, 0/6, 0/6, 2/6, 6/6
13-week study
Ergotamine tartrate Ergotamine Muscular atrophy Speijers et al.,
concentration Females 0, 0.48,1.99, 7.63 1.55 0.61–3.20 0/10, 0/10, 2/10, 7/10 1993
0, 5, 20, 80 mg/kg diet Males 0, 0.42, 1.65, 6.18 1.69 0.63–3.53 1/10, 1/10, 1/10, 7/10
BMD, benchmark dose; BMDS 3.2, US EPA software (https://www.epa.gov/bmds); BMR, benchmark response).
available information on experimental animals from the two 4-week studies on

ergotamine tartrate (Speijers et al., 1992) and on α-ergocryptine (Janssen et al.,
2000a), and from the 13-week study on ergotamine tartrate (Speijers et al., 1993)
(Table 37).
Dose–response modelling on data from experimental animals was
performed by means of the BMD analysis using the US EPA software BMDS
3.23 (Table 37). Models available in BMDS 3.2 for dichotomous (quantal)
response were used as the default set of models and Bayesian MA was used for
the calculation of BMD confidence intervals (BMD-CI) following recent JECFA
guidance (EHC 240 Chapter 5 in FAO/WHO 2020). The lower confidence bound
(BMDL10) corresponding to a BMR10 for extra risk of tail muscular atrophy, was
selected as the reference point for the hazard characterization.
11. Evaluation
The Committee identified the pharmacological effect of ergometrine maleate on
the uterus – causing uterine contractions in humans during late pregnancy and
postpartum – as the critical effect for the evaluation of EAs in the diet.
3
https://www.epa.gov/bmds
205
The Committee established an acute reference dose (ARfD), based on

the following considerations:
1) The lowest oral therapeutic dose of 0.2 mg ergometrine maleate
(equivalent to 2.5 µg/kg bw, expressed as ergometrine) is considered
a pharmacological effect level in the most sensitive individuals, i.e.
those with high absorption.
2) Of the EAs that have been used as drugs, ergometrine is known to
have the highest potency for uterine contractions and its uterotonic
effect increases towards the end of pregnancy.
In selecting an uncertainty factor (UF) for extrapolation from the
pharmacological effect level at the therapeutic dose (LOEL) to a NOEL, the
Committee took into consideration that the data relate to a short-lived, reversible,
pharmacological effect, seen within a very sensitive subpopulation (women
in late pregnancy or postpartum). A UF of 2 was considered appropriate for
extrapolating from a pharmacological LOEL to a NOEL.
To derive an ARfD from a NOEL based on human data, in the absence
of additional information, the default UF would normally be 10. However, for
a substance that reversibly interacts with specific receptors, as is the case here,
with a pharmacological effect that is predominantly dependent on its maximum
plasma concentration (i.e. Cmax), a UF for toxicokinetic differences is considered
unnecessary. The Committee therefore applied the UF of 3.16 to cover possible
interindividual toxicodynamic differences.
Applying a composite UF of 6.3 (2 × 3.16) results in an ARfD of 0.4 µg
ergometrine/kg bw (2.5 ÷ 6.3 = 0.4). The Committee noted that it is appropriate
to establish a group ARfD for EAs but concluded that the available data are
not sufficient to establish toxic equivalency factors (TEFs) for different EAs.
Therefore, the ARfD is established as a group ARfD for the simple sum of total
EAs in the diet.
This ARfD would also be protective for other potentially sensitive

subgroups in the population, such as children, based on similar calculations in
relation to adverse effects (gastrointestinal symptoms) in that group following
unintentional exposure to ergometrine maleate.
Limited data from two 4-week studies on ergotamine tartrate and
α-ergocryptine in rats allowed the determination of a reference point (BMDL10)
of 1.3 mg/kg bw for EAs, based on muscular degeneration in the tail, secondary
to vasoconstriction. The Committee noted that the human pharmacological
effect level of 2.5 µg/kg bw and its derived NOEL provided a much more sensitive
reference point for derivation of an ARfD than the BMDL10 value from a
downstream toxic effect in animals.
206
Ergot alkaloids
As a first approach to establishing a TDI, the Committee considered the

data from repeated-dose animal studies and selected the lowest BMDL10 value of
0.6 mg/kg bw per day calculated for ergotamine, based on tail muscular atrophy,
secondary to vasoconstriction, observed in the 13-week study in rats (Speijers et
al., 1993) as reference point. Applying a default UF of 100 for intra- and inter-
species differences, a UF of 2 for extrapolation from a 13-week study to chronic
exposure and an additional UF of 3 to take into account the limitations of the
available toxicity data would indicate derivation of a TDI of 1 µg/kg bw per day.
The Committee considered that a TDI should not be higher than the
ARfD and decided to establish a group TDI for the sum of total EAs in the diet at
the same value as the group ARfD of 0.4 µg/kg bw per day.
The Committee noted that some estimates of the mean (0.46–0.47 µg/
kg bw per day) and high percentile (0.56–0.86 µg/kg bw per day) chronic dietary
exposure in children and some estimates of the high percentile acute dietary
exposure in children (0.65–0.98 µg/kg bw per day) and in adults (0.49 µg/kg bw
per day) exceeded the EAs group HBGV, and that this may indicate a human
health concern.
11.1 Recommendations
The Committee recommended the following:
■■ additional data on the EAs to allow for the derivation of toxic
equivalency factors (TEFs);
■■ additional data on the occurrence of EAs (at least for the 12 considered
at this meeting) in wheat and wheat-based products and in rye and
rye products from WHO regions and clusters for which no data were
submitted for this evaluation;
■■ the establishment of sampling plans for EAs.
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228
Previous cargoes – solvents and reactants
Abdul Afghan,1 Yiannis Kiparissis1 and Paul Loeven1
1
Bureau of Chemical Safety, Food Directorate, Health Products and Food Branch, Health
Canada / Government of Canada
A. ASSESSMENT OF SUBSTANCES PROPOSED AS PREVIOUS

CARGOES 232
1. Introduction 232
2. Background 233
2.1 Global production and consumption of fats and oils 233
2.2. Regulations affecting fats and oils 234
2.3 Global transport of fats and oils 234
2.4 The interrelationship of national, regional and trade interests 235
2.5 Development of the Codex Code of Practice for storage and transport of
edible fats and oils in bulk 235
3. Development of criteria 237
4. Basis of evaluation 238
4.1 Chemistry/reactivity 238
4.2 Methods of analysis 238
4.3 Dietary exposure assessment for previous cargo chemical substances 239
4.3.1 Exposure estimates in the 2006 criteria document 239
4.3.2 Exposure estimates based on up-to-date consumption data for
adults 240
4.3.3 Exposure estimates for infants and young children 241
4.3.4 Exposure from other dietary sources 241
4.3.5 Conclusion 241
4.4 Approach to toxicological evaluation 242
5. Recommendations 243
B. EVALUATION OF SUBSTANCES 244

Previous cargoes – solvents and reactants (Group 1) 244
I. Acetic anhydride 244
1. Explanation 244
2. Chemical and technical considerations 245
2.1 Manufacture and uses of acetic anhydride 245
2.1.1 Acetic acid process (ketene process) 245
2.1.2 Acetaldehyde oxidation process 246
2.1.3 Carbonylation of methyl acetate process 247
2.2 Impurities and secondary contaminants 247
2.3 Reactivity and reactions with fats and oils 248
229
3.2 Toxicity in experimental animals 249

3.2.2 Short-term and long-term studies of toxicity and carcinogenicity 249
3.2.5 Allergenicity 253
3.2.6 Impurities 253
4. Occurrence and exposure 256
5. Comments 257
5.1 Chemical and technical considerations 257
5.4 Allergenicity 259
5.5 Impurities 259
5.6 Assessment of dietary exposure 260
6. Evaluation 260
II. sec-Butyl acetate 261

1. Explanation 261
2.1 Manufacture and uses of sec-butyl acetate 262

5. Comments 271
5.5 Impurities 273
6. Evaluation 274
III. tert-Butyl acetate 274

1. Explanation 274
2.1 Manufacture and uses of tert-butyl acetate 275
230

5. Comments 288
5.5 Impurities 290
6. Evaluation 291
IV. n-Pentane 291

1. Explanation 291
2.1 Manufacture and uses of n-pentane 292
5. Comments 301
5.5 Impurities 303
6. Evaluation 304
231
V. Cyclohexane 305
1. Explanation 305
2.1 Manufacture and uses of cyclohexane 306
5. Comments 319
5.5 Impurities 322
6. Evaluation 323
7. Recommendations 324
8. References 324
A. ASSESSMENT OF SUBSTANCES PROPOSED AS

PREVIOUS CARGOES
1. Introduction
Fats and oils destined to be used as food are transported and stored in large
volumes. Transportation in large volumes by sea is exempted from many land-
based regulations as it is not practical to have fleets of ships solely dedicated to the
transportation of food in large tanks, since the trade is generally unidirectional
from producer to consumer. Furthermore, the construction and dependency
on the availability of a limited number of single-use carriers would make the
transport of fats and oils extremely expensive. To address the economic realities,
232
certain types of ships are permitted to carry different classes of cargo in their
tanks on their outbound and onward journeys. A non-food item may be carried
in a tank in one direction and a single type of fat or oil on the further voyage.
Since ships are constructed to have several individual tanks, each may contain a
cargo destined for a different location and may be used to carry either a food or
non-food item depending on the contract.
A number of organizations have been involved in the development of
codes of practice, transportation contracts, ship construction, cargo segregation,
environmental issues and food safety. The Codex Alimentarius Commission
(CAC) adopted and published a code of practice for the storage and transport
of edible fats and oils in bulk, which was developed by the Codex Committee
on Fats and Oils (CCFO) in 1987 (CAC, 1987). At that time, CCFO recognized
the need to assess the acceptability of previous cargoes transported in a tank
subsequently used for the transportation of an edible fat or oil. Commercial trade
contracts recognized the need to specify that certain chemicals should never be
acceptable previous cargoes for subsequent cargoes of edible fats or oils. These
substances formed the basis of the “banned lists” of previous cargoes. In 2001, a
combined list of chemicals banned as previous cargoes was developed by CCFO
and adopted by CAC (CAC, 2001); it was added to the Codex code of practice
as Appendix 1. Other substances carried in bulk were considered to pose a low
risk to public health as a contaminant in edible fats or oils; these formed the basis
of “acceptable lists” of previous cargoes. The development of a CCFO acceptable
list of previous cargoes was also based on trade experience. A preliminary list
was reviewed by the Scientific Committee on Food (SCF) and their findings
were reported to CCFO in 1999; 14 substances were identified for which there
were insufficient data to make a safety determination. After further discussion
at subsequent CCFO meetings, a list of 23 potentially safe previous cargoes that
require evaluation was developed. CCFO asked for scientific advice from FAO/
WHO on these 23 substances that lacked safety evaluations. The present evaluation
by JECFA addresses the following solvents and reactants: acetic anhydride, sec-
butyl acetate, tertbutyl acetate, n-pentane and cyclohexane, which are substances
on the current list of chemicals acceptable as previous cargoes by CCFO.
2. Background
2.1 Global production and consumption of fats and oils

The global trade in edible fats and oils is more than 200 million metric tonnes
annually and valued at approximately US$ 120 billion (USDA, 2019). By far the
233
largest contributors are palm (36%) and soybean oil (28%), followed by rapeseed/
canola (14%), sunflower seed (10%), palm kernel (4%), peanut (3%), cottonseed
(3%), coconut (2%) and olive oils (2%).
Many vegetable oils are produced in regions (for example: soybean –
Argentina, Brazil, USA; rapeseed – Australia, Canada; sunflower seed – Ukraine;
palm – Indonesia and Malaysia; and coconut – equatorial latitudes) far from the
major sites of consumption. Olive oil is produced in regions with a Mediterranean
climate in both the northern and southern hemispheres. International trade in
fats and oils uses the most economical method of ocean transportation since
global trade in edible fats and oils is primarily unidirectional. Soybean oil from
Argentina and Brazil, for example, is shipped to both Asian and European
markets, but there is unlikely to be a complementary cargo of fat or oil available
for transportation in the reverse direction. Similarly, oils from tropical regions
are traded globally, often without reciprocal trade in fats and oils.
2.2 Regulations affecting fats and oils

Shipment of fats and oils is described in numerous national and international
regulations and agreements. Land-based transportation is regulated by local and
national guidelines and/or legislation, whereas international trade is subject to
commercial agreements, international shipping regulations and various codes of
practice. The development of banned lists and acceptable lists of previous cargoes
is founded on existing trade contracts.
About 85% of the fats and oils are traded globally using FOSFA (The
Federation of Oils, Seeds and Fats Associations, London) contracts. The balance is
traded under contracts issued by NIOP (National Institute of Oilseed Products) or
other organizations. A contract under “banned list terms” requires that fats and oils
are not shipped in tanks that have contained a substance on the banned list as the
immediate previous cargo. For certain chemicals, this requirement is extended to
the three previous cargoes. Alternatively, a contract may state that “the immediate
previous cargo shall be a product on the FOSFA List of Acceptable Previous
Cargoes”. In this case, the receiver will only accept the cargo if the previous cargo
is on FOSFA’s acceptable list. These two lists only cover a small proportion of the
chemicals transported by sea; thus many substances appear on neither list and their
acceptability as a previous cargo is subject to agreement by the contracting parties.
2.3 Global transport of fats and oils

Transportation by sea is regulated by the International Maritime Organization
(IMO). The International Convention for the Prevention of Pollution from Ships
234
(MARPOL) aims to prevent operational and accidental pollution from ships.

MARPOL limits the carriage of different classes of liquid cargoes to specific
tanker vessels based on ship construction and the class of chemical. Under this
convention, fats and oils may not be transported in vessels designated to carry
cargoes of crude oil, fuel oil, heavy diesel oil or lubricating oil. The International
Code for the Construction and Equipment of Ships Carrying Dangerous
Chemicals in Bulk (IBC Code) lists chemicals carried as bulk liquids, their
pollution category, the type of ship design and any relevant restrictions or
derogations. The previous cargoes under consideration (see Table 1) are in the
medium- or low-risk categories for marine pollutants. The single exception is
propylene tetramer, which is considered a high-risk marine pollutant. MARPOL
also deals with tank washing and material discharge. Pentane falls into an
additional category of oil-like substances requiring additional attention between
cargoes.
2.4 The interrelationship of national, regional and trade interests

The practice of Acceptable List trading was discussed in line with regional
initiatives to protect consumer health. The adoption of the hazard analysis and
critical control point (HACCP) principles and their inclusion in the Codex
Alimentarius approach to the safe trade of food and food products can be applied
to the transport of oils and fats by sea. The CAC adopted the Code of Practice for
the Storage and Transport of Fats and Oils in Bulk developed by CCFO in 1987
(CAC-RCP 36-1987). The Code has been revised periodically and a banned list
of substances was added in 2001. The list of acceptable previous cargoes adopted
by the European Union (EU) and based on existing trade lists, was evaluated by
the European Food Safety Authority (EFSA).
2.5 Development of the Codex Code of Practice for Storage and

Transport of Edible Fats and Oils in Bulk
CCFO discussions highlighted the need for lists of banned and acceptable
previous cargoes. The topic of contamination by previous cargoes led to the
incorporation of the FOSFA and NIOP trade lists into the Code by reference
only. In 2001, CAC adopted the “Banned List” and it appears in the current code
of practice as Appendix 3.
The development of a List of Acceptable Previous Cargoes by CCFO
began with attempts to harmonize the FOSFA and NIOP trade lists with an EU
list. The Acceptable List was further refined in 1999 when CCFO considered a
list of substances proposed by the EU that had been reviewed by the SCF. Having
235
Table 1
List of substances submitted by CCFO for evaluation by JECFA for addition to the list of
acceptable previous cargoes
Substance (synonyms) CAS number Assessment groupa
Acetic anhydride (ethanoic anhydride) 108-24-7 1
1,4-Butanediol (1,4-butylene glycol) 110-63-4 2
Butyl acetate, sec- 105-46-4 1
Butyl acetate, tert- 540-88-5 1
Calcium ammonium nitrate solution 15245-12-2 4
Calcium lignosulfonate liquid (lignin liquor; sulphite lye) 8061-52-7 4
Calcium nitrate (CN-9) solution 35054-52-5 4
Cyclohexane 110-82-7 1
Fatty alcohols
iso Decyl alcohol (isodecanol) 25339-17-7 2
Myristyl alcohol (1-tetradecanol, tetradecanol) 112-72-1 2
iso Nonyl alcohol (isononanol) 27458-94-2 2
iso Octyl alcohol (isooctanol) 26952-21-6 2
Tridecyl alcohol (1-tridecanol) 112-70-9 2
Unfractionated fatty alcohol mixture or mixtures of fatty alcohols from natural 3
oils and fatsb
Methyl tertiary butyl ether (MTBE) 1634-04-4 5
Mineral oil, medium and low viscosity, class II 3
Mineral oil, medium and low viscosity, class III 3
Montan wax 8002-53-7 3
Pentane 109-66-0 1
1,3-Propanediol (1,3-propylene glycol) 504-63-2 2
Propylene tetramer (tetrapropylene, dodecene) 6842-15-5 3
Soybean oil epoxidized 8013-07-08 3
Ethyl tertiary butyl ether (ETBE) 637-92-3 5
a
Group 1 was considered at this meeting. For the evaluations of the other substances see the report of the ninetieth meeting (Annex 1, reference 246).
b
Discussed with Group 2 – alcohols.
developed a list of acceptable previous cargoes, it was determined that there were
14 substances on it that required further evaluation; these 14 substances formed
the basis of the CCFO Proposed Draft List of Acceptable Previous Cargoes, which
was adopted by CAC 34 in 2011. For consideration at the ninetieth and ninety-first
meetings, a list of 23 substances was proposed to FAO/WHO (Table 1) by CCFO
for scientific advice on their suitability as previous cargoes for the carriage of fats
and oils by sea-going vessels upon its evaluation against the four criteria. Each
substance on the list has been assigned to Groups 1–5 (1 – solvents/reactants; 2 –
alcohols; 3 – oils and waxes; 4 – solutions; 5 – butyl ethers). Substances in Group
1 were evaluated at the present meeting.
236
3. Development of criteria
As a result of the CCFO request to FAO/WHO for scientific advice on the
development of criteria for the assessment of the safety of residues of previous
cargoes in the tanks of sea-going vessels carrying edible fats and oils, a technical
meeting was convened (in November 2006) at the Dutch National Institute
of Public Health and the Environment (RIVM). RIVM prepared a technical
background document (FAO/WHO, 2006, Appendix II) and drafted the meeting
report with FAO/WHO (2007).
Discussions were limited to the assessment of previous cargoes in the
transport of edible fats and oils in bulk by sea and the consideration of safety
implications in terms of human health. The experts accepted that the quality of
the fats and oils cargo could change as a result of hydrolysis and oxidation, but
they acknowledged that these changes were already taken into account in trade
contracts.
The experts considered a list of parameters originating from discussions
at CCFO meetings, noting that previous cargoes are generally liquid chemical
substances, slurries of solid particles or aqueous solutions. To further frame the
deliberations, the experts decided to consider only a generic worst-case scenario
since developing criteria to cover every possible combination of previous cargo,
type of tank, cleaning regime and possible further processing of the subsequent
cargo of fat or oil would not be a realistic approach.
The experts developed the following worst-case scenario: the smallest
commercially viable tank size (200 m3), coated with a polymer that absorbs the
previous cargo, is filled to 60% capacity (as required by contract), and the cargo
of fat or oil is not to be further processed or refined. The model also assumed
that the tank and associated pipework has been cleaned according to defined
standards, inspected and considered clean and dry. Under these circumstances,
the maximum level of contamination in the subsequent fat or oil cargo by the
previous cargo was calculated to be 100 mg/kg. This value was used to determine
a single estimate of worst-case human exposure of 0.1 mg/kg bw per day. Based
on this generic exposure value, the experts considered that for the evaluation of
previous cargoes, the acceptable daily intake (ADI) or tolerable daily intake (TDI)
should be greater than or equal to 0.1 mg/kg bw in order to provide sufficient
protection for children and high-intake consumers. Negligent or fraudulent
practices were not considered to be part of the criteria. The experts identified
four criteria necessary to determine the acceptability of a previous cargo (see
FAO/WHO, 2006).
The criteria as adopted by CAC 34 (2011) are listed in Table 2.
237
Table 2
Criteria adopted by CAC 34 and included in RCP-36-1987
1. The substance is transported/stored in an appropriately designed system with adequate cleaning routines, including the verification of
the efficacy of cleaning between cargoes, followed by effective inspection and recording procedures.
2. Residues of the substance in the subsequent cargo of fat or oil should not result in adverse human health effects. The ADI (or TDI) of the
substance should be greater than or equal to 0.1 mg/kg bw per day. Substances for which there is no numerical ADI (or TDI) should be
evaluated on a case-by-case basis.
3. The substance should not be or contain a known food allergen, unless the identified food allergen can be adequately removed by
subsequent processing of the fat or oil for its intended use.
4. Most substances do not react with edible fats and oils under normal shipping and storage conditions. However, if the substance does
react with edible fats and oils, any known reaction products must comply with criteria 2 and 3.
4. Basis of evaluation
4.1 Chemistry/reactivity
Edible fats and oils are normally chemically stable; however, there may be potential
for reactions with residues of previous cargoes that could give rise to products
that are hazardous to human health. Consideration should be given to chemical
substances that can react with edible fats and oils under normal transportation
conditions. Minor oxidation and hydrolysis are normally anticipated in trade
contracts and are not considered a consequence of contact with a previous
cargo, unless accelerated degradation occurs. Although many possible reactions
require the presence of specific catalysts or temperatures well in excess of those
anticipated during transportation, potential reactions of the previous cargo with
triglycerides and free fatty acids or other minor components present in the fat or
oil should still be considered.
4.2 Methods of analysis

In a few cases where contamination is considered critical there has been an
international effort to develop specific analytical methods. Cases of contamination
with diesel fuel (alkanes) and mineral oils (mineral oil saturated hydrocarbons,
MOSH; mineral oil aromatic hydrocarbons, MOAH) led to the development of
relevant international standards. Although many of the substances under review
at the present meeting can be analysed by gas or liquid chromatography using
appropriate detector systems, little progress has been made in the application
238
of these technologies to their contamination of oils and fats. It is assumed that

available methods with suitable modifications will be capable of determining the
maximum anticipated level of 100 mg/kg of previous cargo in the subsequent
cargo of fats or oils.
4.3 Dietary exposure assessment for previous cargo chemical

substances
As a consequence of considering a range of previous cargo chemical substances
at its ninetieth meeting, the Committee concluded that it was appropriate to
review the approach to estimating dietary exposure set out in the 2006 document
Development of criteria for acceptable previous cargoes for fats and oils (criteria
document) (FAO/WHO 2006).
The Committee noted that since the 2006 criteria document was drafted,
newer and better-quality data on the consumption of fats and oils by adults,
infants and young children have become available.
The Committee also noted that some of the previous cargo chemical
substances assessed have additional sources of dietary exposure and expressed
the view that it may be necessary to consider this in the exposure assessment.
4.3.1 Exposure estimates in the 2006 criteria document

Based on the best available data at that time, the 2006 criteria document set out
the following approach to dietary exposure assessment of previous cargo chemical
substances present in fats and oils:
■■ Estimated mean per capita consumption of 0.025 kg/day of a single
type of fat or oil. The value was rounded up from the maximum per
capita consumption of refined soybean oil of 22 g/person per day
from the GEMS/Food cluster diets.
■■ A factor of 2.5 to cover children and high consumers was derived from
a rounded ratio between the mean and 97.5th percentile consumption
of total vegetable oil from a food consumption survey in the United
Kingdom (20 and 52 g/person per day for the population aged
> 18 years). The criteria document also noted that dietary exposure of
children to contaminants is frequently 2.5 times that of adults.
■■ A worst-case concentration of 100 mg/kg for a previous cargo
contaminant in fats or oils.
■■ A body weight of 60 kg.
These data were used to define a worst-case dietary exposure estimate:
239
= 0.1 mg/kg bw per day
Based on the mean per capita consumption of fats and oils, and a factor
of 2.5, there would be no health concern to the general population from exposure
to previous cargoes if the ADI or TDI is sufficiently protective, for example, the
ADI or TDI is greater than, or equal to 0.1 mg/kg bw per day.
4.3.2 Exposure estimates based on up-to-date consumption data for adults

Since 2006, the GEMS/Food cluster diets have been revised, and the FAO/
WHO Chronic Individual Food Consumption – summary statistics database
(CIFOCOss) has become available (WHO, 2017). The 2006 criteria document
noted that food consumption information from dedicated surveys would be more
appropriate than the food consumption estimates from the GEMS/Food cluster
diets (WHO, 2012). However, it used the cluster diets, as food consumption
survey data were only available from a very limited number of countries at that
time. CIFOCOss currently contains food consumption data from 37 countries.
From the current version of CIFOCOss, the maximum mean consump-
tion for a single fat or oil type is 35 g/person per day for consumption of virgin
or extra-virgin olive oil by elderly Italians. The maximum 95th percentile (p95)
consumption of a single fat or oil is 138 g/person per day for edible cottonseed oil
by women (age 15–49 years) from Burkina Faso. This group also has the highest
97.5th percentile consumption of 189 g/person per day.
Based on the protocols currently used by JECFA for veterinary drugs,
the number of consumers of cottonseed oil in the Burkina Faso survey (n = 116)
would suggest that the 95th percentile is the highest reliable percentile (Boobis et
al., 2017; Arcella et al., 2019).

These data suggest that for adults, a mean fat or oil consumption of
35 g/person per day and a high consumption of fat or oil of 140 g/person per day
would be a conservative estimate consistent with available data.
The use of updated food consumption data will result in a revised
estimated worst-case dietary exposure for adults:
240
4.3.3 Exposure estimates for infants and young children

Potentially vulnerable population groups, like infants and young children, were
not specifically considered in the 2006 criteria document. Since then, individual
consumption data for several population groups, including infants and young
children, have become available through CIFOCOss and other sources. Infants
and young children should be considered in the risk assessment because they
could potentially experience high exposure to previous cargo chemical substances
per kg body weight during growth and development.
Information on consumption of food oils by infants and young children
was also available from the US Environmental Protection Agency’s Food
Commodity Intake Database (FCID) (US EPA, 2020), which in turn is based on
data from the US National Health and Nutrition Survey/What We Eat In America,
2005–2010 cycles. The highest oil consumption for infants and young children
based on FCID is comparable to those in the CIFOCOss database; however, oil
consumption information based on FCID takes into account individual body
weights.
The highest reported consumption of a specific fat or oil type was for
palm oil. Estimated mean and p95 consumption by infants and young children
were 7.6 and 19 g/day, respectively. Estimated mean and p95 consumption on a
body weight basis were 1 g/kg bw per day and 3 g/kg bw per day, respectively.
These data were used to define a worst-case dietary exposure estimate for
infants and young children:
4.3.4 Exposure from other dietary sources

For some previous cargo chemical substances potentially present in food oils, there
are additional sources of dietary exposure, such as contamination (for example,
contaminated drinking-water) or food additive uses (Table 3). Dietary exposures
from these different sources should be considered in exposure assessment.
4.3.5 Conclusion
The Committee concluded that, based on up-to-date data on consumption of
single fats and oils in the general population, which have become available since
2006, the generic human exposure value of 0.1 mg/kg bw per day used in the 2006
criterion no. 2 to determine the acceptability of a previous cargo should be revised.
Consequently, the updated, more conservative generic human exposure value of
0.3 mg/kg bw per day should be used in the evaluation of these substances.
241
Table 3
List of substances for evaluation by JECFA arising from the development of a list of
acceptable previous cargoes by the Codex Committee on Fats and Oils: Other sources of
exposure
Substance (synonyms) Other sources of exposure
1,4-Butanediol (1,4-butylene glycol) Used in food contact material
Calcium ammonium nitrate solution Calcium, nitrate and ammonium are ubiquitous in the human diet
Calcium lignosulfonate liquid (lignin liquor; sulfite lye), molecular Calcium lignosulfonate (40-65) is used as a food additive, an additive
weight not specified in animal feed and as an ingredient in pesticides
Calcium nitrate (CN-9) solution Calcium and nitrate are ubiquitous in the human diet
iso Decyl alcohol (isodecanol) None
Myristyl alcohol (1-tetradecanol; tetradecanol) Flavouring agent, formulation agent, lubricant, release agent
iso Nonyl alcohol (isononanol) None
iso Octyl alcohol (isooctanol) Used in food contact material
Tridecyl alcohol (1-tridecanol) Used in food contact material
Unfractionated fatty alcohol mixture or mixtures of fatty alcohols Occurs naturally in foods
from natural oils and fatsa
Methyl tertiary butyl ether (MTBE) Drinking-water
Mineral oil, medium and low viscosity, class II and III Used in food contact material, direct food additive
Montan wax Food additive
1,3-Propylene glycol Used in place of 1,2-propanediol as a food additive
Propylene tetramer (tetrapropylene, dodecene) None
Soybean oil epoxidized Used in food contact material
Ethyl tertiary butyl ether (ETBE) Drinking-water
a
Discussed with Group 2 – Alcohols.
The Committee noted that these estimates of dietary exposure were

derived from a more conservative approach to using data on consumption of
single fats and oils and a worst-case concentration of previous cargo chemicals in
a single fat or oil of 100 mg/kg.
The Committee also concluded that additional sources of dietary
exposure need to be considered in exposure assessment of previous cargo

chemical substances.
4.4 Approach to toxicological evaluation  

The Committee received no submitted data and, therefore, reviewed monographs
from previous evaluations of individual substances conducted by JECFA, WHO,
International Agency for Research on Cancer (IARC), and national and regional
governmental authorities to retrieve additional relevant references for completing
242
the present assessment. The Committee also conducted literature searches. The
details are included in the consideration of individual substances.
At its ninetieth meeting, the Committee revised the generic value for
assumed worst-case human dietary exposure from 0.1 to 0.3 mg/kg bw per day
and used this revised generic exposure value for the evaluation of previous cargoes.
The Committee also considered data on exposure to the substances from sources
other than previous cargoes. Thus, the ADI (or TDI) should be greater than or
equal to the estimated dietary exposure (0.3 mg/kg bw per day plus exposure
from other possible dietary sources) in order to provide sufficient protection for
infants, children and high-intake consumers. In situations where no appropriate
numerical ADI (or TDI) was available from JECFA, the Committee considered
other previously established health-based guidance values or calculated a margin
of exposure (MOE) based on a reference point characterizing the toxicological
hazard (such as a no-observed-adverse-effect level (NOAEL), etc.) identified
from the available data divided by the estimated dietary exposure. Interpretation
of this MOE is a matter of expert judgement that takes into account limitations in
the available toxicological database.
5. Recommendations
The Committee recommended that the CCFO consider revising Criterion no. 2
in RCP-36-1987 as adopted by CAC 34 (2011).
■■ Based on the consumption of fats and oils by infants and young
children, there is no health concern for the general population from
dietary exposure to previous cargo chemical substances if the ADI or
TDI is sufficiently protective, for example, the ADI or TDI is greater
than, or equal to 0.3 mg/kg bw per day. Substances for which there is
no numerical ADI or TDI should be evaluated on a case-by-case basis
(for example, MOE approach).
■■ Where there are additional sources of dietary exposure to the
previous cargo chemical substances, they should be considered in the
exposure assessment.
243
B. EVALUATION OF SOLVENTS AND REACTANTS

This section considers the suitability of certain solvents and reactants as an
immediate previous cargo for a subsequent cargo of an edible fat or oil. These
substances are included on the FOSFA and NIOP acceptable previous (prior)
cargo lists. Separate subsections consider acetic anhydride, sec-butyl acetate, tert-
butyl acetate, n-pentane and cyclohexane.
I. Acetic anhydride
1. Explanation
Acetic anhydride is rapidly hydrolysed to acetic acid in the presence of water.
Consequently, the toxicological information on acetic acid and data from previous
evaluations are considered relevant to the assessment of acetic anhydride.
Acetic anhydride has not been previously considered by JECFA. In 1997
the Scientific Committee on Food (SCF) and EFSA (2012) concluded that since
acetic anhydride will be converted to acetic acid during tank washing, use of
acetic anhydride as a previous cargo for fats and oils is considered acceptable.
Acetic acid was first reviewed by the Committee at its ninth and tenth
meetings in 1967 (Annex 1, references 11 and 13). At those two meetings, the
Committee concluded that for the purposes of evaluation, all sources of acetate
used as food additives should be considered together. Since acetic acid has a
sufficiently acid taste to limit the amount used in foods, it was not considered
necessary to specify an ADI. However, at its seventeenth meeting, the Committee
allocated a group ADI “not specified”,1,2 to acetic acid and its potassium and
sodium salts. The Committee noted established metabolic pathways for acetic
1
Originally the term “not limited” was used; however, this term was replaced by “not specified” at the
Committee’s eighteenth meeting (Annex 1, reference 35).
2
The current term “not specified” is applicable to a food substance of very low toxicity that, on the basis
of the available chemical, biochemical and toxicological data as well as the total dietary intake of the
substance (from its use at the levels necessary to achieve the desired effect and from its acceptable
background in food), does not, in the opinion of the Joint FAO/WHO Expert Committee on Food
Additives, represent a hazard to health. For that reason, and for reasons stated in individual evaluations,
the establishment of an ADI expressed in numerical form is not deemed necessary. An additive meeting
this criterion must be used within the bounds of Good Manufacturing Practice: that is, it should be
technologically efficacious and should be used at the lowest level necessary to achieve this effect, it
should not conceal inferior food quality or adulteration, and it should not create a nutritional imbalance.
(https://apps.who.int/iris/bitstream/handle/10665/44065/WHO_EHC_240_13_eng_Annex1.
pdf;jsessionid=BDA8CB8E9D2770D3A11D7C8AA1FF2AA0?sequence=13).
244
acid and its consumption as a normal constituent of the diet (Annex 1, reference
32). At its forty-ninth meeting in 1998, the Committee re-evaluated the safety
of acetic acid as a flavouring agent and concluded that there were no safety
concerns based on the negative genotoxicity profile, the absence of any adverse
effects at 350 mg/kg bw per day in a short-term toxicity test (63 days) in rats
and a relatively small contribution from use as a flavouring agent (Annex 1,
reference 131). The Committee also noted that acetic acid can be predicted to
undergo complete metabolism to endogenous products via the fatty acid and
tricarboxylic acid pathways. In the opinion of the Committee the endogenous
levels of metabolites from these substances would not give rise to perturbations
outside the physiological range.
For the present evaluation, previous assessments (monographs)
completed by JECFA, SCF or EFSA, and national and regional governmental
authorities were identified by searching their respective websites. This was
followed by a comprehensive search to identify any critical new data for the
assessment of human health risk on PubMed and PubChem. The search terms
used were acetic anhydride and synonyms (for example, acetyl acetate and
acetanhydride), CAS number (108-24-7), toxicity and toxicokinetics. Given the
paucity of information relevant to the oral toxicity of acetic anhydride, secondary
searches for relevant information on its hydrolysis product (acetic acid) were also
conducted to supplement this assessment. The cut-off date for inclusion in this
report was 29 December 2020.
2. Chemical and technical considerations

The physicochemical characteristics of acetic anhydride are listed in Table 4.
2.1 Manufacture and uses of acetic anhydride

The following manufacturing processes for industrial synthesis of acetic
anhydride are described in the literature.
2.1.1 Acetic acid process (ketene process)

The ketene process for the production of acetic anhydride was the main
manufacturing process up to the 1970s. It proceeds in two steps: the thermal
cleavage of acetic acid to form ketene (at 730–750 oC, 15–20 kPa) and the reaction
of ketene with acetic acid.
245
Table 4
Physical and chemical characteristics of acetic anhydride
Chemical name Acetic anhydride
Synonyms Ethanoic anhydride; acetyl acetate; acetic acid, 1,1’-anhydride; ethanoyl ethanoate; acetic acid anhydride;
acetyl acetate; acetyl oxide; acetic oxide
CAS number 108-24-7
Chemical structure
H2C CH3
Molecular formula; molar C4H6O3; 102.09 g/mol
mass
Description Colourless liquid with a strong, pungent, vinegar-like odour
Melting point −73.1 °C
Boiling point 139.5 °C
Solubility Soluble in water. In aqueous solution, acetic anhydride hydrolyses and acetic acid is formed
CH3COOH → CH2=C=O + H2O

CH2=C=O + CH3COOH → (CH3CO)2O
Triethyl phosphate is commonly used as a dehydration catalyst for the

water formed in the first step. Aqueous 30% ammonia is employed as solvent in
the second step (Held et al., 2000; Wagner, 2002).
2.1.2 Acetaldehyde oxidation process

Liquid-phase catalytic oxidation of acetaldehyde can be directed by appropriate
catalysts, such as transition metal salts of cobalt or manganese, to produce
anhydride. Either ethyl acetate or acetic acid may be used as reaction solvent.
Acetaldehyde oxidation generates peroxyacetic acid, which then reacts with more
acetaldehyde to yield acetaldehyde monoperoxyacetate (CAS No. 7416-48-0).
Under sufficient pressure to permit a liquid phase at 55–56 oC, the acetaldehyde
monoperoxyacetate decomposes nearly quantitatively into anhydride and water

in the presence of copper.
CH3CHO + O2 → CH3COOOH
CH3COOOH + CH3CHO → CH3COOOCH(OH)CH3
CH3COOOCH(OH)CH3 → (CH3CO)2O + H2O
The catalyst is a mixture of three parts copper acetate to one part cobalt
acetate by weight (Wagner, 2002).
246
2.1.3 Carbonylation of methyl acetate process

Acetic anhydride is produced by carbonylation of methyl acetate:
CH3CO2CH3 + CO → (CH3CO)2O
This process involves the conversion of methyl acetate to methyl iodide

and an acetate salt. Carbonylation of the methyl iodide in turn yields acetyl iodide,
which reacts with acetate salts or acetic acid to give the product. Rhodium and
lithium iodides are employed as catalysts. Because acetic anhydride is not stable
in water, the conversion is conducted under anhydrous conditions (Zoeller et al.,
1992). The catalyst system for the methyl acetate carbonylation process involves
rhodium chloride trihydrate (CAS No. 13569-65-8), methyl iodide (CAS No. 74-
88-4), chromium metal powder and an aluminium oxide support or a nickel
carbonyl complex with triphenylphosphine, methyl iodide and chromium
hexacarbonyl (Wagner, 2002).
The chief industrial application of acetic anhydride is for acetylation
reactions in the production of cellulose acetates (The Merck Index, 2000;
Wagner, 2002). Acetic anhydride is also used in the manufacture of fibres,
plastics, pharmaceuticals, dyes and explosives (PubChem online database). In
food applications it is used in the starch industry as an acetylation compound in
production of modified starches: INS No. 1414, INS No. 1420 and INS No. 1422
(Annex 1, references 230 and 231).
2.2 Impurities and secondary contaminants

Technical-grade acetic anhydride (maximum 97%), often contains colour bodies,
heavy metals, phosphorus and sulfur compounds. Anhydride manufactured
by acetic acid pyrolysis sometimes contains ketene polymers, for example,
acetylacetone, diketene and dehydroacetic acid, and particulate carbon or soot
is occasionally encountered. Polymers of allene, or its equilibrium mixture,
methylacetylene-allene, are reactive impurities which, if exposed to air, slowly
autoxidize to dangerous peroxidic compounds (Wagner, 2002).
The Committee noted that fats and oils for human consumption have to
conform to the maximum levels for heavy metals in the Codex General Standard
for Contaminants and Toxins in Food and Feed, CXS-193-1995, and it is therefore
not necessary to estimate exposures to heavy metals in food oils due to carryover
from previous cargoes of acetic anhydride. Additional impurities in acetic
anhydride, depending on the method of manufacture, include ketene polymers
(for example, acetylacetone, diketene, dehydroacetic acid), polymers of allene or
its equilibrium mixture, methylacetylene-allene, and particulate carbon or soot.
247
The quantities of these impurities in acetic anhydride are unknown; therefore,

exposure to ketene polymers, polymers of allene (or its equilibrium mixture), and
particulate carbon or soot cannot be estimated.
2.3 Reactivity and reactions with fats and oils

On contact with water (for example, during tank washing) acetic anhydride
hydrolyses to acetic acid (SCF, 1997). Acetic anhydride acetylates free hydroxyl
groups without a catalyst, but esterification is more complete in the presence of
acids, so acetic anhydride and acetic acid could react with alcohols (for example
mono- and diglycerides) forming acetates (Wagner, 2002).

No test methods for analysing acetic anhydride in fats and oils were found in
the literature. Residues of acetic acid can be detected in cleaning water and wipe
samples via liquid chromatography with tandem mass spectrometry (LC-MS/MS)
or ion chromatography (IC). The claimed best detection limit of the IC method
with conductivity detection for acetic acid was reported to be around 0.012–
0.6 ppm (Krata et al., 2009; Hodgkins et al., 2011). The method for analysis of
trace levels of acetic acid in wastewater samples by ultra-high-performance liquid
chromatography (UHPLC) with a high sensitivity photodiode array detector
(PDA) (Nexera X2 system, SPD-M30A, HS capillary flow cell) with a limit of
detection (LOD) of 0.3 ppm and limit of quantification (LOQ) of 1.0 ppm is
described by Shimadzu (2014).
3. Biological data

Acetic anhydride readily hydrolyses to acetic acid in the presence of water. The
washing of cargo containers between transport of different cargoes, and moisture
within the edible oil are likely to transform almost all residual acetic anhydride to
acetic acid. Any traces of acetic anhydride remaining are expected to be readily
hydrolysed to acetic acid when ingested. Consequently, for the purposes of this
assessment, systemic exposure is expected to be predominantly to acetic acid.
Acetic acid is a product of normal metabolism in humans, and it is expected
to be readily absorbed, metabolized and rapidly excreted. In a study on human
248
volunteers, Smith, Jeukendrup & Ball (2007) reported that ingestion of a solution
containing carbon-13-labelled sodium acetate (120 mg/kg bw acetate) resulted
in a transient increase in plasma and urinary acetate concentrations. Plasma
acetate concentrations peaked at 60 minutes and returned to baseline levels
between 90 and 120 minutes following ingestion. Based on analysis of the breath
and urine following ingestion, approximately 80% of the administered acetic
acid is expected to be exhaled and less than 1% is expected to be excreted in
the urine. Similarly, rats exposed to radiolabelled acetic acid in the diet were
reported to excrete 50% as CO2 (EC, 2012). In dogs exposed to sodium acetate via
intravenous injection, acetic acid was rapidly eliminated with elimination half-
lives of 3.0 ± 0.5, 4.0 ± 0.3, 4.1 ± 0.4 and 5.0 ± 0.5 minutes following doses of 3,
4, 5 and 6 mmol/kg (equivalent to approximately 183, 240, 300 and 360 mg/kg),
respectively (Freundt, 1973). Elimination from the cerebrospinal fluid (CSF) is
slightly slower (after administration of 6 mmol/kg an elimination rate of 14.6 ±
5.9 minutes was recorded in dogs; Freundt, 1973).
3.2 Toxicity in experimental animals

Owing to its chemical reactivity, acetic anhydride is highly corrosive (PubChem1)
and the reported median lethal dose (LD50) values in rats range from 630 mg/
kg bw (ECHA, 2020) up to 1800 mg/kg bw (Smyth, Carpenter & Weil, 1951;
OECD, 1997). According to the rat study summarized in ECHA (2020),
symptoms reported following acute oral exposure are dyspnoea, apathy, lying
down, staggering, shaking, trembling, diarrhoea, hypertension, arched back and
general poor condition. Postmortem examinations of animals that died during
the study revealed acute dilation in the heart, hardening in the stomach lining
and bloody ulceration. Although exposure to acetic acid at high concentrations is
also irritating and corrosive, acute oral toxicity of acetic acid in rats and mice is
low with estimated LD50 values >3000 mg/kg bw (Annex 1, reference 132).
3.2.2 Short-term and long-term studies of toxicity and carcinogenicity

No short-term or long-term studies investigating the oral toxicity of acetic
anhydride were identified.
Acetic acid
JECFA (Annex 1, references 33 and 132), EFSA (2013), EC (2012) and Environment
and Climate Change Canada and Health Canada (2019) briefly summarized the
1
https://pubchem.ncbi.nlm.nih.gov/compound/Acetic-anhydride
249
results of repeated-dose oral toxicity studies in various experimental animals.

However, none of the raw data for the studies summarized by these agencies
are readily accessible and the studies were performed before Good Laboratory
Practice (GLP) and other guidelines were established. Brief summaries of the
available information are provided below.
Hemmingway & Sparrow (1942) reported on rats exposed to doses of
approximately 4200 to 4800 mg/kg bw per day sodium acetate (approximately
3074 to 3514 mg/kg bw acetate) via gavage for 14 days. The effects observed
were decreased body weight, blistering paws, reddened noses and death. Leung
& Paustenbach (1990) and the EC (2012) mentioned the results of a study by
Mori (1952), indicating that rats exposed to 4500 mg/kg bw per day acetic acid
in the diet for 30 days experience gastric lesions. However, Mori (1952) noted
that, owing to its volatility, quantifying the acetic acid ingested by the rats was
impossible. Mori (1952) reported that rats were exposed to glacial acetic acid at
a concentration of 50 cm³/kg of rice. In addition to the 30-day experiment, Mori
(1952) investigated the lesions in the stomach of albino rats maintained on diets
of rice containing glacial acetic acid. For the chronic portion of the study, five
male rats were exposed to acetic acid at a concentration of 10 cm³/kg of rice for
18 days followed by 20 cm³/kg for 157 days and finally 50 cm³/kg for up to an
additional 177 days (between 200 and 352 days in total). Four out of five of the
chronically exposed male rats showed gastric lesions. In a separate study, three
out of three female and one out of two male rats exposed to 50 cm³/kg for 166
days showed gastric lesions and five out of five female rats exposed to 50 cm³/kg
for 200 days showed gastric lesions.
Kondo et al. (2001) administered 6% acetic acid (~290 mg/kg bw per
day) to groups of six male “spontaneously hypertensive” rats via the diet using
a prepared acetic acid solution, a commercial rice vinegar product or water
(control) for 8 weeks. Changes in blood pressure, heart rate, body weight, food
and water consumption, renin, angiotensin II, aldosterone and prostaglandin
E2 levels were monitored. According to the authors, acetic acid significantly
decreased blood pressure and renin activity and no adverse effects were reported.
Environment and Climate Change Canada and Health Canada (2019) used these
results to establish a no-observed-adverse-effect level (NOAEL) of 290 mg/kg bw
per day.
The summary of the study by Wysokinska (1952; Polish language article),
provided by JECFA (Annex 1, reference 33), indicates that daily exposure of rats
by gavage to 3 mL of a 10% solution of acetic acid (equivalent to approximately
1000 mg/kg bw per day) for 90 days results in decreased haemoglobin and
erythrocyte counts in rats. Based on the dose and concentration of acetic acid
used in this gavage study, it is likely that these effects are a result of haemorrhage
from severe irritation at the site of first contact.
250
The United States Environmental Protection Agency (US EPA, 2010)

summarized the results of an unpublished 90-day repeated-dose oral toxicity test
(experimental animal species not reported). No toxicological effects regarding
mortality, clinical observations, neurotoxicity assessment, haematology, clinical
chemistry, organ weights or macroscopic or microscopic observations were
noted. Based on a decrease in body weight following exposure to 390 mg/kg bw
per day, a NOAEL of 195 mg/kg bw per day was identified. The US EPA (2010)
suggested that the reduction in weight gain was probably due to reduced appetite
and a corresponding reduction in feed consumption.
Similarly, Sollamann (1921)1 reported that rats exposed to acetic acid in
drinking water at concentrations of 0.5% (equivalent to approximately 410 mg/kg
bw per day) for 9 to 15 weeks showed reduced body weight gain, loss of appetite
and decreased food consumption (previously reviewed by the Committee in
1966/1967 (Annex 1, reference 12). Administration of acetic acid in the drinking
water at doses of between 8 and 210 mg/kg bw per day did not induce changes in
food and water consumption.
Pardoe (1952) used sodium acetate as a vehicle control for evaluating the
effects of lead acetate: male rats were exposed to 350 mg/kg bw per day sodium
acetate (approximately 256 mg/kg bw per day acetate) via gavage for 135 days.
Since the purpose of Pardoe’s (1952) study was to investigate the toxicity of lead
acetate, no control group was included as a basis for evaluating the effects of
acetic acid specifically. Nevertheless, according to the available information, no
significant effects were reported. JECFA (Annex 1, reference 132) used the results
from this study to identify a NOAEL2 of 350 mg/kg bw per day for acetic acid.
EFSA’s (2013) assessment of acetic acid briefly mentions the results of
an 8-month gavage study in rats (no original citations provided) in which rats
exposed to 150 mg/kg bw (three doses per week) demonstrated hyperplasia of the
forestomach and oesophagus without evidence of tumour formation.
Lamb & Evvard (1919) reported that dietary exposure of pigs to acetic
acid at successive doses of 0, 240, 720, 960 and 1200 mg/kg bw per day for 30-day
periods each (total of 150 days exposure) resulted in no significant differences in
growth rate, weight gain, early morning urinary ammonia and terminal blood pH
when compared to controls (previously reviewed by the Committee in 1966/1967
(Annex 1, reference 12). Based on a 6-month dietary study in pigs EFSA (2013)
identified a NOAEL of 450 mg/kg bw per day (no additional details given).
According to EFSA (2013) and Environment and Climate Change
Canada and Health Canada (2019), acetic acid shows no carcinogenic potential.
1
Note: Similar results were reported by EC (2012) and cited as: Henschler D (1973). Toxikologisch-
arbeitsmedizinische Begründungen von MAK-Werten (Maximale Arbeitsplatz-Konzentrationen), 20.
Lieferung, Essigsäure. Weinheim: VCH Verlagsgesellschaft.
2
At the time called NOEL.
251
However, Alexandrov et al. (1989) reported hyperplasia in the forestomach and

oesophagus in outbred white male rats exposed to 0.5 mL of 3% acetic acid
solution (~ 38 mg/kg bw per day assuming a body weight of 0.4 kg and 3% =
30 mg/mL), via gavage, three times per week, for 8 months.
Following co-administration with n-nitrososarcosin ethyl ester, a known
oesophageal carcinogen in rats, acetic acid promoted tumour formation in the
oesophagus (Alexandrov et al., 1989). Rotstein & Slaga (1988) reported that
although acetic acid is a very weak tumour promoter in the multistage mouse
skin model, it was very effective at enhancing tumour progression following
initiation with dimethylbenz[a]anthracene (Rotstein & Slaga, 1988).

There are no data on the reproductive or developmental toxicity of acetic anhydride
following oral exposure but there are data on the effects of inhalation exposure.
The OECD (1997) summarized the results of a developmental toxicity study in
pregnant rats exposed to vapour concentrations of 0, 25, 100 or 400 ppm acetic
anhydride for 6 hours/day during gestation days (GD) 6 to 15. Fetotoxicity (details
not specified) associated with maternal effects (severe respiratory tract irritation
and body weight reductions) were observed at 100 ppm. Based on “substantial
irritation of the respiratory tract”, a lowest-observed-adverse-effect-level (LOAEL)
for maternal effects of 25 ppm was identified. Since no fetal effects were noted at the
LOAEL, a NOAEL for developmental effects of 25 ppm was identified. The OECD
(1997) summary also mentions that following 13 weeks of exposure to 0, 1, 5 or
20 ppm acetic anhydride vapour for 6 hours/day and 5 days/week, no adverse
effects on the reproductive organs of male or female rats were observed.
Reproductive and developmental toxicity of acetic acid

Developmental toxicity following oral exposure to acetic acid has been investigated
in mice, rats and rabbits (Food and Drug Research Labs, 1974). Female CD-1
mice, Wistar rats and Dutch-belted rabbits were administered doses of 0, 16, 74.3,
345 and 1600 mg/kg bw per day apple cider vinegar (table strength 5%) by oral
gavage from GD 6 to 15 (mice and rats) or days 6 to 18 (rabbits) and euthanized
on GD 17 (mice), 20 (rats) or 29 (rabbits). In pregnant mice, the body weights of
the dams dosed with 345 and 1600 mg/kg bw per day were decreased compared
to the negative control group. Although there were no effects on fetal weights,
an increase in the number of litters containing dead fetuses and fetuses with
incomplete ossification of the sternebrae were observed following exposure of
dams to 1600 mg/kg bw per day. Consequently, a maternal NOAEL of 74.3 mg/
kg bw per day and a developmental toxicity NOAEL of 345 mg/kg bw per day
was identified. No toxicologically relevant effects or abnormalities were observed
252
in rats at any dose and a NOAEL of 1600 mg/kg bw per day for reproductive and
developmental toxicity was identified. Although no evidence of developmental
toxicity was observed in rabbits up to doses of 1600 mg/kg bw per day, maternal
toxicity (reduced body weight in animals given 1600 mg/kg bw per day) and
evidence of reproductive toxicity (decreased pregnancy rate, smaller numbers
of live fetuses, corpora lutea and implant sites, and increased resorptions) was
observed at doses as low as 74.3 mg/kg bw per day. However, given the sensitivity
of rabbits to the bactericidal effects of acetic acid on the gastrointestinal flora
(EFSA, 2013; Environment and Climate Change Canada and Health Canada,
2019), the Committee considered the data on rabbits as not relevant to human
health risk assessment.
3.2.4 Genotoxicity
Acetic anhydride has produced negative results in in vitro and in vivo genotoxicity
testing. The equivocal results reported by Seifried et al. (2006), were probably due
to the acidic nature of acetic anhydride, as it has been demonstrated that acetic
acid reduces the pH of the culture medium and produces false-positive results in
vitro (Morita et al., 1990). Table 5 summarizes the available information on the
genotoxicity of acetic anhydride.
Acetic acid genotoxicity

Acetic acid is considered non-genotoxic (Annex 1, reference 132); however, due
to its acidic nature, chromosomal aberrations have been observed in vitro when
the pH/osmolality of the culture medium is not controlled. Acidic substances
that decrease the pH of the culture medium have been shown to induce false-
positive results in in vitro systems (Morita, Takeda & Okumura, 1990). Table 6
summarizes the available information on the genotoxicity of acetic acid.
3.2.5 Allergenicity
The Committee did not identify any studies in animals that indicated that acetic
anhydride elicits an allergic response upon oral exposure. There are also no data
available that indicate that it contains a known food allergen. However, there is
information on humans that indicates that acetic anhydride and acetic acid are
capable of inducing allergic reactions (see section 3.3).
3.2.6 Impurities
Some of the impurities identified in section 2.2 are reactive and potentially
genotoxic (for example, diketene).1
1
As determined using the online Toxtree software: https://apps.ideaconsult.net/data/ui/toxtree
253
Table 5
Summary of some in vitro and in vivo studies on the genotoxicity of acetic anhydride
Test system and testing conditions Treatment Result Reference
Gene mutation in Salmonella Typhimurium Not reported Negative (S9− and S9+) OECD (1997)
(TA98, TA1537, TA1538, TA100, TA1535) Cytotoxic: 0.1 µL/plate (S9+); 1.0 µL/
plate (S9−)
Gene mutation in Salmonella Typhimurium 8 to 5000 µg/plate Negative (S9− and S9+) OECD (1997) Seifried et
(TA98, TA1537, TA1538, TA100, TA1535) Cytotoxic: 3333 µg/plate (S9+); 667 µg/ al. (2006)
plate (S9−)
Gene mutation in Salmonella Typhimurium 3.3 to 1000 µg/plate Negative (S9− and S9+) Mortelmans et al. (1986)
(TA98, TA1537, TA100, TA1535) Cytotoxic: 900 µg/plate (S9+); 190 µg/ as cited in OECD (1997)
plate (S9−)
Gene mutation in Salmonella Typhimurium Not reported Negative (S9− and S9+) McMahon, Cline &
(G46, C3076, D3052, TA98, TA1537, TA1538, Thompson (1979)
TA100) and Escherischia coli (WP2 and
WP2 uvrA)
Gene mutation in mouse lymphoma cells 0.04 to 0.3 µL/mLa Negative (S9+) OECD (1997) Seifried et
(L5178Y TK+/−) Cytotoxic: 0.05 µL/mL (S9+); 0.1 µL/ Equivocal (S9−) al. (2006)
mL (S9−)
In vivo rat micronucleus test following 13 0, 1, 5 or 20 ppm for 6 hrs/day, 5 days/ Negative OECD (1997)
weeks of inhalation exposure week for 13 weeks
a
Seifried et al. (2008) corrected the units reported in Seifried et al. (2006) for the results of the mouse lymphoma assay.
Table 6
Summary of some in vitro genetic toxicity studies on acetic acid
Gene mutation in Salmonella Typhimurium 100–6666 µg/plate Negative (S9− and S9+) Zeiger (1992)
(TA98, TA97, TA100, TA1535)
Sister chromatid exchange in human 2.5–10 mM (~150–601 mg/L) Positive (S9−)a Morita, Takeda &
lymphocytes Okumura (1990)
Chromosome aberration in Chinese 4–14 mM (~240–841 mg/L) Positive (S9− and S9+) Mohammadzadeh-
hamster ovary K1 cells 10–30 mM (~601–1802 mg/L) Positive (S9− and S9+)b Aghdash et al. (2018)
DNA fragmentation in human umbilical 487.71 µM sodium acetate (IC50) No fragmentation Abd-Elhakim et al.
vein endothelial cells (2018)

Comet assay with male rat peripheral 50, 100 and 200 mmol/L Positive OECD (1997) Seifried et
lymphocytes al. (2006)
a
Only a slight increase was observed above 5 mM, which was correlated with a decrease in pH of the culture medium.
b
Positive results were only observed at the highest concentrations evaluated in the absence and presence of S9 after buffering the culture medium and the authors
concluded that the clastogenic effect of acetic acid in cultured cells is attributable to acidification of the culture medium.

Acute exposure of humans to acetic anhydride causes severe eye, skin and
respiratory tract irritation, and death at high concentrations (Sinclair, 1994;
OECD, 1997). Additionally, Yokota, Takeshita & Morimoto (1999) reported that
254
amines and acetic anhydrides are capable of inducing both IgE-mediated asthma
and IgG-mediated late respiratory responses in sensitized chemical workers.
EFSA (2012) suggested that the hypersensitivity reactions reported in sensitized
workers may be due to acetic anhydride’s ability to react with amino acids and
potentially form haptens. However, EFSA (2012) concluded that there is currently
no indication that acetic anhydride is an allergen or an adjuvant when present in
liquid, such as when used as a previous cargo.
Acetic acid
Acetic acid is highly irritating to the skin and mucous membranes. Leung &
Paustenbach (1990) reported blackening and hyperkeratosis of the skin of the
hands, conjunctivitis, pharyngitis and erosion of the exposed teeth in workers
exposed to acetic acid vapour at concentrations of 80 to 200 ppm over 7 to 12
years. Furthermore, the German Senate Commission for the Investigation of
Health Hazards of Chemical Compounds in the Work Area (German MAK
Commission, 2011) and EC (2012) reported that accidental ingestion of 20 and
50 g (equivalent to approximately 286 to 714 mg/kg bw) of concentrated acetic
acid is lethal in humans, with survivors showing signs of oesophageal constriction.
EFSA (2013) suggested that adverse effects in humans following exposure to
acetic acid are related to the corrosive action of acetic acid at high concentrations
(for example, ulceration of skin and mucous membranes, gastrointestinal and/or
respiratory tract damage) and that the sensitization potential appears to be low
based on human experience. The EC (2012) suggest that, based on occupational
exposures, individuals may become accustomed to the irritating effects of acetic
acid over time.
Boehncke & Gall (1996) reported a case of a type I hypersensitivity-
like reaction in a 68-year-old individual who, shortly following consumption of
ethanol (for example, from medication, beer, wine or other alcoholic beverages)
or salad dressings containing acetic acid, consumed on separate occasions,
experienced conjunctivitis, angioedema, dyspnoea and urticarial rashes. The
authors observed that the patient’s serum IgE was significantly elevated (327 U/
mL versus <150 U/mL) and that following a standardized skin prick test, positive
reactions were noted for acetic acid concentrations ranging from 9.6 to 1.2% but
not for ethanol. An extensive list of other possible allergens all produced negative
results in skin prick testing.1 To rule out the possibility of irritation, skin prick
testing with acetic acid at the same concentrations was conducted in five healthy
volunteers. None of the five volunteers showed positive reactions to acetic acid.
1
i.e. Acetaldehyde, several seasonal and perennial allergens, meat, fish, crustaceans, fruits, vegetables,
nuts, cereals (malt, barley, rice, rye, wheat, as well as hop and brewer's yeast), food preservatives (sodium
salicylate 5%, hydroxybenzoic acid 1%; both diluted in distilled water and in pharmaceutical quality),
tartrazine, and several brands of beer and red and white wine.
255
Przybilla & Ring (1983) reported the case of a healthy 22-year-old woman who
experienced generalized itching, facial flushing, dizziness, abdominal pain,
numbness in the mouth, and dyspnoea and collapse on several occasions after
drinking wine, beer, rum or vinegar. Skin prick tests were negative for ethanol,
wine and beer; however, positive results were obtained for vinegar and acetic acid
(9.6 and 0.96%). In contrast, skin prick tests for 10 healthy volunteers were negative
for acetic acid. Following oral challenge with 1 mL of ethanol or 50 mL of beer the
patient experienced urticaria, facial flushing, itching of the mucous membranes,
hoarseness, dyspnoea, tachycardia and painful uterine cramps. Serum IgE levels
in this patient were significantly elevated (i.e. 690 kU/L). Boehncke & Gall (1996)
proposed that, owing to its size, acetic acid itself is unlikely to function directly as
an antigen, but more likely as a hapten.
Kondo et al. (2009) conducted a parallel-group, randomized, double-
blind, placebo-controlled trial in a cohort of 175 obese Japanese people aged 25
to 60 years to investigate the effects of acetic acid on body fat mass. Following
exposure to 0, 15 or 30 mL of apple vinegar (equivalent to 0, 750 or 1500 mg
acetic acid) for 12 weeks, Kondo et al. (2009) reported decreased body weight,
body mass index, visceral fat area, waist circumference and serum triglyceride
levels in both treatment groups compared to controls. Additionally, the high-
dose group showed significantly lower systolic blood pressure at the 12-week
measurement. EFSA (2011) reported decreased systolic blood pressure in
another Japanese study with 750 mg/kg bw per day acetic acid following 2 weeks
of consumption (original article in Japanese). According to the US National
Library of Medicine’s clinical trials database, a 12-week trial1 in pre-hypertensive
Americans aged 30–65 years has been planned to investigate the blood pressure
lowering effects of a fruit-flavoured beverage containing diluted Mizkan rice
vinegar with and without 750 mg acetic acid. Although there appears to be some
evidence for the blood pressure lowering effects of acetic acid in animals (Kondo
et al., 2001) and humans, EFSA (2011) concluded that a sustained effect on blood
pressure is unlikely since acetic acid is rapidly absorbed and excreted following
consumption.
4. Occurrence and exposure

Acetic anhydride is approved in some countries as an acetylation agent
for use in the preparation of modified food starches and acetylated mono-
glycerides. Acetic anhydride is rapidly hydrolysed to acetic acid, a constituent
1
https://clinicaltrials.gov/ct2/show/study/NCT03596099
256
present naturally in vinegar (at up to 15%) and other foods (EFSA, 2018).
Environment and Climate Change Canada and Health Canada (2017) determined
that the dietary exposure to acetic anhydride from food additive uses, if any, is
likely to be negligible.
Acetic anhydride may also be used in the manufacture of paper food
packaging or paper trays. However, Environment and Climate Change Canada
and Health Canada (2017) determined that exposure to acetic anhydride from
packaging uses is not expected since there are negligible residual levels in the
finished packaging materials.
No data were found on concentrations of acetic anhydride in food oils due
to carryover from previous cargoes. A worst-case concentration of 100 mg/kg
has been assumed for all previous cargo substances.  Worst-case exposures to
previous cargo substances in food oils have been estimated at 0.3 mg/kg bw per
day, based on a worst-case concentration of 100 mg/kg and an oil intake of 3 g/
kg bw per day by infants and young children who are high consumers (see section
A4.3).   It is not expected that exposure to acetic acid present due to hydrolysis of
acetic anhydride in carryover from previous cargoes would add significantly to
total acetic acid exposures, estimated at 2.1 g/day (equivalent to 35 mg/kg bw per
day for adults) based on Life Sciences Research Office (LSRO) data (LSRO, 1977).
5. Comments
5.1 Chemical and technical considerations

The chemical and technical considerations for acetic anhydride are summarized
in Table 7.

Acetic anhydride readily hydrolyses to acetic acid in the presence of water. The
washing of containers after transporting a cargo, and moisture within the edible
oil is likely to transform almost all residual acetic anhydride to acetic acid. Any
traces of acetic anhydride remaining are expected to be readily hydrolysed to
acetic acid when ingested. Acetic acid is a product of normal metabolism in
humans. It is expected to be readily absorbed, metabolized and rapidly excreted
(Freundt, 1973; Smith, Jeukendrup & Ball, 2007).
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Table 5
Chemical and technical considerations for acetic anhydride
Name: Acetic anhydride (ethanoic anhydride)
CAS number Alternative CAS numbers
108-24-7 None
Chemical details Acetic anhydride; acetyl acetate; acetic acid, 1,1’-anhydride; ethanoyl ethanoate; acetic acid anhydride;
acetyl acetate; acetyl oxide; acetic oxide
Colourless liquid with a strong, pungent, vinegar-like odour
H2C CH3
Molar mass: 102.09 g/mol

Melting point: −73.1 °C
Boiling point: 139.5 °C
Soluble in water. Hydrolyses to acetic acid in aqueous solution

Route(s) of synthesis Manufactured by different processes: 1) acetic acid process (ketene process) from acetic acid to form a
ketene and the further reaction of the ketene with acetic acid; 2) acetaldehyde oxidation process; and 3)
carbonylation of methyl acetate process.
Composition Technical quality acetic anhydride (max. 97%) often contains colour bodies, heavy metals, phosphorus
and sulfur compounds. Acetic anhydride manufactured by the ketene process sometimes contains ketene
polymers, e.g. acetylacetone, diketene, dehydroacetic acid, and particulate carbon or soot. Polymers of
allene, or its equilibrium mixture, methylacetylene-allene, are reactive impurities which slowly autoxidize
to peroxides if exposed to air.
Uses The primary industrial application of acetic anhydride is for acetylation reactions in the production
of cellulose acetates. Acetic anhydride is used in the manufacture of fibres, plastics, pharmaceuticals,
dyes and explosives. In food applications it is used in the starch industry as an acetylation compound in
production of modified starches.
Analytical methods None reported for previous cargoes. Residues of acetic acid can be detected in cleaning water and wipe
samples with LC-MS/MS, IC or UHPLC.
Potential reaction(s) with a On contact with water (e.g. during tank washing) acetic anhydride hydrolyses to acetic acid. Acetic
subsequent cargo of fat or oil anhydride acetylates free hydroxyl groups without a catalyst, but esterification is more complete in the
presence of acids; acetic anhydride and acetic acid could react with alcohols (for example mono- and
diglycerides) forming acetates.
LC-MS/MS, liquid chromatography–tandem mass spectrometry; IC, ion chromatography; UHPLC, ultra-high-performance liquid chromatography.

Since ingested acetic anhydride is highly corrosive to the mucous membrane
of the gastrointestinal tract, the available toxicity database for oral toxicity in
laboratory animals is restricted. The reported oral median lethal dose (LD50) in
rats ranges from 630 mg/kg bw (ECHA, 2020) up to 1800 mg/kg bw (OECD, 1997).
Acetic acid is highly irritating to the skin and mucous membranes and following
ingestion, early signs of toxicity can be attributed to its irritating properties at
high concentrations. The Committee had previously noted no effects at a dose of
350 mg/kg bw per day acetic acid from a study in male rats administered sodium
258
acetate via oral gavage for 63 days (Annex 1, reference 132; Pardoe, 1952). In
a developmental toxicity study in mice, a maternal NOAEL of 74.3 mg acetic
acid/kg bw per day (based on decreased body weight gain at 345 mg/kg bw per
day) and a developmental toxicity NOAEL of 345 mg acetic acid/kg bw per day
were identified (based on increases in the number of litters containing dead
fetuses and incomplete ossification at 1600 mg/kg bw per day). No evidence of
developmental toxicity was observed in rats up to doses of 1600 mg acetic acid/
kg bw per day (Food and Drug Research Labs, 1974).
The Committee concluded that the available in vitro and in vivo
information on acetic anhydride and acetic acid does not raise concerns for
genotoxicity. Although acetic acid has been associated with tumour promotion
(Rotstein & Slaga, 1988; Alexandrov et al., 1989), the Committee considered that
the tumour promoting and tumour progression effects reported are likely to be
the result of the site-of-contact irritating/cytotoxic potential of acetic acid at high
concentrations and would not be of concern at the low concentrations that occur
in fats or oils following its transport as a previous cargo.
5.4 Allergenicity
The Committee concluded that, considering the widespread presence of acetic
acid in the diet, it is unlikely that acetic anhydride present in low concentrations
such as when transported as a previous cargo will produce an allergic response.
5.5 Impurities
The Committee noted that fats and oils for human consumption have to conform
to the maximum levels for heavy metals set out in the Codex General Standard
for Contaminants and Toxins in Food and Feed, CXS-193-1995, and it is therefore
not necessary to estimate exposures to heavy metals in food oils due to carryover
from acetic anhydride previous cargoes. Additional impurities in acetic
anhydride, depending on the method of manufacture, include ketene polymers
(for example, acetylacetone, diketene and dehydroacetic acid), polymers of allene
or its equilibrium mixture, methylacetylene-allene and particulate carbon or soot.
The quantities of these impurities in acetic anhydride are unknown; exposure to
ketene polymers, polymers of allene (or its equilibrium mixture) and particulate
carbon or soot, therefore, cannot be estimated.
259
5.6 Assessment of dietary exposure

Acetic anhydride is approved in some countries as an acetylation agent
for use in the preparation of modified food starches and acetylated mono-
glycerides. Acetic anhydride is rapidly hydrolysed to acetic acid, a constituent
present naturally in vinegar (at up to 15%) and other foods (EFSA, 2018).
Environment and Climate Change Canada and Health Canada (2017) determined
that the dietary exposure to acetic anhydride from food additive uses, if any, is
likely to be negligible.
Acetic anhydride may also be used in the manufacture of paper food
packaging or paper trays. However, Environment and Climate Change Canada
and Health Canada (2017) determined that exposure to acetic anhydride from
packaging uses is not expected due to negligible residual levels in the finished
packaging materials.
No data were found on concentrations of acetic anhydride in food
oils due to carryover from previous cargoes. A worst-case concentration of
100 mg/kg has been assumed for all previous cargo substances.  Worst-case
exposures to previous cargo substances in food oils have been estimated at
0.3 mg/kg bw per day, based on a worst-case concentration of 100 mg/kg and
an oil intake of 3 g/kg bw per day by infants and young children who are high
consumers (see section 3.4.3 Occurrence and exposure). It is not expected that
exposure to acetic acid present due to hydrolysis of acetic anhydride in carryover
from previous cargoes would add significantly to total acetic acid exposures,
estimated at 2.1 g/day (equivalent to 35 mg/kg bw per day for adults) based on
Life Sciences Research Office (LSRO) (1977) data.
6. Evaluation
No information regarding the short-term and long-term toxicity of acetic
anhydride was identified. However, upon evaluation of the available information,

the Committee noted that it had previously allocated a group ADI “not specified”
to acetic anhydride’s immediate hydrolysis product, i.e. acetic acid and its
potassium and sodium salts (Annex 1, reference 32). Since acetic anhydride is
anticipated to be rapidly hydrolysed to acetic acid during tank washing, within
the edible oil cargo and after ingestion, the group ADI “not specified” for acetic
acid and its potassium and sodium salts is considered directly relevant for this
assessment of acetic anhydride. The US National Research Council estimated
that mean exposure to acetic acid from all food sources is 2.1 g/day for persons
above 2 years of age (LSRO, 1977), which is equivalent to 35 mg/kg bw per day for
adults based on a body weight of 60 kg. It is not expected that exposure to acetic
260
acid present due to hydrolysis of acetic anhydride in carryover from previous

cargoes would add significantly to total exposures to acetic acid. Therefore, acetic
anhydride at the generic human dietary exposure value for previous cargoes of
0.3 mg/kg bw per day would only contribute marginally to the overall dietary
exposure to acetic acid and is not expected to result in adverse effects on human
health.
The Committee concluded that considering the widespread presence
of acetic acid in the diet, it is unlikely that acetic anhydride present in low
concentrations such as when transported as a previous cargo will produce an
allergic response.
Acetic anhydride acetylates free hydroxyl groups without a catalyst,
but esterification is more complete in the presence of acids, so acetic anhydride
and acetic acid could react with alcohols (for example mono- and diglycerides)
forming acetates (Wagner, 2002). Reaction rates are likely to be slow at ambient
temperature.
Although exposure to acetic anhydride and acetic acid as a result
of transporting acetic anhydride as a previous cargo does not appear to be a
health concern, there is uncertainty concerning the purity or “grade” of acetic
anhydride that is transported as a previous cargo. Since acetic anhydride may
contain impurities (for example, diketene and dehydroacetic acid), which are
potentially genotoxic, the Committee could not reach a conclusion on the safety
of transporting acetic anhydride as a previous cargo for edible fats and oils until
the nature and quantities of these impurities have been clarified.
II. sec-Butyl acetate
1. Explanation
The Committee had not previously evaluated sec-butyl acetate; however, at its
eleventh and forty-ninth meetings, in 1968 and 1999, it evaluated one of its
isomers, n-butyl acetate (Annex 1, references 14 and 131). SCF (1997) considered
sec-butyl acetate acceptable as a previous cargo for edible fats and oils primarily
on the basis that it is easily removed by tank cleaning. More recently, the EFSA
(2012a) Panel on Contaminants in the Food Chain (CONTAM Panel) concluded
that sec-butyl acetate meets the criteria for acceptability as a previous cargo
for edible fats and oils on the basis that the metabolites (i.e. acetic acid and
2-butanone) were previously assessed by the Panel as acceptable previous cargoes
for edible fats and oils. In addition, the primary metabolite, sec-butanol, was not
261
found to be genotoxic and did not represent a toxicological concern at exposure

levels that might occur in fats and oils from the transport of sec-butyl acetate as
a previous cargo.
For the present assessment, previous assessments (monographs)
completed by JECFA, EFSA, SCF, the WHO International Programme on
Chemical Safety (WHO/IPCS), and national and regional governmental
authorities were identified by searching their respective websites. This was
followed by a comprehensive search to identify any critical new data for the
assessment of human health risk on PubMed and PubChem. The search terms
used were sec-butyl acetate and synonyms (for example, 2-butyl acetate), CAS
number (105-46-4) and toxicity. Given the paucity of relevant information
concerning the oral toxicity of sec-butyl acetate, secondary searches for relevant
information were performed on the metabolites of sec-butyl acetate (i.e. acetic
acid, sec-butanol and 2-butanone) in an effort to supplement the toxicological
information on this compound. The cut-off date for inclusion in this report was 4
January 2021. The data concerning acetic acid are summarized in section I.

The physicochemical characteristics of sec-butyl acetate are listed in Table 8.
2.1 Manufacture and uses of sec-butyl acetate

The most common process for manufacturing sec-butyl acetate is esterification of
acetic acid with sec-butanol using sulfuric acid as a catalyst. The removal of water
by azeotropic separation forces the reaction to completion. Common azeotropic
agents are cyclohexane, benzene and toluene. The acid catalyst is neutralized and
the ester purified by distillation (Lewis, 2007; Cheung at al., 2012). Esterification
of sec-butanol with acetic anhydride offers an alternative route for producing sec-
butyl acetate. Owing to its high reactivity, acetic anhydride readily forms esters
with alcohols. The reaction is irreversible and the esterification goes to completion
without eliminating products or water (O'Neil, 2001; Cheung at al., 2012). sec-
Butyl acetate can also be prepared from 2-butene under pressure and heated with
an excess of glacial acetic acid containing 10% sulfuric acid. Instead of sulfuric
acid, boron trifluoride-diethyl ether may be used as a catalyst (Cheung at al.,
2012).
sec-Butyl acetate is used as a solvent in nitrocellulose lacquers,
thinners, enamels, leather finishes, acyclic polymers and vinyl resins (Cheung
at al., 2012; PubChem online). In the EU, sec-butyl acetate is approved for use
262
Table 8
Physical and chemical characteristics of sec-butyl acetate
Chemical name Butyl acetate, sec-; sec-butyl acetate
Synonyms Butan-2-yl acetate; 2-butyl acetate; sec-butyl ethanoate
CAS number 105-46-4
Chemical structure CH3
CH3
H3C
mass
Description Colourless liquid with a fruity scent. It produces irritating vapour. Liquid and vapour are highly flammable
Melting point −99 °C
Boiling point 112 °C
Flash point 17 °C (closed cup)
Solubility Slightly soluble in water, soluble in ethanol and diethyl ether
as a flavouring substance for food (FL No. 09.323) (Regulation (EC) 1334/2008;
Commission Implementing Regulation (EU) No 872/2012).

sec-Butyl acetate may contain acetic acid up to 0.2% as an impurity (Kodak, 1985).

Hydrolysis of sec-butyl acetate results in formation of acetic acid and sec-
butanol, which in the presence of acid may undergo transesterification with
lipids producing a mixture of glycerol and fatty acid sec-butyl esters. However,
these reactions are slow and require excess alcohol and temperatures above
100 °C (Schuchardt et al., 1998).

No test methods for analysing sec-butyl acetate in fats and oils were found in the
literature. sec-Butyl acetate can be analysed by gas chromatography with flame
ionization detection (GC-FID) or GC-MS (PubChem, SciFinder – CAS online
databases).
263
3. Biological data

There are no studies on the biochemical aspects of sec-butyl acetate following oral
exposure and no physiologically-based pharmacokinetic models were identified.
However, based on its physicochemical properties (molecular weight = 116.16 g/
mol; log n-octanol/water partition coefficient (Kow) = 1.72; PubChem), sec-butyl
acetate is expected to be readily absorbed into the systemic circulation following
oral exposure. Once absorbed, sec-butyl acetate is expected to be rapidly
hydrolysed (within minutes) to acetic acid and sec-butanol in the blood, liver,
small intestine and respiratory tract (WHO/IPCS, 2005). Sec-butanol is then
expected to undergo rapid metabolism by alcohol dehydrogenase primarily to
2-butanone and to be excreted either by exhalation or in the urine, or to undergo
further metabolism to 3-hydroxy-2-butanone and 2,3-butanediol (Traiger &
Bruckner, 1976; Dietz et al., 1981; WHO/IPCS, 1987). Peng et al. (1995) also
demonstrated that in vitro, sec-butyl acetate undergoes hydroxylation to an
unstable hemiketal (2-hydroxy-2-acetoxybutane) followed by a non-hydrolytic
cleavage to 2-butanone by cytochrome P450 2B4.
Dietz et al. (1981) demonstrated that the concentrations of 2-butanone
and its metabolites in rats orally dosed with sec-butanol are comparable to
concentrations obtained after oral dosing with 2-butanone. Therefore, it is expected
that sec-butanol and 2-butanone will exhibit similar toxicological profiles.

Sec-Butyl acetate exhibits low acute oral toxicity with a reported oral LD50 of
3200 mg/kg bw in rats (WHO/IPCS, 2005). According to a US National Institute
for Occupational Safety and Health Pocket guide to chemical hazards,1 symptoms
possibly induced by sec-butyl acetate include eye irritation, headache, drowsiness,
upper airway dryness, dry skin and narcosis.
Sec-butanol/2-butanone
Similarly, sec-butanol exhibits low acute oral toxicity, with reported oral LD50
values of 2193 mg/kg bw in rats and 6480 mg/kg bw in rabbits (ECETOC, 2003).
According to the German MAK Commission,2 acute exposure to sec-butanol
1
https://www.cdc.gov/niosh/npg/npgd0073.html
2
https://onlinelibrary.wiley.com/doi/full/10.1002/3527600418.mb7892e0019
264
(all routes) leads to central nervous system depression and typical symptoms of
acute alcohol intoxication/narcosis. The severity of intoxication is greater for sec-
butanol than for ethanol. 2-Butanone also exhibits low acute oral toxicity in mice
and rats, with reported LD50 values of > 2000 mg/kg bw (US EPA, 2003).

No relevant oral toxicity studies were identified for sec-butyl acetate, sec-butanol
or 2-butanone.

No reproductive or developmental toxicity data were identified for sec-butyl
acetate. However, some toxicological information is available on the reproductive
and developmental toxicity of sec-butanol and 2-butanone.
Sec-butanol
A two-generation reproductive and developmental oral toxicity study with
the primary metabolite sec-butanol was conducted in rats (Cox et al., 1975, as
cited in US EPA, 2003a; unpublished study). In the US EPA (2003a) evaluation
of the original study, various deficiencies were highlighted (for example, lack
of measurements of estrous cyclicity, sperm parameters, weights of uterus,
epididymides, seminal vesicles and brain; and incomplete clinical chemistry,
haematology and histopathology). Nevertheless, this study was chosen as the
critical study in the derivation of the US EPA (2003a) reference dose (RfD) for
2-butanone. Since a copy of the original study was not available for the present
assessment, details of the US EPA (2003a) evaluation are provided below.
Weanling FDRL-Wistar stock male and female rats (F0; 30 per sex and
group) were exposed to sec-butanol at concentrations of 0, 0.3, 1.0 or 3.0% for
8 weeks in drinking water prior to mating, and during gestation of two separate
F1 generations. The doses received by the F0 generation up to day 10 after
birth (postnatal day (PND) 10) of the F1A pups were reported as 538, 1644 or
5089 mg/kg bw per day in males and 594, 1771 and 4571 mg/kg bw per day
in females. Maternal toxicity (reduced body weight gain) accompanied by
developmental effects in the F1A pups (increased fetal mortality and reduced
fetal and pup body weights) were reported following exposure to the highest
concentration in drinking water (3% solution; doses of 5089 mg/kg bw per day in
males and 4571 mg/kg bw per day in females). Therefore, F0 generation parents
in the high-dose group and F1A offspring were given drinking water instead of
sec-butanol between PND 10 and PND 21 of lactation. Dosing was resumed
after PND 21 at a lower concentration (2.0% instead of 3.0%) for 2 weeks post-
lactation. The estimated average daily intake was 3384 mg/kg bw per day for
265
males and 3122 mg/kg bw per day for females. Dosing at the other drinking water
concentrations (0.3 and 1.0%) continued as per the experimental protocol. Two
weeks post-lactation of F1A, the F0 generation was re-mated to produce a second
set of first-generation offspring (F1B). The pregnancies of 20 dams were terminated
on gestational day 20 and the F1B fetuses were evaluated for reproductive and
developmental toxicity. Dosing continued for selected F1A pups (8 pups per litter
and dose) from all dose groups postweaning, at drinking water concentrations
of 0, 0.3, 1.0 or 2.0% sec-butanol. At 12 weeks of age F1A animals were mated to
produce the second generation (F2) litters. On PND 21, the F1A parents and F2
offspring were sacrificed.
A reduction in body weight gain (~15%) was reported in the F0 generation
at the highest dose (3.0% concentration) compared to controls. There was also an
increased incidence of the number of F0 male rats that failed to copulate with F0
females (0% (1/30), 0.3% (2/30), 1% (0/30), and 3% (6/30) in the highest dose
group (3.0%) compared to controls (20% versus 3%). The US EPA analysed the
data for F0 male copulatory success rate, which suggested the highest dose (3%) of
sec-butanol had a possible impact on male performance. Data were not available
to determine the copulatory failure rate for the other generations in this study. No
other toxicologically relevant adverse effects on reproductive parameters were
noted in the F0 generation.
Adverse developmental effects observed in the F1A and F1B offspring from
the F0 generation consisted of:
■■ increased early and late fetal death at the highest dose (3.0%) for the
F1A generation when compared to controls;
■■ a significant reduction in F1A pup body weights at the highest dose
(3.0%) on PND 4 (~22%) and PND 21 (~39%) and at the 1.0% dose
on PND 21 compared to controls; and
■■ decreased F1B fetal weight (10%; not statistically significant) at the
2.0% dose with a dose-related trend.
No toxicologically significant increases in the incidence of skeletal

malformations were noted in the F1B offspring. Toxicologically relevant
histopathological findings in the adult F1A generation after approximately
23 weeks (for example, 3 weeks in utero, 3 weeks indirectly via lactation, 9 weeks
maturation, 2 weeks mating, 3 weeks gestation, 3 weeks weaning) of exposure
were limited to abnormalities in the kidneys (i.e. tubular cast formation and foci
of tubular degeneration and regeneration) of males from all dose groups. Similar
tubular effects were not observed in females. Similar to the F1A pups, reduced
body weights were reported in the F2 offspring from the highest dose group
(2.0%) on PND 4 (5%) and 21 (13%) compared to controls. No body weight
266
changes for F2 pups in the other dose groups (0.3% and 1.0%) were observed.
A NOAEL of 594 mg/kg bw per day (0.3% sec-butanol) was identified based on
decreased pup body weight in the F1A generation at a dose of 1771 mg/kg bw per
day (1% sec-butanol). Based on the decreased body weights measured in F1A pups
on PND 21, the US EPA (2003a) calculated a lower 95% confidence limit on the
benchmark dose for a 5% response (BMDL05) of 657 mg/kg bw per day using US
EPA’s Benchmark Dose Software (BMDS, version 1.3.1).
In another developmental toxicity study, reduced fetal weights were also
observed in the offspring of rats following inhalation exposure of the dams to
maternally toxic doses of sec-butanol (Nelson et al., 1989). In this study, pregnant
Sprague-Dawley rats (15 per group) were exposed (whole body) to concentrations
of 0, 3500, 5000 or 7000 ppm sec-butanol via the inhalation route for 7 hours per
day from gestational days 1 to 19. Maternal toxicity was observed at all doses
based on reduced food intake and decreased body weight gain. Exposure of dams
to concentrations of 5000 ppm and 7000 ppm led to significantly reduced fetal
body weights. A significant increase in resorptions per litter, decreased number of
live fetuses per litter, and an increased number of fetuses with skeletal variations
were also observed in the offspring of dams exposed to 7000 ppm. The study
authors described the skeletal variations as typical of fetotoxicity, in particular
reduced ossification. A no-observed-adverse-effect-concentration (NOAEC) for
developmental toxicity of 3500 ppm was identified.
2-Butanone
No relevant reproductive or developmental oral toxicity studies on 2-butanone
were identified. However, developmental toxicity in rats and mice has been
observed following inhalation exposure to 2-butanone. Inhalation exposure
(whole body) of pregnant Sprague-Dawley rats to approximately 3000 ppm
2-butanone, for 7 hours per day during gestational days 6 to 15 resulted in
decreased weight gain in dams and increased incidence of skeletal variations
in offspring (Deacon et al., 1981). In Swiss CD-1 mice, inhalation exposure
(whole body) to approximately 3000 ppm 2-butanone for 7 hours per day during
gestational days 6 to 15 led to increased relative liver weights in the dams, a small
but statistically significant decrease in male fetal body weights, and an increased
incidence of misaligned sternebrae (Schwetz et al., 1991). In neither study were
adverse effects on dams and development observed at concentrations of 400
and 1000 ppm 2-butanone. Based on the results of these studies, a NOAEC of
1000 ppm for maternal and developmental toxicity in rats and mice was identified.
267
Table 9
Summary of selected in vitro genotoxicity studies on sec-butanol
Studies based on in vitro systems
Gene mutation in Salmonella Typhimurium (TA98, TA100, Up to 10 000 µg/plate Negative (S9− and S9+a) ECETOC (2003)
TA1535, TA1537, TA1538) Plate incorporation
Gene mutation in Salmonella Typhimurium (TA98, TA100, Up to 4000 µg/plate Negative (S9− and S9+a) Brooks, Meyer & Hutson
TA1535, TA1537, TA1538) and Escherichia coli (WP2uvrA/ Plate incorporation (1988)
pKM 101)
Gene mutation in Saccharomyces cerevisiae JD1 10–5000 µg/mL Negative (S9− and S9+a) Brooks, Meyer & Hutson
(1988)
Chromosome aberration in Chinese hamster ovary cell line Up to 5000 µg/mL Negative (S9− and S9+) Brooks, Meyer & Hutson
(1988)
a
Aroclor 1254-induced rat liver S9 mix.
3.2.4 Genotoxicity
No studies on the genotoxicity of sec-butyl acetate were identified. Sec-butanol
did not raise concerns for genotoxicity based on the available information from
in vitro studies (Table 9). There is some evidence that 2-butanone (Table 10)
elicits aneuploidy in yeast assays (not part of the standard battery of genotoxicity
tests listed in WHO/IPCS, 2020) without metabolic activation. However, the
findings were inconsistent with other studies evaluating similar end-points using
the standard battery of in vitro and in vivo genotoxicity tests. The Committee
concluded that based on the available in vitro and in vivo information, sec-butyl
acetate does not raise concerns for genotoxicity.
3.2.5 Allergenicity
There were no studies identified on the allergenic potential of sec-butyl acetate.
Sec-butanol and 2-butanone did not induce skin sensitization reactions in the
guinea-pig maximization tests (ECETOC 2003; ECHA, 2020a,b).
3.2.6 Impurities
No impurities of concern were identified.

No data on the effects of sec-butyl acetate on humans were identified.
268
Table 10
Summary of selected in vitro and in vivo genotoxicity studies on 2-butanone
Gene mutation in Salmonella Typhimurium (TA102) Up to 5000 µg/plate Negative (S9− and S9+) Jung et al. (1992)
Gene mutation in Salmonella Typhimurium (TA98, TA100, 0.05–32 µL/plate Negative (S9− and S9+) O’Donoghue et al. (1988)
TA1535, TA1537, TA1538)
Gene mutation in Salmonella Typhimurium (TA97, TA98, 100–10 000 µg/plate Negative (S9− and S9+) Zeiger et al. (1992)
TA100, TA1535, TA1537)
Gene mutation in Escherichia coli (WP2 and WP2uvrA) 31.25–4000 µg/plate Negative (S9− and S9+) ATSDR (2020)
Gene mutation in Saccharomyces cerevisiae 10–5000 µg/plate Negative (S9− and S9+) ATSDR (2020)
Induction of mitotic aneuploidy in Saccharomyces cerevisiae 3.54% Positive (S9−) WHO/IPCS (1993)
Zimmermann et al.
(1985)
Chromosomal aberrations in rat liver cells (RL4) 250–1000 µg/mL Negative (S9−) ATSDR (2020)
No data (S9+)
Unscheduled DNA synthesis in rat hepatocytes 0.1–5.0 µL/mL Negative (S9−) O’Donoghue et al. (1988)
No data (S9+)
Morphological transformation in BALB/3T3 9–18 µL/mL Negative (S9−) O’Donoghue et al. (1988)
No data (S9+)
Gene mutation in mouse lymphoma cells (L5178Y TK+/−) 0.67–12 µL/mL Negative (S9− and S9+) O’Donoghue et al. (1988)
Micronucleus frequency in V79 Chinese hamster fibroblasts 280 mM Negative (S9−) Kreja & Seidel (2002)
(32 525 µg/mL) No data (S9+)
DNA damage (Comet assay) in V79 Chinese hamster 112 and 560 mM Negative (S9−) Kreja & Seidel (2002)
fibroblasts (13 009 and 65 050 No data (S9+)
µg/mL)
DNA damage (Comet assay) in A549 cells 112 and 560 mM Negative (S9−) Kreja & Seidel (2002)
No data (S9+)
Studies based on in vivo systems
Bone marrow micronucleus test in CD-1 mouse 1.96 mL/kg via Negative O’Donoghue et al. (1988)
intraperitoneal
injection
Bone marrow micronucleus test in Chinese hamsters 411 mg/kg via Negative Basler (1986)
intraperitoneal
injection

sec-Butyl acetate is naturally present in vinegar at concentrations up to 67 mg/
kg (UNEP/ILO/WHO, 2005). No data were identified on exposure to sec-butyl
acetate from vinegar consumption. However, it may be estimated using EFSA
(2018) data on acetic acid exposure from vinegar consumption, which were
based on an assumed concentration of 6% acetic acid in the vinegar. The EFSA
(2018) estimates of mean acetic acid exposure from vinegar consumption range
269
across surveys from 0–2.8 mg/person per day for infants to 2.3–123.3 mg/
person per day for the elderly. Therefore, the maximum mean intake of vinegar is
0.05 g/day for infants (2.8 mg acetic acid/day × 1 g/1000 mg × 100 g vinegar/6 g
acetic acid) and 2 g/day for the elderly population. Assuming a vinegar intake of
0.05 g/day by infants, a sec-butyl acetate concentration of 67 mg/kg in vinegar,
and a body weight of 5 kg for infants (the body weight indicated for infants aged
0–12 months by EFSA, 2012c), maximum mean exposure to sec-butyl acetate
from vinegar consumption is 0.01 mg/kg body weight per day. Assuming a
vinegar intake of 2 g/day for the elderly, sec-butyl acetate concentrations of
67 mg/kg in vinegar, and a body weight of 60 kg, maximum mean exposure
to sec-butyl acetate from vinegar consumption by the elderly is 0.03 mg/kg body
weight per day.
sec-Butyl acetate has a fruity odour (UNEP/ILO/WHO, 2005), and
is approved for use as a flavouring agent (09.323) in Europe (EFSA, 2008,
2017). The maximized survey-derived daily intake (MSDI) of sec-butyl acetate,
calculated based on production volumes (corrected by a factor of 0.6 to account
for incomplete survey data), was estimated at 0.0012 µg/capita per day (EFSA,
2017). Based on the MSDI of 0.0012 µg/day and an adult body weight of 60 kg, the
exposure to sec-butyl acetate from use as a flavouring agent in Europe is equivalent
to 2 E-08 mg/kg bw per day. A modified theoretical added maximum daily intake
(mTAMDI) for sec-butyl acetate present in foods as a flavouring agent, calculated
on the basis of standard portion sizes and normal use levels in beverages and
foods, was estimated at 3900 µg/person per day (EFSA, 2017). Based on
the mTAMDI of 3900 µg/day and on an adult body weight of 60 kg, the exposure
to sec-butyl acetate from flavouring agent uses in Europe is equivalent to 0.07 mg/
kg bw per day. No data are available on exposure of infants and young children
to sec-butyl acetate from its use as a flavouring agent, but these exposures are
expected to be low given that most infant formulas and foods for young children
do not contain fruity flavouring.
No data were found on concentrations of sec-butyl acetate in food
oils due to carryover from previous cargoes. A worst-case concentration of

100 mg/kg has been assumed for all previous cargo substances.  Worst-case
exposures to previous cargo substances in food oils have been estimated at
0.3 mg/kg bw per day, based on a worst-case concentration of 100 mg/kg and
an oil intake of 3 g/kg bw per day by infants and young children who are high
consumers (see section A4.3).   Exposures to sec-butyl acetate from vinegar and
from its use as a flavouring agent are not expected to add significantly to the
estimated sec-butyl acetate exposure of 0.3 mg/kg bw per day from previous
cargoes for infants and young children.
270
Table 11
Chemical and technical considerations for sec-butyl acetate
Name: Butyl acetate, sec- (sec-butyl acetate)
105-46-4 None
Chemical details Sec-butyl acetate; butan-2-yl acetate; 2-butyl acetate; sec-butyl ethanoate
Colourless liquid with a fruity scent. It produces highly flammable, irritating vapour.
CH3
CH3
H3C

Melting point: −99 °C
Boiling point: 112 °C
Slightly soluble in water, soluble in ethanol and diethyl ether

Route(s) of synthesis The most common process for manufacturing sec-butyl acetate is esterification of acetic acid with sec-
butanol using sulfuric acid as a catalyst. An alternative production route is esterification of sec-butanol
with acetic anhydride.
Composition May contain acetic acid up to 0.2% as an impurity. No impurities of concern have been identified.
Uses Used as a solvent in nitrocellulose lacquers, thinners, enamels, leather finishes, acyclic polymers and vinyl
resins, and as a flavouring substance for food.
Analytical methods None reported for previous cargoes. Potential methods for its determination in fats and oils include GC-FID
and GC-MS.
Potential reaction(s) with a Hydrolyses to acetic acid and sec-butanol, which, in the presence of acid, may participate in
subsequent cargo of fat or oil transesterification with lipids, producing a mixture of fatty acid sec-butyl esters and glycerol. However, the
reactions are slow, requiring an excess of alcohol and temperatures above 100 °C.
GC-FID, gas chromatography with flame ionization detection; GC-MS, gas chromatography–mass spectrometry.
5. Comments

The chemical and technical considerations for sec-butyl acetate are summarized
in Table 11.

Following oral exposure, sec-butyl acetate is expected to be rapidly absorbed into
the systemic circulation, then hydrolysed within minutes to acetic acid and sec-
butanol in the blood, liver, small intestine and respiratory tract (WHO/IPCS,
2005). sec-Butanol is then expected to undergo rapid metabolism by alcohol
dehydrogenase, primarily to 2-butanone, and be excreted by either exhalation
271
or in the urine, or to undergo further metabolism to 3-hydroxy-2-butanone and

2,3-butanediol (Traiger & Bruckner, 1976; Dietz et al., 1981; WHO/IPCS, 1987).

The acute toxicity of sec-butyl acetate after oral exposure is low. In rats, an oral
LD50 of 3200 mg/kg bw was reported (WHO/IPCS, 2005). For sec-butanol, the
oral LD50 was greater than 2000 mg/kg bw in rats and rabbits (ECETOC, 2003).
No short-term or long-term oral toxicity data are available for sec-butyl acetate.
Due to the lack of information on the reproductive and developmental
toxicity and short-term and long-term oral toxicity of sec-butyl acetate, the
Committee also considered the summary results of a two-generation reproductive
and developmental toxicity study on the primary metabolite, sec-butanol (Cox et
al., 1975, as cited by the US EPA, 2003a; original study unpublished). Male and
female rats were given sec-butanol in drinking-water at concentrations of 0, 0.3,
1.0 or 3.0% for 8 weeks prior to mating, and during gestation of two separate F1
generations. The doses of the F0 generation up to day 10 after the birth of the F1A
pups were reported based on average daily intakes as 538, 1644 or 5089 mg/kg
bw per day in males and 594, 1771 and 4571 mg/kg per day in females (intakes
were not reported for subsequent weeks). Maternal toxicity (reduced body
weight gain) accompanied by developmental effects (increased fetal death, and
reduced fetal and pup body weight) and possible effects on male reproductive
performance (i.e. effects on male copulatory success), was reported following
exposure to the highest drinking-water concentration (doses of 5089 mg/kg per
day in males and 4571 mg/kg per day in females). Although reduced pup body
weights were observed in F1A pups at the 1% dose on PND 4 and 21, reduction
in pup body weights at the same dose in the F2 generation was not observed.
Based on these results, the Committee identified a NOAEL of 594 mg/kg per day
(0.3% sec-butanol) based on decreased pup body weight in the F1A generation.
In a review in 2003, the US EPA, using its Benchmark Dose Software (BMDS,
version 1.3.1), calculated a lower 95% confidence limit on the BMDL05 of 657 mg/
kg bw per day based on decreased pup weights in the F1A pups on PND 21 (Cox
et al., 1975, as cited in US EPA, 2003; unpublished study).
The Committee concluded that sec-butyl acetate, sec-butanol and
2-butanone are non-genotoxic in vitro and in vivo.
272
5.4 Allergenicity
The Committee did not identify any reports of allergenicity upon oral exposure
to sec-butyl acetate that would indicate that this substance is or contains a known
food allergen.
sec-Butanol and 2-butanone did not induce skin sensitization reactions
in the guinea-pig maximization tests (ECETOC, 2003; ECHA, 2020a,b).
5.5 Impurities

sec-Butyl acetate is naturally present in vinegar at concentrations up to 67 mg/
kg (WHO/IPCS, 2005). Maximum mean exposure to sec-butyl acetate from
vinegar consumption, calculated based on data provided by EFSA (2018), was
estimated to range from 0.01 mg/kg bw per day for infants to 0.03 mg/kg bw per
day for the elderly.
sec-Butyl acetate has a fruity odour (WHO/IPCS, 2005) and is approved
for use as a flavouring agent (09.323) in Europe (EFSA, 2008, 2017). Exposure
to sec-butyl acetate from it use as a flavouring agent in Europe was estimated to
be 0.07 mg/kg bw per day for adults. No data are available on exposure of infants
and young children to sec-butyl acetate from use as a flavouring agent, but these
exposures are expected to be low given that most infant formulas and foods for
young children do not contain fruity flavouring.
No data were found on concentrations of sec-butyl acetate in food oils due
A4.3). Exposures to sec-butyl acetate from vinegar and from flavouring agent uses
are not expected to add significantly to the estimated sec-butyl acetate exposure
of 0.3 mg/kg bw per day from previous cargoes, for infants and young children.
273
6. Evaluation
No information regarding the short-term and long-term toxicity of sec-butyl
acetate was identified; however, for sec-butanol, the Committee identified
a BMDL05 of 657 mg/kg bw per day based on reduced offspring body weight
from a two-generation reproductive and developmental toxicity study in rats
(summarized by US EPA, 2003). sec-Butyl acetate is naturally present in vinegar
and is approved for use as a flavouring agent in Europe. The Committee estimated
that exposure to sec-butyl acetate from vinegar consumption and its use as a
flavouring agent is approximately 0.1 mg/kg bw per day. A comparison of the
BMDL05 of 657 mg/kg bw per day for sec-butanol with the generic human dietary
exposure value for previous cargoes of 0.3 mg/kg bw per day for sec-butyl acetate
as a previous cargo plus its presence in the diet (0.1 mg/kg bw per day) yields a
margin of exposure (MOE) of 1643, which is considered sufficient to address the
uncertainties in the database.
There are no data on allergenicity upon oral exposure to sec-butyl acetate
that indicate that it is or it contains a known food allergen.
sec-Butyl acetate hydrolyses to acetic acid and sec-butanol, which in the
presence of acid may participate in transesterification with lipids, producing a
mixture of fatty acid sec-butyl esters and glycerol. However, the reactions are
slow, requiring an excess of alcohol and temperatures above 100 °C.
Therefore, sec-butyl acetate meets the criteria for acceptability as a
previous cargo for edible fats and oils.
III. Tert-butyl acetate
1. Explanation
The Committee has not previously evaluated tert-butyl acetate; however, at its
eleventh and forty-ninth meetings, the Committee evaluated butyl acetate (not
specifically tert-butyl acetate) (Annex 1, references 14 and 131). SCF (1997)
considered tert-butyl acetate acceptable as a previous cargo for edible fats and oils
primarily on the basis that it is easily removed by tank cleaning. More recently,
EFSA (2012a) concluded that tert-butyl acetate meets the criteria for acceptability
as a previous cargo for edible fats and oils on the basis that the available data
on tert-butyl acetate and its primary metabolites (i.e. acetate and tert-butanol),
do not give rise to concerns about systemic toxicity, developmental toxicity,
genotoxicity and allergenicity.
274
Table 12
Physical and chemical characteristics of tert-butyl acetate
Chemical name Butyl acetate, tert-; tert-butyl acetate
Synonyms Acetic acid tert-butyl ester; t-butyl acetate; tert-butyl ethanoate
CAS number 540-88-5
CH3
H3C CH3
mass
Description Colourless liquid with a fruity odour. It produces highly flammable irritating vapour.
Melting point 97.8 °C
Boiling point 16.6–22.2 °C (closed cup)
Boiling point Practically insoluble in water, soluble in ethanol, ethyl ether, chloroform and acetic acid.
Solubility Soluble in water. In aqueous solution, acetic anhydride hydrolyses and acetic acid is formed
For the present assessment, previous assessments (monographs) completed

by JECFA, EFSA, SCF, WHO/IPCS, and national and regional governmental
authorities were identified by searching their respective websites. This was followed
by a comprehensive search to identify any critical new data for the assessment of
human health risk on PubMed and PubChem. The search terms used were tert-
butyl acetate and synonyms (for example, t-butyl acetate), CAS number (540-88-
5), toxicity and toxicokinetics. The results were screened for relevance, specifically
concerning the oral route of exposure. Given the paucity of relevant information
concerning the oral toxicity of tert-butyl acetate, a secondary search for relevant
information on metabolites (i.e. tert-butanol and acetic acid) was conducted to
supplement this assessment. The cut-off date for inclusion in this report was 29
December 2020. The data concerning acetic acid are summarized in section I.

The physicochemical characteristics of tert-butyl acetate are listed in Table 12.
2.1 Manufacture and uses of tert-butyl acetate

tert-Butyl acetate is produced from isobutylene reacting with acetic acid in the
liquid phase with vanadium pentoxide-impregnated silica as catalyst and with heat
to increase the yield (SciFinder – CAS, PubChem online databases). Esterification
of tert-butanol and acetic acid produces tert-butyl acetate. The catalyst is sulfuric
275
acid. The removal of water by azeotropic separation forces the reaction to

completion. Common azeotropic agents are cyclohexane, benzene and toluene.
The acid catalyst is neutralized and the ester purified by distillation (Lewis, 2007;
Cheung at al., 2012).
tert-Butyl acetate is used as a solvent in adhesives, sealants and paints,
and as a gasoline additive. There is no information on its use in the food industry.

No impurities of concern have been identified in tert-butyl acetate.

Hydrolysis of tert-butyl acetate results in formation of acetic acid and tert-
butanol, which in the presence of acid may undergo transesterification with lipids
producing a mixture of glycerol and fatty acid tert-butyl esters. The reactions,
however, are slow and require excess alcohol and temperatures above
100 °C (Schuchardt et al., 1998).

No test methods for analysing tert-butyl acetate in fats and oils have been found
in the literature. tert-Butyl acetate can be analysed by GC-FID or GC-MS
(PubChem, SciFinder – CAS online databases).
3. Biological data

No studies on the biochemical aspects of tert-butyl acetate following oral
exposure and no physiologically-based pharmacokinetic models were identified.
However, based on its physicochemical properties (molecular weight = 116.16 g/
mol; log Kow = 1.76; PubChem1), tert-butyl acetate is expected to be readily
absorbed systemically following oral exposure. In contrast to other butyl acetate
isomers, tert-butyl acetate is not expected to be readily hydrolysed in the blood,
liver, small intestines and respiratory tract, and is a poor substrate for alcohol
1
https://pubchem.ncbi.nlm.nih.gov/compound/Tert-butyl-acetate
276
dehydrogenase (WHO/IPCS, 2005; EFSA, 2012b). For example, the hydrolysis

half-life of tert-butyl acetate is 270 minutes when exposed to human blood and
300 minutes when exposed to the blood of Sprague-Dawley rats; compared with
12 and 4 minutes respectively for n-butyl acetate (Essig, Groth & Freundt, 1989).
Essig, Groth & Freundt (1989) suggest that this differential rate of hydrolysis is
due to the steric hindrance of the tert-butyl moiety. Since limited kinetic data
are available for tert-butyl acetate following oral exposure, the data reported
following inhalation exposure are briefly summarized to provide a qualitative
description of tert-butyl acetate’s likely metabolic and elimination pathways.
In an inhalation study in Sprague-Dawley rats, Cruzan & Kirkpatrick
(2006) demonstrated that the predominant route of excretion is via the urine
(i.e. 89.2% of the radioactivity was recovered in the urine versus 2.7 and 4.8%
in the faeces and air, respectively). Cruzan & Kirkpatrick (2006) also suggested
that following exposure to low concentrations of tert-butyl acetate (for example,
100 ppm), conversion to 2-hydroxymethylisopropyl acetate is higher than
conversion to tert-butanol, whereas at high concentrations of tert-butyl acetate
(for example, 1000 ppm), conversion to tert-butanol prevails over conversion to
2-hydroxymethylisopropyl acetate. Additionally, Groth & Freundt (1994), in a
study on Sprague-Dawley rats, showed elevated blood concentrations of both
tert-butyl acetate and tert-butanol following inhalation exposure (via tracheal
cannula) to 900 ppm tert-butyl acetate. tert-Butanol concentrations in the
blood exceeded concentrations of tert-butyl acetate near the end of the 5-hour
exposure period. Similar results (i.e. decreasing tert-butyl acetate and increasing
tert-butanol concentrations in blood over time) were reported by Essig, Groth &
Freundt (1989).
According to Cruzan & Kirkpatrick (2006), following inhalation exposure
of rats to 100 ppm tert-butyl acetate, 24.2% of 2-hydroxymethylisopropyl acetate
and 13.8% of tert-butanol is excreted in the urine after 6 hours, whereas 57.1%
is excreted as 2-hydroxyisobutyric acid. Since both 2-hydroxymethylisopropyl
acetate and tert-butanol are capable of being converted to 2-hydroxyisobutyric
acid (the metabolite with the highest percentage of excretion in the urine) the
exact relative contribution of hydroxylation of the tert-butyl moiety versus
hydrolysis cannot be determined. Hydroxylation and glucuronidation of the
acetate moiety is considered a minor metabolic pathway because only 1.3 and
0.6% of tert-butyl-2-hydroxyacetate glucuronide is measured in the urine 6 hours
after inhalation exposure to 100 and 1000 ppm tert-butyl acetate, respectively.
Bernauer et al. (1998) suggest that metabolism of tert-butanol in rats is different
to that in humans. For example, following oral administration of 5 mg/kg bw
carbon-13-labelled tert-butanol, a human volunteer excreted primarily 2-methyl-
1,2-propanediol and 2-hydroxyisobutyric acid in the urine. Based on parallel
data in rats, Bernauer et al. (1998) suggested that tert-butyl alcohol sulfate is a
277
major urinary metabolite in rats following exposure to tert-butanol, whereas

2-hydroxyisobutyric acid was the major metabolite excreted in human urine.
Nevertheless, based on the kinetic data in rats exposed by inhalation
to tert-butyl acetate, the metabolism of tert-butyl acetate probably occurs by
both the carboxylesterase and cytochrome P450 pathways resulting primarily
in the production of tert-butanol. Based on the data summarized by Cruzan
& Kirkpatrick (2006), the Office of Environmental Health Hazard Assessment
(OEHHA) (2018) suggested that 71% of tert-butyl acetate is metabolized to tert-
butanol (i.e. 13.8% urinary tert-butanol + 57.1% urinary 2-hydroxyisobutyric
acid). The OEHHA (2018) and EFSA (2012a) also suggest that tert-butyl acetate
is widely distributed following absorption and is eliminated primarily via the
urine as metabolites.

Tert-butyl acetate exhibits low acute oral toxicity in rats, with estimated LD50
values ≥3420 mg/kg bw (WHO/IPCS, 2005; ECHA, 2020c). Clinical signs of
toxicity (for example, lethargy, ataxia, flaccid muscle tone, dyspnoea, loss of
righting reflex, prostration, and piloerection, tremors and coma) associated with
acute oral exposure to a high dose (for example >2000 mg/kg bw) are probably
related to irritation and central nervous system depression.
Tert-butanol exhibits low acute oral toxicity with estimated LD50 values
≥3500 mg/kg bw in rats and rabbits (National Toxicology Program; NTP, 1997).
The critical acute effects observed following oral exposure were signs of “alcoholic
intoxication” and hepatotoxicity with the sedative and intoxicating effects of tert-
butanol being reported to be similar but more potent than those of ethanol (NTP,
1997).

No information on short-term or long-term oral toxicity of tert-butyl acetate is
available. The inhalation toxicity studies with tert-butyl acetate are considered
to be of limited use quantitatively. However, in the absence of more robust
information on the short-term and long-term effects following oral exposure to
tert-butyl acetate, effects observed following repeated-dose inhalation exposure
are considered further below.
Faber et al. (2014) describe the results of two 13-week inhalation
toxicity studies in male and female rats (Crl:CD(SD)) and male and female mice
(Crl:CD1(IGR)) exposed to 0, 100, 400 or 1600 ppm tert-butyl acetate via whole-
body exposure for 6 hours per day and 7 days per week. In rats, exposure to
278
1600 ppm tert-butyl acetate for 13 weeks induced increased locomotor activity
(but no change in functional observation battery in week 3), decreased body
weight, decreased food consumption, increased liver, adrenal gland and kidney
weights, and histopathological lesions in the kidney. Except for the kidney effects
observed in all treated male rats, no other toxicologically relevant effects were
noted in rats exposed to doses of 100 and 400 ppm. Hyaline droplet accumulation
and basophilic tubules appeared to be related to accumulation of α-2u-globulin
(as measured using immunohistopathology and enzyme-linked immunosorbent
assay (ELISA) methods) and are considered by the Committee as a male rat
specific effect that is not relevant to human health risk assessment. In the 13-week
inhalation toxicity study in mice, no effects on the kidney were noted. Treatment-
related effects were limited to hyperactivity (but no change in functional
observation battery on days 62 and 63) in the groups that received the 400 and
1600 ppm doses. Furthermore, liver effects (increased absolute and relative liver
weights in males and females; centrilobular hepatocellular hypertrophy in 1/10
females) were noted in the group exposed to 1600 ppm. Male mice also showed
decreased T4, without any change in TSH or T3 or histopathological lesions in the
thyroid, an effect that may have been a result of increased liver enzyme activity.
Tert-butanol (metabolite)
A 13-week oral (drinking water) toxicity study of tert-butanol was conducted
with male and female F344 rats (10 per sex and dose) at doses of 0, 2.5, 5, 10, 20
or 40 mg/mL administered ad libitum in deionized water for 94 to 95 days. Doses
equal to 0, 235, 496, 804, 1599 or 3589 mg/kg bw per day in males and 261, 503,
7658, 1452 or 3500 mg/kg bw per day in females were achieved. Deaths were
recorded of all males dosed with 3589 mg/kg bw per day and 6/10 females dosed
with 3500 mg/kg bw per day. Statistically significant decreases in body weight
were observed at all doses except in males in the lowest dose group and in females
in the highest dose group. The dose-related clinical signs of toxicity observed in
males and females were ataxia, hypoactivity and blood in the urine, with ataxia
and hypoactivity being the earliest signs of toxicity. Increases in absolute and
relative kidney and liver weights were noted in both males and females in the
highest dose group and in males in the second highest dose group. Similarly,
gross necropsy revealed treatment-related renal lesions (urinary tract calculi,
dilation of the ureter and renal pelvis, or thickening of the urinary bladder
mucosa) in both males and females from the high-dose group and males from
the second highest dose group. Microscopically, the gross lesions were related
to increased severity of nephropathy in males and increased mineralization of
the kidney in females at all doses. Additionally, inflammation and hyperplasia of
the transitional epithelium of the urinary bladder were observed in both males
279
and females in the highest dose groups and in males in the second highest dose
group. Based on increased mineralization in the kidneys of females at all doses, a
LOAEL of 261 mg/kg bw per day was identified (NTP, 1995).
In a parallel 13-week study in male and female B6C3F1 mice (10 per
sex and dose), tert-butanol was administered ad libitum via the drinking water
at concentrations of 0, 2.5, 5, 10, 20 or 40 mg/mL. In male mice this resulted in
doses of approximately 0, 350, 640, 1590, 3940 or 8210 mg/kg bw per day. In
female mice, doses of approximately 0, 500, 820, 1660, 6430 or 11 620 mg/kg bw
per day were achieved. Over the course of the 13-week study, two males and one
female from the highest dose group died as a result of tert-butanol exposure and
animals in the high-dose group showed emaciation. Males given the high dose
also exhibited ataxia and hypoactivity. Significantly reduced body weights (up to
25% lower) were noted in males in the two highest dose groups and females in
the highest dose group. Apart from some evidence of slight dehydration (slightly
increased haemoglobin and haematocrit) no other toxicologically relevant changes
were noted from clinical chemistry or haematological observations. At terminal
necropsy, the absolute and relative kidney weights of females from the high-dose
group were significantly increased. Hyperplasia and chronic inflammation were
also observed in the urinary bladder of males from the two highest dose groups
and females from the highest dose group. No other toxicologically significant
effects were noted. A NOAEL of 1590 mg/kg bw per day was identified based on
significantly decreased body weights and hyperplasia and chronic inflammation
of the urinary bladder in males that received the higher doses (NTP, 1995).
In the long-term rat study, male F344 rats (50 per dose) were exposed to
0, 1.25, 2.5 or 5 mg/mL in drinking water (approximately equal to 90, 200 or 420
mg/kg bw per day) for 2 years. Female F344 rats (50 per dose) were exposed to
slightly higher concentrations of 0, 2.5, 5 or 10 mg/mL (equal to approximately
180, 330 or 650 mg/kg bw per day). An additional subgroup of 10 animals per dose
and sex was exposed for 15 months and sacrificed for interim evaluation. Survival
of male and female rats from the highest dose groups was significantly lower
than that of control animals. Also, decreased mean terminal body weights were
observed in the males from all treatment groups and in females from the highest
dose group. Percentage probability of survival at the end of the study was 20, 12,
8 or 2% in males given doses of 0, 90, 200 or 420 mg/kg bw per day, respectively.
In females, the percentage survival at the end of the study was 56, 48, 44 or 24%
at doses of 0, 180, 330 and 650 mg/kg bw per day, respectively. The terminal mean
body weights of treated males were 85, 83 and 76% in the 90, 200 and 420 mg/
kg bw per day dose groups, respectively. In females, the figures were 98, 96 and
79% for the 180, 330 and 650 mg/kg bw per day dose groups, respectively. At the
15-month interim evaluation, a dose-responsive increase in absolute and relative
kidney weights was noted in treated males and females accompanied by increased
280
severity of nephropathy (NTP, 1995; Hard et al., 2019). Statistically significant

increases in relative kidney weights following 15 months of exposure were noted
in males dosed with 200 and 420 mg/kg bw per day and statistically significant
increases in absolute and relative kidney weights were observed in females from
all of the treatment groups. In addition, a renal tubule adenoma was observed in
one male from the high-dose group at the interim sacrifice. Following terminal
necropsy at 2 years, the incidence of renal tubule adenoma or carcinoma in all
treated males had increased above concurrent control and historical control
values (8% versus 0–2%) and analysis of additional step sections from the males
revealed a dose-responsive increase in proliferative lesions (hyperplasia and
neoplasia). Proliferative effects in the renal tubules of females were limited to
hyperplasia in females from the highest dose group; however, the severity of
nephropathy was significantly increased in females from all dose groups and in
males from the highest dose group. Severity grades of 1.6, 1.9, 2.3 and 2.9 for
chronic progressive nephropathy were assigned for females dosed with 0, 180,
330 and 650 mg/kg bw per day, respectively (statistical significance was noted
for all treatment groups). The incidence rates for hyperplasia were 0/50, 0/50,
3/50 and 17/50 for females dosed with 0, 180, 330 and 650 mg/kg bw per day,
respectively (statistical significance was only reached at the highest dose). The
proliferative changes were also accompanied by inflammation (suppurative) of
the kidneys in females from the two highest dose groups. The incidence rates
were 2/50, 3/50, 13/50 and 17/50 for females dosed with 0, 180, 330 and 650 mg/
kg bw per day, respectively. Although tert-butanol did not induce carcinogenicity
in females, it did induce renal tubule adenomas or carcinomas (combined) in
males exposed to doses ≥90 mg/kg bw per day (NTP, 1995). Based on the renal
effects observed in both male and female animals, LOAELs of 90 mg/kg bw per
day for males and 180 mg/kg bw per day for females were identified (NTP, 1995).
In the chronic mouse study, male and female B6C3F1 mice (60 per
sex and dose) were exposed to much higher concentrations in drinking water
(0, 5, 10 or 20 mg/L) than in the rat study. The resulting doses were 540, 1040
or 2070 mg/kg bw per day in male mice and 510, 1020 or 2110 mg/kg bw per
day in female mice. Survival of the males in the high-dose group and terminal
body weights in females given the high dose were significantly decreased. The
percentage probability of survival was 45, 62, 58 and 28% in males dosed with
0, 540, 1040 or 2070 mg/kg bw per day, respectively. Terminal body weights of
the treated females were 98, 97 and 88% of the control body weights for animals
in the 510, 1020 and 2110 mg/kg bw per day dose groups, respectively. Male
and female mice from the highest dose groups also showed increased incidence
of chronic inflammation of the urinary bladder. An associated statistically
significant increase in the incidence of transitional epithelial hyperplasia of
the urinary bladder of male mice from the high-dose group was also observed.
281
Additionally, male mice in the highest dose group showed an increased incidence
of fatty changes in hepatocytes and follicular cell hyperplasia was significantly
increased in all treated male mice. Female mice also showed a statistically
significantly increased incidence of follicular cell hyperplasia at doses of 1020
and 2110 mg/kg bw per day. The incidence rates for follicular cell hyperplasia
in males were: 5/60, 18/59, 15/59 and 18/57 at doses of 0, 540, 1040 or 2070 mg/
kg bw per day, respectively. In females, the incidence rates were: 19/58, 28/60,
33/59 and 47/57 at doses of 0, 510, 1020 or 2110 mg/kg bw per day, respectively.
Follicular cell hyperplasia was accompanied by an increased incidence of thyroid
follicular cell adenoma in females exposed to 2110 mg/kg bw per day, whereas
male mice showed only a marginally increased incidence of thyroid follicular cell
adenoma or carcinoma at doses ≥1040 mg/kg bw per day. Based on the increased
incidence of follicular cell hyperplasia, a LOAEL of 540 mg/kg bw per day was
identified for males and a NOAEL of 510 mg/kg bw per day was identified for
females (NTP, 1995).
Based on the results of the NTP (1995) drinking water studies summarized
above, the critical non-neoplastic effects associated with repeated oral exposure
to tert-butanol are lesions in the urinary tract, with rats appearing more sensitive
than mice and males appearing more sensitive than females. Similar to the
conclusions of Faber et al. (2014) regarding the inhalation toxicity of tert-butyl
acetate, the observed non-neoplastic histopathological changes in the kidney of
male rats exposed to tert-butanol are considered a male rat specific effect related
to α-2u-globulin, with limited relevance to human health risk assessment (US
EPA, 2017; Hard et al., 2019). The Committee noted that the proliferative effects
in the thyroid glands of mice were not observed following 13 weeks of exposure
to a high dose of tert-butanol (NTP, 1995; 1997) or tert-butyl acetate (Faber et
al., 2014). Furthermore, Blanck et al. (2010) suggested that the proliferation of
thyroid follicular cells in mice is due to a non-genotoxic mode of action associated
with increased liver enzyme activity at high doses.
In addition to the subchronic (13-week) and chronic (2-year) drinking
water studies, the NTP (1997) conducted 18-day and 13-week inhalation toxicity
studies in mice and rats with target tert-butanol concentrations ranging from 113
to 7000 ppm. Target concentrations were 113, 225, 450, 900 or 1750 ppm in rats
and mice exposed for 13 weeks; and 450, 900, 1750, 3500 or 7000 ppm in rats and
mice exposed for 18 days. The critical effects noted by the NTP (1997) following
13 weeks of inhalation exposure to tert-butanol were increased kidney weights
and associated increased severity of chronic nephropathy in male F344 rats. No
treatment-related histopathological lesions were noted in the female F344 rats or
in the male and female B6C3F1 mice. However, the liver weights of female mice
in the two highest dose groups (1080 and 2100 ppm) in the 13-week studies were
significantly increased.
282

In a developmental toxicity study by Yang et al. (2007), female Sprague-Dawley
rats (22 animals per dose) were administered tert-butyl acetate via oral gavage
at doses of 0, 400, 800 or 1600 mg/kg bw per day during gestational day (GD)
6 to 19 (corn oil was used as the vehicle). In a preliminary dose range finding
study, significant systemic toxicity (maternal death, decreased maternal body
weight gain and decreased fetal weight) was observed at doses greater than
1000 mg/kg bw per day. In the main experiment, 2 out of 22 dams in the high-
dose group died 1 to 3 days following initiation of dosing. Animals in the high-
dose group also exhibited decreased body weight gain, piloerection, abnormal
gait, decreased locomotor activity, loss of fur, vocalization, reddish tears from
the eyes, reddish vaginal discharge, nasal haemorrhage and coma throughout the
study. At terminal necropsy (gestational day 20), surviving dams from the high-
dose group showed one case each of congestion/haemorrhage of the duodenum
and atrophy of the spleen. The terminal organ weights of the adrenal gland
(absolute and relative) and liver (relative) were increased and that of the thymus
(absolute) decreased in animals from the high-dose group. The two dams that
died prior to gestational day 10 showed expansion of the stomach, hypertrophy
of the liver, and congestion/haemorrhage of the small intestine. Although a dose-
dependent tendency towards reduced body weights was apparent (not statistically
significant), dams exposed to 400 or 800 mg/kg bw per day appeared normal
throughout the study period and no significant treatment-related effects were
noted during terminal necropsy. Statistically significant effects on the fetus were
observed in offspring of dams from the middle and high-dose groups. Reduced
body weight was seen in the fetuses of females in the high-dose group and
increases in the incidence of skeletal variations (supernumerary rib and retarded
ossification) in fetuses of females in both the middle and high-dose groups. Based
on the effects summarized above, the NOAEL for maternal toxicity was 800 mg/
kg bw per day. For embryo-fetal development, the NOAEL was 400 mg/kg bw per
day (Yang et al., 2007).
In a non-guideline study, female Sprague-Dawley rats (Crl:CD(SD);
22 per group) were exposed to 0 (corn oil), 400, 800, 1000 or 1600 mg/kg bw
per day tert-butyl acetate via oral gavage from GD 6 to 20 (Faber et al., 2014).
Effects related to irritation and central nervous system alteration (for example,
rocking, lurching or swaying while ambulating, walking on tiptoes, splayed
hindlimbs, circling and/or retropulsion) were observed shortly after dosing in
dams exposed to doses ≥800 mg/kg bw per day. Additionally, one female from
the high-dose group was found dead shortly after exposure on GD 8 and other
animals given the high dose showed hypoactivity, prostration, impaired use of
right and/or left hindlimb, dragging body and hunched posture. Lacrimation
283
and salivation were also noted before and after dosing in all treatment groups.
Slight and transient decreases in feed consumption and body weight gains were
observed in dams exposed to doses ≥800 mg/kg bw per day. Following gross
necropsy of the surviving animals, findings were restricted to the dams from
the high-dose group, namely, increased weights of the adrenal gland (absolute
and relative), liver (relative) and kidney (relative), and decreased weight of
the thymus (absolute and relative). Except for severely distended ureters and a
severely dilated renal pelvis in the moribund female from the high-dose group, no
adverse histopathological findings were noted in the treated dams. Indicators of
reproductive toxicity such as mean gravid uterine weights, mean number of viable
fetuses per litter, intrauterine growth and survival, mean numbers of corpora
lutea and implantation sites, and the mean litter proportions of preimplantation
loss were similar across all groups. Small but statistically significant reductions
in body weight were observed in fetuses from all treated groups, with a dose-
related trend. The authors of the study commented, based on historical control
data, that the unusually small average litter size in the controls resulted in higher
average fetal body weights in this group and concluded that there was no effect on
fetal body weight attributable to the treatment. The NOAEL for maternal toxicity
from this study was 400 mg/kg bw per day. Since the effect on fetal weight is
unclear and the fetuses were not examined for morphological abnormalities, the
Committee concluded that a NOAEL for developmental toxicity could not be
identified from this study.
In the reproductive and developmental inhalation toxicity study by Faber
et al. (2014), male and female Sprague-Dawley rats (Crl:CD(SD); 10 per sex and
dose) were exposed to 0, 100, 400 or 1600 ppm of tert-butyl acetate via whole-
body exposure, for 70 consecutive days prior to mating, throughout mating, and
until GD 20 (109–110 days for males and 108–119 days for females). Following
this period, inhalation exposure of the females was discontinued and the males
were euthanized. Inhalation exposure of the dams was re-initiated on PND 5
and continued until euthanasia postweaning. F1 offspring were euthanized, apart
from one pup per sex from each litter, on PND 24. A selected group of pups were
exposed to the same whole-body inhalation concentration as their parents for an
additional 6 days postweaning and euthanized on PND 27. Except for transient
decreases in body weight gain and food consumption, no evidence of reproductive
or systemic toxicity was observed in the parental generation. Furthermore, apart
from slightly decreased body weight and body weight gain from PNDs 22 to 23 in
pups from the high-dose group, there was no evidence of developmental toxicity
in the F1 generation (Faber et al., 2014).
284
Daniel & Evans (1982) reported that tert-butanol produced developmental delay
in postnatal physiological and psychomotor performance in Swiss-Webster mice
whose mothers were fed a liquid diet containing 0, 0.5, 0.75 or 1.0% tert-butanol
(equal to doses between 2830 and 8721 mg/kg bw per day) during GD 6 to 20.
Grant & Samson (1982) reported decreased absolute and relative brain weights in
neonatal Long-Evans rats exposed to tert-butanol in milk formula during PNDs
4 to 7 (onset of the “brain growth spurt”), via a feeding tube at doses between
600 and 2690 mg/kg bw. However, in contrast to ethanol, tert-butanol did not
induce alterations in myelin formation and protein production (Grant & Samson,
1982). Faulkner et al. (1989) observed an increased number of resorptions per
litter in CBA/J and C57ABL/6J mice exposed to 10.5 mmol/kg bw (equal to
~780 mg/kg bw) twice daily during GD 6 to 18. Nelson et al. (1989) reported
reduced fetal weight in Sprague-Dawley rats exposed to tert-butanol via whole-
body inhalation of 0, 2000, 3500 and 5000 ppm during GD 1 to 19 (statistically
significant reductions in male and female fetal weights were recorded at all
concentrations; LOAEC = 2000 ppm).
The NTP (1995) reported that mice exposed to 11 620 mg/kg bw per
day tert-butanol in drinking water for 13 weeks showed a significantly increased
estrous cycle length. In contrast, no significant effect on estrous cycles was
observed in rats and no significant differences in sperm morphology or motility
were observed in mice or rats following 13 weeks of exposure via drinking water
(NTP 1995; study summaries previously provided). Additionally, the NTP (1997)
did not observe any adverse effects on reproductive parameters in male or female
rats or mice exposed to tert-butanol via inhalation for 18 days and 13 weeks
(studies briefly summarized previously).
The Committee concluded that developmental toxicity occurs at
high doses of tert-butanol and that other more sensitive end-points have been
identified.
3.2.4 Genotoxicity
According to the available information, tert-butyl acetate is non-genotoxic in
vitro and in vivo. The genotoxicity studies on tert-butyl acetate are summarized
in Table 13.
Using the standard battery of genotoxicity tests, tert-butanol shows negative results
(NTP, 1995). Notably, tert-butanol did not induce micronuclei in erythrocytes of
male and female mice exposed to extremely high doses (up to 11 620 mg/kg bw
per day) in drinking water for 13 weeks.
285
Table 13
Summary of some in vitro and in vivo genotoxicity studies on tert-butyl acetate
Gene mutation in Salmonella Typhimurium (TA98, 50–5000 µg/plate Negative (S9− and S9+) OEHHA (2018)
TA100, TA1535, TA1537, TA102) and Escherichia coli
(WP2uvrA/pKM 101)
Chromosome aberration in human lymphocytes 290–1160 µg/mL Negative (S9− and S9+) OEHHA (2018)
Bone marrow micronucleus test in Sprague-Dawley 100–1600 ppm via nose-only inhalation Negative OEHHA (2018)
rats
Positive results have been reported for tert-butanol from genotoxicity

tests using non-standard testing methodology. Sgambato et al. (2009) suggested
that the positive results in the Comet assay are due to indirect mechanisms (i.e.
oxidative stress) as indicated by substantial increases in the levels of 8-hydroxy-
2'-deoxyguanosine (8-OHdG), an important marker of DNA oxidative damage.
The positive results reported for S. Typhimurium strain TA102 provide further
indications of an oxidative stress mechanism (Levin et al., 1982; Williams-Hill et
al., 1999; McGregor et al., 2005). Tert-butanol has been shown to bind to DNA in
the liver, lungs and kidneys of mice following oral gavage exposure to single doses
of between 0.1 and 997 µg/kg bw, using a sensitive analytical technique (Yuan
et al., 2007). In view of the negative results in a range of genotoxicity tests in
vitro and in vivo, the Committee considered these findings to have questionable
relevance for the genotoxicity of tert-butanol. Table 14 summarizes the available
information on genotoxicity.
3.2.5 Allergenicity
No studies regarding the allergenic effects of tert-butyl acetate on humans
following oral exposure were identified. The German MAK Commission (2016)
briefly mentioned negative results from a Buehler skin sensitization test in
guinea-pigs. No other relevant information was identified.
Tert-butanol is generally considered a mild skin irritant and there is one case
report of allergic contact dermatitis from its use in sunscreen (Edwards &
Edwards, 1982).
3.2.6 Impurities
No impurities of concern were identified for tert-butyl acetate.
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Table 14
Summary of some in vitro and in vivo genotoxicity studies on tert-butanol
Gene mutation in Salmonella Typhimurium (TA98, TA100, 100–10 000 µg/plate Negative (S9− and S9+) NTP (1995)
TA1535, TA1537, TA1538)
Gene mutation in Salmonella Typhimurium (TA102) 5–5 000 µg/plate Equivocal (S9−) McGregor et al. (2005);
(non-GLP) Positive (S9+) Williams-Hill et al. (1999)
Gene mutation in mouse lymphoma cells (L5178Y TK+/−) 625–5 000 µg/mL Negative (S9− and S9+) NTP (1995)
Sister chromatid exchange in Chinese hamster ovary cells 160–5 000 µg/mL Negative (S9− and S9+) NTP (1995)
Chromosome aberration in Chinese hamster ovary cells 160–5 000 µg/mL Negative (S9− and S9+) NTP (1995)
DNA damage (modified Comet assay) in rat fibroblasts 0.44 mM (~33 µg/mL) Positive (S9−) Sgambato et al. (2009)
Micronucleus test in B6CF1 mouse peripheral blood cells 350–11 620 mg/kg bw Negative NTP (1995)
per day for 13 weeks via
drinking water
Bone marrow micronucleus test in F344 rats 39–2 500 mg/kg bw via Negativea NTPb
intraperitoneal injection
DNA binding in mouse (male Kunning) liver, lung and ~0.1–997 µg/kg bw via Positive Yuan et al. (2007)
kidney oral gavage
GLP, good laboratory practice.
a
Summary results published by NTP [online] (https://ntp.niehs.nih.gov/whatwestudy/testpgm/status/ts-10402-n.html?utm_source=direct&utm_medium=
prod&utm_campaign=ntpgolinks&utm_term=ts-10402-n.)
b
https://tools.niehs.nih.gov/cebs3/views/index.cfm?action=main.download&bin_id=10265&library_id=13672&fileIdsSelected=1de240ff6604d3e201660c
33ea8a0308

The German MAK Commission (2016) described a study in humans investigating
the odour threshold (0.008 mL/m3) and irritation potential (eye; 177 mL/m3)
of airborne tert-butyl acetate. It concluded that tert-butyl acetate is about as or
possibly somewhat less irritating to the eyes than n-butyl acetate.

No data were found on concentrations of tert-butyl acetate in food from any
source. A worst-case concentration of 100 mg/kg has been assumed for all
previous cargo substances.  Worst-case exposures to previous cargo substances
in food oils have been estimated at 0.3 mg/kg bw per day, based on a worst-case
concentration of 100 mg/kg and an oil intake of 3 g/kg bw per day by infants and
young children who are high consumers (see section A4.3).  
287
Table 14
Chemical and technical considerations for tert-butyl acetate
Name: Butyl acetate, tert- (tert-butyl acetate)
540-88-5 None
Chemical details tert-Butyl acetate; acetic acid tert-butyl ester; t-butyl acetate; tert-butyl ethanoate
Colourless liquid with a fruity odour. It produces highly flammable irritating vapour.
CH3
CH3
H3C CH3

Practically insoluble in water; soluble in ethanol, ethyl ether, chloroform and acetic acid.
Route(s) of synthesis Produced from isobutylene reacting with acetic acid with vanadium pentoxide impregnated silica as
catalyst or by esterification of tert-butanol and acetic acid.
Composition No impurities of concern have been identified. May contain acetic acid up to 0.2% as an impurity.
Uses Used as a solvent in adhesives, sealants and paints and as a gasoline additive.
Analytical methods None reported for previous cargoes. Potential methods for its determination in fats and oils include GC-FID
and GC-MS.
Potential reaction(s) with a Hydrolyses to acetic acid and tert-butanol, which in the presence of acid may participate in
subsequent cargo of fat or oil transesterification with lipids producing a mixture of fatty acid tert-butyl esters and glycerol. However, the
reactions are slow, requiring an excess of alcohol and temperatures above 100 °C.
5. Comments

The chemical and technical considerations for tert-butyl acetate are summarized
in Table 15.

There are no studies on the biochemical aspects of tert-butyl acetate following oral
exposure and no physiologically-based pharmacokinetic models were identified;
however, based on its physicochemical properties, tert-butyl acetate is expected
to be readily absorbed and distributed systemically following oral exposure
(WHO/IPCS, 2005). Since limited kinetic data are available for tert-butyl acetate
following oral exposure, the available kinetic data following inhalation exposure
were considered.
288
Based on the kinetic data in rats following inhalation exposure to tert-

butyl acetate (Cruzan & Kirkpatrick, 2006), metabolism of tert-butyl acetate is
likely to occur by both carboxylesterase and cytochrome P450 pathways resulting
primarily in the production of tert-butanol and acetic acid. The biochemical
aspects as well as the toxicological data on tert-butanol were also considered
in the evaluation of methyl tertiary butyl ether (MTBE) as a previous cargo for
edible fats and oils at the ninetieth meeting of the Committee.

The acute toxicity of tert-butyl acetate following oral exposure is low. The oral
LD50 in rats was reported to be 3420 mg/kg bw (WHO/IPCS, 2005). No short-
term or long-term oral toxicity data are available for tert-butyl acetate. According
to the results of a developmental toxicity study in rats by Yang et al. (2007), tert-
butyl acetate exhibits developmental toxicity (reduced fetal body weight and
increased incidence of skeletal variations) at doses ≥800 mg/kg bw per day. On
the basis of this study, the Committee identified a NOAEL for maternal toxicity
of 800 mg/kg bw per day and a NOAEL of 400 mg/kg bw per day for embryo-fetal
development.
Due to the lack of information on the short-term and long-term oral
toxicity of tert-butyl acetate, the Committee also reviewed selected information
on the oral toxicity of tert-butanol. Following chronic oral exposure via drinking-
water (NTP, 1995), tert-butanol produces significant effects in the kidneys of male
(renal tubule adenoma or carcinoma) and female rats (dose responsive increase
in severity of chronic progressive nephropathy) at doses as low as 90 and 180 mg/
kg bw per day, respectively. In female rats, dose responsive increases in absolute
kidney weights at 15 months, inflammation (suppurative) of the kidneys and
transitional epithelial hyperplasia were also observed. In male rats, these renal
effects are most likely attributable to the binding of tert-butanol to α-2u-globulin in
the kidneys (NTP, 1995; US EPA IRIS, 2017; Hard et al., 2019) and the Committee
considered this mechanism to be male rat-specific and not relevant to humans.
The Committee considered that although chronic progressive nephropathy
(CPN) is a commonly diagnosed rat-specific condition of questionable relevance
to humans (WHO, 2015), the lesions associated with chronic progressive
nephropathy in the female rats (tubular degeneration, glomerular sclerosis, etc.)
also occur in the human kidney (US EPA, 2017). Moreover, there is evidence of
a dose responsive increase in the incidence of nephropathy following only 13
weeks of exposure to tert-butanol, in relatively young female rats (NTP, 1995). At
significantly higher concentrations in drinking-water, and long-term exposure,
tert-butanol also induces non-neoplastic effects in the liver of male mice (fatty
289
changes in hepatocytes at 2070 mg/kg bw per day) and in the thyroid of male
(follicular cell hyperplasia at 1040 mg/kg bw per day) and female mice (follicular
cell hyperplasia at 1020 mg/kg bw per day) (NTP, 1995). Associated increases
in neoplasia in the thyroid of male (follicular cell adenoma or carcinoma at
1040 mg/kg bw per day) and female mice (follicular cell adenoma at 2070 mg/kg
bw per day) were also noted.
The Committee concluded that the available in vitro and in vivo
information on tert-butyl acetate does not raise concerns for genotoxicity and
that the neoplastic effects observed in rats and mice exposed to tert-butanol via
drinking-water probably occur via a non-genotoxic mode of action and at much
higher doses than those expected from oral exposure to tert-butyl acetate as a
previous cargo. The Committee noted that the neoplastic effects in the thyroid
occur at much higher doses than the non-neoplastic (i.e. renal toxicity) effects in
rats. This conclusion is supported by observations from the 13-week drinking-
water studies in mice and rats where clear evidence of renal toxicity was observed
in male and female animals without any evidence of thyroid toxicity at much
higher doses (NTP, 1995).
5.4 Allergenicity
The Committee did not identify any reports that indicated that tert-butyl acetate
or tert-butanol elicits an allergenic response upon oral exposure. There are also
no data available that indicate that tert-butyl acetate would contain a known food
allergen.
5.5 Impurities

No data were found on concentrations of tert-butyl acetate in food from any
source. A worst-case concentration of 100 mg/kg has been assumed for all
previous cargo substances.  Worst-case exposures to previous cargo substances
in food oils have been estimated at 0.3 mg/kg bw per day, based on a worst-case
concentration of 100 mg/kg and an oil intake of 3 g/kg bw per day by infants and
young children who are high consumers (see section A4.3).  
290
6. Evaluation
No information regarding the short-term and long-term toxicity of tert-butyl
acetate was identified; however, the Committee identified a LOAEL of 180 mg/kg
bw per day (NTP, 1995) based on renal effects observed in female rats chronically
exposed to a metabolite of tert-butyl acetate (i.e. tert-butanol) in drinking-water.
The LOAEL for tert-butanol is lower than the NOAEL of 400 mg/kg bw per day of
tert-butyl acetate for developmental toxicity and represents a conservative metric
for risk assessment of tert-butyl acetate. No data were found on concentrations
of tert-butyl acetate in food from any source. A comparison of the LOAEL of
180 mg/kg bw per day with the generic human dietary exposure value for previous
cargoes of 0.3 mg/kg bw per day for tert-butyl acetate as a previous cargo yields
a MOE of 600, which is considered sufficient to address the uncertainties in the
database.
There are no data on allergenicity upon oral exposure to tert-butyl acetate
tert-Butyl acetate hydrolyses to acetic acid and tert-butanol, which in the
presence of acid may participate in transesterification with lipids producing a
mixture of fatty acid tert-butyl esters and glycerol. However, the reactions are
slow, requiring an excess of alcohol and temperatures above 100 °C.
Therefore, tert-butyl acetate meets the criteria for acceptability as a
previous cargo for edible fats and oils.
IV. n-Pentane
1. Explanation
The Committee has not previously evaluated n-pentane. SCF considered n-pentane
acceptable as a previous cargo in 1997 based on a previous decision concerning
the use of n-pentane in the manufacture of plastic materials intended to come in
contact with foodstuffs (SCF, 1995). In the SCF (1995) opinion, n-pentane was
considered acceptable as a food contact material due to its volatile nature and
the unlikelihood of its presence in the finished product. EFSA (2012) concluded
that n-pentane meets the criteria for acceptability as a previous cargo for edible
fats and oils, based on evidence suggesting that it exhibits a low systemic toxicity
potential, and lacks mutagenic and carcinogenic potential. However, EFSA also
concluded that there were inadequate data in humans and animals regarding the
oral toxicity of n-pentane to establish a health-based guidance value (HBGV).
291
Table 16
Physical and chemical characteristics of pentane
Chemical name Pentane
Synonyms n-pentane; pentan; amyl hydride
CAS number 109-66-0
H3C
Molecular formula; molar C5H12; 72.15 g/mol
mass
Description Clear colourless liquid with a petroleum-like odour. Forms highly flammable vapour–air mixtures
Melting point −129.7 °
Boiling point 36 °C
Flash point −49 °C (closed cup)
Solubility Insoluble in water, soluble in most organic solvents

completed by JECFA, SCF or EFSA were identified by searching their respective
websites. This was followed by a comprehensive search to identify any critical
new data for the assessment of human health risk on PubChem and PubMed and
considering previous reviews by national and regional governmental authorities.
The search terms used were the common name (n-pentane), CAS number
(109-66-0), toxicity and toxicokinetics. The results were screened for relevance,
specifically in regard to the oral route of exposure. In an effort to supplement
the toxicity information on short-term and long-term oral exposure, a secondary
search was conducted for isopentane (CAS No. 78-78-4), an isomer of n-pentane.
The cut-off date for inclusion in this report was 29 December 2020.

The physicochemical characteristics of pentane are listed in Table 16.
2.1 Manufacture and uses of n-pentane

Pentane is produced by distillation from natural gasoline or naphtha. Natural
gasoline contains approximately 7% and catalytic cracker naphtha contains 1%
pentanes (Mears & Eastman, 2005). Pentane is also produced by dehydration and
subsequent hydrogenation of 2- and 3-pentanol and from 2-bromopentane by
Grignard reaction (The Merck Index, 2000).
292
Pentane is used as an additive in motor and aviation fuel, as a propellent

and solvent in cosmetics, and as a blowing agent to make foamed food packaging
materials (Mears & Eastman, 2005; PubChem online database).

Pentane may contain residues of sulfur (< 0.5 mg/kg), naphthenes (< 1 mg/
kg), benzene (< 3 mg/kg), toluene (< 3 mg/kg), aromatics (benzene, toluene
and xylene) (< 5 mg/kg) and n-hexane (0.1 mg/kg) (Shell Chemicals Online).
Technical grade pentane may contain branched and cyclic hydrocarbons of
similar molecular mass, which are not expected to be of concern (EFSA, 2012a).
The major route of exposure to benzene is inhalation rather than diet
(WHO, 2000; Duarte-Davidson et al., 2001). Estimated absorbed exposures of
inhaled benzene in the United Kingdom have been estimated to range from
0.71 µg/kg bw per day for rural children to 14.12 µg/kg bw per day for urban
adult smokers who work adjacent to busy roads (Duarte-Davidson et al., 2001).
Estimates of dietary exposure range from 1.4–2.8 µg per day (WHO, 2000),
equivalent to 0.02–0.05 µg/kg bw per day for adults weighing 60 kg. The estimated
maximum dietary exposure to benzene present in fats and oils when n-pentane
is carried as a previous cargo, 0.0009 µg/kg bw per day, is minimal compared to
total exposure to benzene.
The estimated maximum exposure to toluene present in fats and oils
when n-pentane is carried as a previous cargo, 0.0009 µg/kg bw per day, is less
than 1% of the 0.119 µg/kg bw per day mean total dietary exposure to toluene
estimated for the Belgian population (Vinci et al., 2015). Presence of sulfur as an
impurity in n-pentane is not a safety concern, as sulfur is present in methionine,
an essential amino acid, and is ubiquitous in the diet (Ingenbleek & Kimura,
2013).
Naphthenes (< 1%) and n-hexane (< 0.1%) may also be present in
n-pentane (Shell Chemicals Online). Maximum naphthene and hexane exposures
associated with exposure to n-pentane at a concentration of 0.3 mg/kg bw per
day from previous cargoes are 0.003 mg/kg bw per day and 0.0003 mg/kg bw
per day, respectively. No estimates of exposure to naphthenes were identified.
The exposure to n-hexane present in fats and oils when n-pentane is carried as
a previous cargo is expected to be low compared to other sources (for example
see Environment and Climate Change Canada, 2009). Upper bound estimates of
total exposure to n-hexane from all sources in Canada ranged from 31.6 µg/kg
bw per day for people aged 60+ years to 95.5 µg/kg bw per day for children aged
0.5–4 years (Environment and Climate Change Canada, 2009).
293

No reaction products of concern are expected with edible fats and oils (EFSA,
2012a). The fundamental chemical reactions for pentane are sulfonation to form
sulfonic acids, chlorination to form chlorides, nitration to form nitropentanes,
oxidation to form various compounds, and cracking to form free radicals (Mears
& Eastman, 2005).

No test methods for analysing pentane in fats and oils have been found in the
literature. Residues of pentane may be analysed by GC-FID or GC-MS (PubChem
online database).
3. Biological data
No studies on the biochemical aspects of n-pentane following oral exposure were
identified.
Using an enclosed chamber system, male Sprague-Dawley rats were
exposed to n-pentane via inhalation (approximately 0.34 µmol of [l,5-14C]
n-pentane plus 1.0 mL of 10.1 µmol/mL unlabelled pentane gas; 99.0% purity),
and chamber air concentrations of n-pentane were measured at 5-minute
intervals for up to 8 hours. In the first experiment, radioactivity in whole blood
and various tissues (liver, kidney, lung, testes, brain, muscle, heart, small and large
intestine, spleen and fat) of six rats were measured at necropsy (8 hours), whereas
in the second experiment, urine and blood samples were collected from four rats.
At the end of the experiment, approximately 4.7% of the n-pentane remained in
the chamber system, and approximately 50% of the total [14C] activity added to
the chamber system was recovered as CO2 (in both experiments). Results from
the first experiment showed that liver, small intestine and kidneys contained the
highest concentration of radioactivity. However, the muscle and liver showed the
largest proportion of total [14C] activity recovered (6.98 and 3.37%, respectively
with a total recovery of 14.92% in all tissues). In the second experiment, recovery
of [14C] activity in blood was 2.1%, whereas the recovery in urine was 7.6%.
When the authors combined the results from both experiments, approximately
78.9% of the administered [14C]-pentane was accounted for, predominantly as
CO2 (Daugherty, Ludden & Burk, 1988).
Chiba & Oshida (1991) reported that in rats exposed by inhalation to
5% n-pentane for 1 hour, n-pentane is metabolized to 2-pentanol, 3-pentanol
and 2-pentanone. Similar results have been observed in vitro. For example,
294
2-pentanol (major metabolite, 83–89%) and 3-pentanol (minor metabolite,

11–16%) were detected when rat and rabbit liver microsomes were incubated
with n-pentane; and 2-pentanol, 3-pentanol and 2-pentanone were detected
following incubation with mouse liver microsomes (Frommer, Ulrich &
Staudinger, 1970). Chiba & Oshida (1991) also demonstrated that pretreatment
of rats with phenobarbital increased the concentration of 2-pentanol, 3-pentanol
and 2-pentanone in the blood of exposed rats, providing evidence that hepatic
microsomes play a critical role in the metabolism of n-pentane. There is also
evidence that alcohol dehydrogenase may be involved in pentane metabolism in
rats, since administration of ethanol or 4-methyl-pyrazole diminished pentane
clearance (Burk, Ludden & Lane, 1983; Allerheiligen, Ludden & Burk, 1987).
Filser et al. (1983) studied the pharmacokinetics of n-pentane (99.7%
purity) following inhalation exposure of rats in closed desiccator jar chambers
to a wide range of n-pentane concentrations. According to Filser et al. (1983),
n-pentane showed first order kinetics at low exposure concentrations (<100
ppm) and saturation kinetics at high concentrations (>100 ppm). In this study,
an elimination half-life for n-pentane of approximately 8 minutes was estimated.
The rapid metabolism and excretion of n-pentane offers little potential for tissue
accumulation (McKee et al., 1998).
Perbellini et al. (1985) studied the partitioning of n-pentane in human
tissues/air and human blood/air ex-vivo. The tissues were collected from two men
(aged 30 and 40 years) who had died suddenly from a heart attack, and stored
human blood was obtained from a hospital blood bank. The tissue/air partitions
were: liver – 2.1; kidney – 0.6; brain – 2.2; fat – 39.6; muscle – 0.7; heart – 0.2; and
lung – 0.5. The blood/air partition was 0.38. Based on the estimated partitions,
n-pentane preferentially partitions to fat.

n-Pentane exhibits low acute oral toxicity in rats, with an estimated LD50 of
> 2000 mg/kg bw (PubChem1). McKee et al. (1998) indicated that following oral
exposure to 2000 mg/kg bw, Crl:CDBR rats (five per sex) exhibited no treatment-
related mortality or persistent signs of systemic toxicity 14 days after exposure.
On the day of treatment, Mckee et al. (1998) observed that all of the exposed rats
showed oral and nasal discharge, swollen abdomen, anogenital staining and/or
soft or mucoidal stool. Based on the available animal data, inhalation of n-pentane
at high concentrations (>30 000 ppm) may also cause neurobehavioural and
neurotoxic effects (ECB, 2003; German MAK Commission, 2015).
1
https://pubchem.ncbi.nlm.nih.gov/compound/8003#section=NIOSH-Toxicity-Data
295

Halder et al. (1985) and, more extensively, the US EPA (2009), summarized
the results of a repeated-dose oral toxicity screening study that specifically
investigated the nephrotoxicity potential of various hydrocarbon solvents,
including n-pentane. In this study, male Fischer 344 rats (n = 10 per group) were
administered n-pentane via oral gavage at doses of 0, 500 or 2000 mg/kg bw per
day (5 days per week) for 4 weeks. Although gross examination of tissues and
organs from all animals was conducted, histopathological observations were
restricted to the kidneys and consisted of grading for:
■■ hyaline droplet accumulation in the proximal convoluted tubules;
■■ regenerative epithelium in the renal cortex; and
■■ intratubular cast formation in the renal medulla.
No treatment-related clinical signs were noted; however, mortality

(incidence rates of 2/10 and 4/10, respectively; cause uncertain), weight loss
(>14%) and decreased absolute kidney weights (8% and 9%, respectively)
were observed in animals given doses of 500 and 2000 mg/kg bw per day.
Histopathological examination of the kidneys did not reveal any effects commonly
associated with hydrocarbon-induced nephropathy (for example, hyaline droplet
changes, regenerative epithelium formation or tubular dilatation). No other
significant changes were noted, except for lesions in the forestomach consisting
of raised, pale, white or dark foci, most notably among rats from the high-dose
group.
Halder et al. (1985) also investigated the effects of isopentane (a structural
analogue of n-pentane). According to the limited results reported, isopentane
was lethal in 90% of the rats administered a dose of 2000 mg/kg bw per day and
10% of the animals that received 500 mg/kg bw per day. No histopathological
evidence of neuropathy was observed following exposure to isopentane. Given
the limitations in study design (for example, males only; limited clinical
observations; no results on haematology or clinical chemistry; histopathology

restricted to kidneys) and reporting, the results of these assays are considered
inadequate for hazard characterization.
In a 90-day guideline-compliant (OECD 413) inhalation study, Sprague-
Dawley rats (10 per sex and concentration) were exposed via whole-body
inhalation to n-pentane vapours (>95% purity) with nominal concentrations of
0, 5000, 10 000 or 20 000 mg/m3 for 13 weeks (6 hours/day, 5 days/week). There
were no clinical signs of toxicity, and mortality was not evident in either controls
or treated animals. There were no treatment-related changes in haematology or
clinical chemistry among treated rats (data not available) and body weight was
similar among all groups. Ophthalmological, gross and histological examinations
296
did not reveal any adverse effects of n-pentane exposure. A statistically significant
increase in the absolute liver weight was noted in animals in the low-dose group;
however, it was not concentration-dependent. Based on the absence of evidence
for systemic effects, a NOAEC of 20 483 mg/m3 (measured value), the highest
concentration tested, was identified (McKee et al., 1998; ECHA, 2020d).
In an inhalation study, Crl:CD rats (10 males per group) were exposed
to 0, 1000, 3000 or 10 000 ppm of n-pentane (or 3003, 9009 and 30 030 mg/
m3) for 2 weeks (6 hours/day, 5 days/week). Five rats per group were killed at
day 14 and the remaining five rats per group were killed after a 14-day recovery
period. Overall, no clinical signs of toxicity were evident in animals in any of
the treatment groups; body weight was similar to the controls, and clinical
pathological and histopathological findings were unremarkable. The functional
observation battery test did not reveal any abnormal behavioural responses to
pentane exposure. Statistically significant increases in serum calcium (11.6 and
12.1 mg/L; versus 11.1 mg/L in controls) and phosphorus concentrations (10.4
and 11.0 mg/L; versus 9.8 mg/L in controls) were seen in rats exposed to 3000
or 10 000 ppm, respectively. However, these changes were reversible during
the 2-week recovery period. The authors of the study proposed a conservative
NOAEC of 1000 ppm based on reversible clinical chemistry changes occurring at
3000 and 10 000 ppm (Stadler et al., 2001).
Takeuchi et al. (1981) studied the neurotoxicity of n-pentane (purity
over 99%), n-hexane and n-heptane in male Wistar rats (7 per group) exposed to
3000 ppm in an exposure chamber for 12 hours/day, 7 days/week for 16 weeks. The
conduction velocity of the tail nerves was measured as an indicator of peripheral
neurotoxicity. Rats exposed to n-pentane behaved normally throughout the
experiment, but transiently decreased body weights were observed after 4 weeks.
No significant differences from controls in nerve conduction were observed
in animals in the n-pentane- and n-heptane-treated groups whereas n-hexane
significantly disturbed nerve conduction. The results reported by Takeuchi et al.
(1981) are supported by the lack of neurotoxicity observed by Frontali et al. (1981)
who exposed male Sprague-Dawley rats (six to nine) to 3000 ppm n-pentane
(purity 99%) for 9 hours/day, 5 days/week for 30 weeks.

In a range-finding developmental toxicity study, pregnant Sprague-Dawley rats (7
per group) were administered n-pentane in corn oil via gavage at a dose of 0, 250,
500, 750 or 1000 mg/kg bw per day, once daily from GD 6 to 15. Animals were
sacrificed at GD 21. Decreased body weight gain of animals in the group exposed
to 1000 mg/kg bw per day (in relation to controls) was the only maternal effect that
was considered attributable to n-pentane exposure. Treatment with n-pentane did
297
not cause any significant changes in fetal body weight or sex ratio. No biologically
significant external malformations were observed (McKee et al., 1998).
In the follow-up study, pregnant rats (25 per group) were administered
n-pentane in corn oil via gavage at doses of 0, 100, 500 and 1000 mg/kg bw
per day during GD 6 to 15. Maternal toxicity was not evident at any dose level.
Most of the dams survived until scheduled terminal sacrifice on GD 21 and
were free of clinical or postmortem effects that could be attributed to treatment.
Mean body weight, body weight change, uterine weight, corrected body weight
and uterine implantation data were similar between treated and control dams.
Among the fetuses there were no statistically significant differences in mean
fetal body weight or mean skeletal ossification sites between treated and control
groups. Furthermore, there were no statistically significant differences in total or
individual variations or malformations (external, visceral or skeletal) between
treated and control groups. Based on these results, a NOAEL of 1000 mg/kg bw
per day, the highest dose tested, was identified for maternal and developmental
toxicity (McKee et al., 1998).
In a developmental toxicity study summarized in Hurtt & Kennedy
(1999), female Crl:CD®BR rats were exposed to n-pentane vapours at target
concentrations of 0, 1000, 3000 or 10 000 ppm for 6 hours/day during GD 6
to 15. No evidence of developmental or maternal toxicity was observed in the
developmental toxicity study and a NOAEC of at least 10 000 ppm was identified.
Overall, the results described here suggest that exposure to n-pentane via
the oral and inhalation routes is not hazardous to the developing rat fetus.
Isopentane
In the ECHA (2020d) registration dossier for n-pentane, toxicity data on
n-pentane’s isomer (isopentane) was considered as read-across. According to the
German MAK Commission (2015) for pentane (all isomers) “the toxic effects and
mode of action of isopentane and tert-pentane are similar to those of n-pentane”.
Consequently, for the purposes of assessing the oral toxicity of n-pentane, the
results of the one-generation reproductive toxicity study with isopentane are
summarized below.
Yu et al. (2011) summarized the results of a guideline-compliant (OECD
415; GLP) one-generation reproductive toxicity study with isopentane. In this
study, isopentane was administered via oral gavage in corn oil to male and female
Sprague-Dawley rats (24 per sex and treatment group) at doses of 0, 100, 300 or
1000 mg/kg bw per day. F0 generation males were treated for 10 weeks prior to
mating and during mating (total 12 weeks of exposure). F0 generation females
were treated starting from 2 weeks prior to mating to PND 21 of the F1 generation
(10 weeks total exposure). No treatment-related evidence of reproductive or
298
developmental toxicity was observed at any dose. Effects were limited to the F0
generation and consisted of transient salivation immediately following dosing in
males administered 300 and 1000 mg/kg bw per day and in females administered
a dose of 1000 mg/kg bw per day (probably due to the irritating properties of
isopentane). Male F0 generation rats treated with 1000 mg/kg bw per day also
exhibited decreased body weight gain and slightly reduced food consumption.
Following terminal necropsy, increased absolute and relative adrenal gland
weights were observed in males and females treated with 1000 mg/kg bw per day.
Male rats treated with 1000 mg/kg bw per day also exhibited increased relative
brain, liver, kidney and testes weights. Although no histopathological lesions
were noted in female rats, male rats treated with 1000 mg/kg bw per day showed
an increased incidence of renal tubular degeneration/regeneration (the incidence
rates in the 0, 100, 300 and 1000 mg/kg bw per day dose groups were 13/24,
10/24, 11/24 and 18/23, respectively). No other effects were reported in the other
F0 generation males or females. Based on effects observed in the F0 generation
rats exposed to 1000 mg/kg bw per day, the Committee identified a NOAEL of
300 mg/kg bw per day.
3.2.4 Genotoxicity
In an in vitro clastogenicity test in Chinese hamster ovary (CHO) cells, n-pentane
produced a dose-related increase in the percentage of aberrant cells following
20 hours harvest in the presence of S9 with statistically significant increases being
noted at 1200 and 1500 µg/mL. However, no increases were observed in the initial
20 hours harvest treatment with and without S9 and no increases were observed
in either the initial or repeat 44 hours harvest in the presence and absence of
S9. Additionally, n-pentane has produced negative genotoxicity results in vitro
and in vivo (Table 17) and lacks structural alerts for mutagenicity/genotoxicity.
Consequently, the weight of evidence suggests that n-pentane does not raise
concerns for genotoxicity.
3.2.5 Allergenicity
n-Pentane did not induce skin sensitization reactions in the guinea-pig
maximization test (Mckee et al., 1998).
3.2.6 Impurities
Some of the impurities identified in section 2.2 are associated with carcinogenicity
(for example, benzene (IARC, 2018)). However, the estimated maximum dietary
exposure to benzene present in fats and oils when n-pentane is carried as a
previous cargo, 0.0009 µg/kg bw per day, is minimal compared to total benzene
exposure.
299
Table 17
Summary of some in vitro and in vivo genotoxicity studies on n-pentane
Gene mutation in Salmonella Typhimurium 1 to 10% (v/v%) Negative (S9− and S9+) Kirwin, Thomas &
(TA98, TA100, TA1535, TA1537, TA1538) Cytotoxic at vapour concentrations of Simmon (1980)
25 and 50%
Gene mutation in Salmonella Typhimurium 0.2 mL Negative (S9− and S9+) ECB (2003)
(TA98, TA100, TA1535, TA1537, TA1538)
Chromosomal aberrations in Chinese 600 to 1 500 µg/mL Negative (S9−) McKee et al. (1998)
hamster ovary cells (using vials with Equivocal (S9+)
limited headspace)
Bone marrow micronucleus test in 5 000 to 20 000 mg/m3 Negative McKee et al. (1998)
Sprague-Dawley rats following 13 weeks of
inhalation exposure (6 hours/day)

There are no published reports on the toxicity of n-pentane after oral admin-
istration to humans. However, human volunteers (3 to 6) exposed to 5000 ppm
n-pentane (76.5% n-pentane, 20.8% isopentane, 1.4% hexane and 1.3% butane)
for 10 minutes did not exhibit any adverse symptoms and, in particular, did not
demonstrate mucous membrane irritation or vertigo (Galvin & Marashi, 1999;
Carreón & Herrick, 2012; German MAK Commission, 2015). Galvin & Marashi
(1999) reported neurotoxic effects associated with exposure to n-pentane as a
component of a solvent mixture used in the occupational environment; however,
these results are confounded by the presence of other hydrocarbons (for example,
n-hexane) and consequently, are not considered relevant for this assessment.

Pentane may be used as a blowing agent in the production of foamed plastic
food packaging (21 CFR 178.3010). However, no data were identified on pentane
concentrations in polystyrene packaging or on migration of pentane from
packaging into food.
No data were found on concentrations of pentane in food oils due to
carryover from previous cargoes. A worst-case concentration of 100 mg/kg has
been assumed for all previous cargo substances. Worst-case exposures to previous
cargo substances in food oils have been estimated at 0.3 mg/kg bw per day, based
300
Table 18
Chemical and technical considerations for n-pentane
Name: n-Pentane
109-66-0 None
Chemical details n-Pentane; pentan; amyl hydride
Clear colourless liquid with a petroleum-like odour. Forms highly flammable vapour–air mixtures.
H3C CH3

Melting point: −129.7 °C
Boiling point: 36 °C
Insoluble in water; soluble in most organic solvents.

Route(s) of synthesis Produced by distillation from natural gasoline or naphtha, by dehydration and subsequent hydrogenation
of 2- and 3-pentanol and from 2-bromopentane by Grignard reaction.
Composition May contain residual sulfur, benzene and other aromatics. Technical grade pentane may contain branched
and cyclic hydrocarbons of similar molecular mass.
Uses Used as an additive in motor and aviation fuel, as a propellant and solvent in cosmetics and as a blowing
agent to make foamed food packaging materials.
Analytical methods None identified for previous cargoes. Pentane may be analysed by GC-FID or GC-MS.
Potential reaction(s) with a It is not expected to react with edible fats and oils.
subsequent cargo of fat or oil
on a worst-case concentration of 100 mg/kg and an oil intake of 3 g/kg bw per

day by infants and young children who are high consumers (see section A4.3).
5. Comments

The chemical and technical considerations for n-pentane are summarized in
Table 18.

No studies on the biochemical aspects of n-pentane following oral exposure
were identified. However, kinetic data in rats following whole-body exposure
via inhalation indicated that n-pentane is readily absorbed and distributed
301
systemically to various tissues, with a higher affinity to fat. It is rapidly metabolized

to pentanols and pentanone, and exhaled as carbon dioxide (Filser et al. 1983;
Daughtery et al. 1988; Chiba & Oshida, 1991). Considering the rapid metabolism
and excretion of n-pentane, there is little potential for tissue accumulation
(McKee et al., 1998).

n-Pentane exhibits low acute oral toxicity in rats, with an estimated LD50 of
> 2000 mg/kg bw (McKee et al., 1998). Only one study, with limited reliability,
which investigated the short-term toxicity of n-pentane was identified. No other
studies on the short-term or long-term toxicity of n-pentane were identified. In
a developmental toxicity study, pregnant rats (25 per group) were administered
n-pentane via gavage at doses of 0, 100, 500 and 1000 mg/kg bw per day during
GD 6 to GD 15 (McKee et al., 1998). Maternal and developmental toxicity was
not evident at any dose. Based on these results, the Committee identified a
NOAEL of 1000 mg/kg bw per day, for the maternal and developmental toxicity
of n-pentane.
To address the limitations in the short-term toxicity database for
n-pentane, the Committee also considered the results of a one-generation
reproductive toxicity test with isopentane (an isomer of n-pentane). In this study,
male and female rats were administered 0, 100, 300 or 1000 mg/kg bw per day
via oral gavage (Yu et al., 2011). No evidence of treatment-related reproductive
or developmental toxicity was observed at any dose. Effects were limited to the
F0 generation. Transient salivation immediately following dosing was observed
in males given doses of 300 and 1000 mg/kg bw per day and in females given
the 1000 mg/kg bw per day dose (probably due to the irritating properties of
isopentane). Male F0 generation rats treated with 1000 mg/kg per day also
exhibited decreased body weight gain and slightly reduced food consumption.
Following terminal necropsy, increased absolute and relative adrenal gland

weights were observed in males and females in the 1000 mg/kg bw per day dose
group. Male rats treated with 1000 mg/kg bw per day exhibited increased relative
weights of the brain, liver, kidneys and testes. Although no histopathological
lesions were noted in female rats, male rats treated with 1000 mg/kg bw per day
showed an increased incidence of renal tubular degeneration or regeneration. No
other effects were reported in the other F0 generation males or females. Based
on effects observed in the F0 generation rats at the highest dose, the Committee
identified a NOAEL of 300 mg/kg bw per day for isopentane.
The Committee concluded that n-pentane is non-genotoxic in vitro and
in vivo.
302
5.4 Allergenicity
The Committee did not identify any reports that indicated that n-pentane elicits
an allergic response upon oral exposure. There are also no data available that
indicate that n-pentane would contain a known food allergen.
5.5 Impurities
Total aromatics (benzene, toluene and xylene) may be present as impurities in
n-pentane at < 5 mg/kg; benzene and toluene may each be present at < 3 mg/
kg, and sulfur may be present at < 1 mg/kg (Shell Chemicals Online). Assuming
that the maximum concentrations of these substances are present as impurities
in n-pentane, exposures associated with a pentane exposure of 0.3 mg/kg bw per
day in oil as carryover from previous cargoes (see ssection A4.3) are 0.0015 µg/
kg bw per day for aromatics, 0.0009 µg/kg bw per day for benzene or toluene, and
0.0003 µg/kg bw per day for sulfur.
The major route of exposure to benzene is inhalation, rather than diet
(WHO, 2000; Duarte-Davidson et al., 2001). Estimated exposures to inhaled
benzene in the United Kingdom range from 0.71 µg/kg bw per day for children
living in rural areas to 14.12 µg/kg bw per day for adult smokers in urban areas
who work adjacent to busy roads (Duarte-Davidson et al., 2001). Estimates of
dietary exposure range from 1.4 to 2.8 µg per day (WHO, 2000), equivalent to
0.02–0.05 µg/kg bw per day for adults weighing 60 kg. The estimated maximum
dietary exposure to benzene present in fats and oils when n-pentane is carried as
a previous cargo, 0.0009 µg/kg bw per day, is minimal compared to total benzene
exposure.
The estimated maximum exposure to toluene present in fats and oils
when n-pentane is carried as a previous cargo, 0.0009 µg/kg bw per day, is less
than 1% of the 0.119 µg/kg bw per day mean total dietary exposure to toluene
estimated for the Belgian population (Vinci et al., 2015). The presence of sulfur
as an impurity in n-pentane is not a safety concern, as sulfur is present in
methionine, an essential amino acid, and is ubiquitous in the diet (Ingenbleek &
Kimura, 2013).
Naphthenes (< 1%) and n-hexane (< 0.1%) may also be present in
n-pentane (Shell Chemicals Online). Maximum exposures to naphthene and
hexane associated with an n-pentane exposure of 0.3 mg/kg bw per day from
previous cargoes are 0.003 mg/kg bw per day and 0.0003 mg/kg bw per day,
respectively. No estimates of exposure to naphthenes were identified. The
exposure to n-hexane present in fats and oils when n-pentane is carried as a
previous cargo is expected to be low compared to exposure from other sources
(for example see Environment and Climate Change Canada, 2009).
303

n-Pentane may be used as a blowing agent in the production of foamed plastic food
packaging (21 CFR 178.3010). However, no data were available on n-pentane
concentrations in polystyrene packaging or on migration of n-pentane from
packaging into food.
No data were found on concentrations of n-pentane in food oils due to
carryover from previous cargoes. A worst-case concentration of 100 mg/kg has
been assumed for all previous cargo substances. Worst-case exposures to previous
cargo substances in food oils have been estimated at 0.3 mg/kg bw per day, based
on a worst-case concentration of 100 mg/kg and an oil intake of 3 g/kg bw per day
by infants and young children who are high consumers (section A4.3).
6. Evaluation
No reliable information regarding the short-term and long-term toxicity of
n-pentane was identified; however, the Committee identified a NOAEL of
1000 mg/kg bw per day for n-pentane based on developmental toxicity testing
in rats. The Committee also identified a NOAEL of 300 mg/kg bw per day for
an isomer (isopentane) following short-term oral exposure in a one-generation
toxicity test in rats (12 and 10 weeks of exposure in males and females, respectively).
A comparison of the NOAEL of 300 mg/kg bw per day for isopentane with the
generic human dietary exposure value for previous cargoes of 0.3 mg/kg bw per
day yields a MOE of 1000, which is sufficient to address the uncertainties in the
database.
There are no data on allergenicity upon oral exposure to n-pentane that
indicate that it is or it contains a known food allergen.
n-Pentane as a previous cargo is not expected to react with edible fats and
oils to form any reaction products.
Exposure to impurities in n-pentane is not anticipated to contribute

significantly to background exposures. Therefore, n-pentane meets the criteria
for acceptability as a previous cargo for edible fats and oils.
304
V. Cyclohexane
1. Explanation
The Committee evaluated cyclohexane as an extraction solvent for foodstuffs
in 1980 and noted a paucity of toxicological data relating to long-term oral
exposure of animals and humans and that the available data indicated that it
had a low order of toxicity (Annex 1, reference 50). No toxicological monograph
was prepared; however, since early findings of haemopoietic injury described
in the literature may be attributed to benzene contamination, specifications
were prepared (Annex 1, reference 50). SCF (1981) evaluated cyclohexane as an
extraction solvent in foodstuffs and could not establish an ADI for humans. In
1996, SCF evaluated cyclohexane as a previous cargo and considered it acceptable
on the basis that it was previously approved as an extraction solvent for flavouring
agents (SCF, 1997). EFSA (2012a) concluded that cyclohexane meets the criteria
for acceptability as a previous cargo for edible fats and oils based on low systemic
toxicity via all routes, negative genotoxicity results in vitro and in vivo, and
absence of allergenic potential.
completed by JECFA, SCF or EFSA were identified by searching their respective
websites. This was followed by a comprehensive search to identify any critical
new data for the assessment of human health risk on PubChem and PubMed and
considering previous reviews by national and regional governmental authorities.
The search terms used were the common name (cyclohexane), CAS number
(110-82-7), toxicity and toxicokinetics. In an effort to supplement the toxicity
information on short-term and long-term oral exposure, a similar comprehensive
literature search was conducted for methylcyclohexane (a structural analogue of
cyclohexane). The cut-off date for inclusion in this report was 29 December 2020.

The physicochemical characteristics of cyclohexane are listed in Table 19.
305
Table 19
Physical and chemical characteristics of cyclohexane
Chemical name Cyclohexane
Synonyms Hexamethylene; hexanaphthene; hexahydrobenzene
CAS number 110-82-7
Chemical structure
Molecular formula; molar C6H12; 84.16 g/mol

mass
Description Colourless, clear, flammable liquid, with characteristic odour
Melting point 4–7 °C
Boiling point 80.7 °C
Flash point −18 °C (closed cup)
Solubility Insoluble in water; soluble or miscible with ethanol, methanol, diethyl ether, acetone, benzene and
carbon tetrachloride
2.1 Manufacture and uses of cyclohexane

Cyclohexane is produced by hydrogenation of benzene in either the liquid or
the vapour phase in the presence of hydrogen. Several cyclohexane processes
requiring nickel, platinum or palladium catalysts have been developed.
Usually, the catalyst is supported, for example, on aluminium oxide, but at least
one commercial process utilizes Raney nickel (PubChem online database).
Cyclohexane is also obtained by fractional distillation of petroleum where it
occurs in amounts between 0.5 and 1.0%. In distillation of petroleum, the C4
boiling range naphthas are fractionated to obtain C5-naphtha containing 10–14%
cyclohexane which, upon further fractionation yields an 85% concentrate (which
is sold as such). Further purification of 85% concentrate cyclohexane necessitates
isomerization of pentanes to cyclohexane, heat cracking to remove open chain
hydrocarbons, and sulfuric acid treatment to remove aromatic compounds (The

Merck Index, 2000).
Cyclohexane is used as a solvent for lacquers and resins, as a paint and
varnish remover and for extraction of essential oils. In the EU, cyclohexane
is approved as an extraction solvent in the preparation of flavourings from
natural flavouring materials with the maximum residue limit in food of 1 mg/kg
(Directive 2009/32/EC). In the United States, cyclohexane is approved for use as
a diluent in colour additive mixtures (21 CFR 73.1). It is also used in industrial
recrystallization of steroids and in the manufacture of adipic acid (INS No. 355)
and caprolactam, which is used in the production of polyamide (nylon) for food
packaging materials.
306

Cyclohexane produced by hydrogenation of benzene may contain residues of
benzene, whereas cyclohexane from petroleum contains residues of hydrocarbons
of similar volatility, mainly C5–C7, possibly including benzene. According to
FOSFA, the cyclohexane transported as a previous cargo is unlikely to contain
more than 0.1% benzene (EFSA, 2012a). Cyclohexane may contain residues of
sulfur and polycyclic aromatic hydrocarbons (PAH) (JECFA specifications).
The estimated exposure to benzene present at a concentration of 0.1%
in cyclohexane carried as a previous cargo is 0.3 µg/kg bw per day. The major
route of exposure to benzene is inhalation rather than diet (WHO, 2000; Duarte-
Davidson et al., 2001). Estimated quantities of inhaled benzene absorbed by
people in the United Kingdom have been estimated to range from 0.71 µg/kg bw
per day for rural children to 14.12 µg/kg bw per day for urban adult smokers who
work adjacent to busy roads (Duarte-Davidson et al., 2001). Estimates of dietary
exposure range from 1.4–2.8 µg/day (WHO, 2000), equivalent to 0.02–0.05 µg/kg
bw per day for adults weighing 60 kg. The estimated maximum dietary exposure
to benzene if present in fats and oils when cyclohexane is carried as a previous
cargo, 0.3 µg/kg bw per day, is below the range of inhalation exposures, although
it is high compared to estimated total dietary exposures.
Cyclohexane may also contain PAH residues, but these residues have
neither been characterized nor quantified, and exposures to PAH substances in
cyclohexane carried as a previous cargo therefore cannot be estimated.

Cyclohexane is not expected to react with edible fats and oils (EFSA, 2012a).

Residues of cyclohexane can be analysed by GC-FID or GC-MS. Yousefi &
Hosseini (2017) analysed the hexane content in 40 samples of different types
of edible vegetable oils using solid phase microextraction-gas chromatography
(SPME)-GC-FID. The LOD and the LOQ were 3 µg/kg and 5 µg/kg, respectively.
307
3. Biological data

Cyclohexane and methylcyclohexane share a 6-membered saturated aliphatic
ring structure and exhibit similar physicochemical properties.1 Based on the
toxicokinetic information presented for rabbits following oral exposure (Elliott,
Parke & Williams, 1959; Elliott, Tao & Williams, 1965), both compounds
undergo hydroxylation and excretion in the urine as glucuronide conjugates and
both compounds are expired as CO2 or unchanged parent compound. Potentially
due to a lower vapour pressure, it is estimated that, compared to cyclohexane,
significantly less unchanged methylcyclohexane is excreted in the expired air
following ingestion (Elliott, Tao & Williams, 1965). In all species, the amount
of cyclohexanone produced following exposure to cyclohexane is limited (ECB,
2004). Based on the physicochemical properties of cyclohexane, crossing of the
blood–brain and placental barriers is possible (ECB, 2004).
Female Chinchilla rabbits (four per sex and dose) were administered
single oral doses by gavage of 0.3, 100, 330, 360 or 390 mg [14C]-cyclohexane/
kg bw. Total residues in tissue at termination of the experiments (3 to 6 days
after administration) showed that only 2.5% of the initial dose was present.
Unchanged [14C]-cyclohexane in exhaled air accounted for 0, 38 and 24.5% at
doses of 0.3, 360 and 390 mg/kg bw, respectively; and exhaled [14C]O2 accounted
for an additional 5.5, 8.5 and 10% of the dose, respectively. Cyclohexanone and
cyclohexanol were not detected in exhaled air. Urinary excretion accounted for
98, 59, 33, 41 and 56% of the radioactivity at doses of 0.3, 100, 330, 360 and
390 mg [14C]-cyclohexane/kg bw, respectively. The major urinary metabolites
were glucuronic acid conjugates of cyclohexanol and (±) trans-cyclohexane-1,2-
diol. Faecal excretion of total [14C] was minimal. Total radioactivity accounted
for in this study ranged from 93.3 to 96.3% (Elliott, Parke & Williams, 1959).
Methylcyclohexane was excreted mainly via the urine (65%) and expired
air (15%) following oral administration of approximately 200 to 245 mg/kg bw
methylcyclohexane to rabbits. The main urinary metabolites were glucuronide
conjugates of trans-4-methylcyclohexanol, cis-3-methylcyclohexanol and trans-
3-methylcyclohexanol, whereas the main components in exhaled air were
unchanged methylcyclohexane (~10%) and CO2 (~5%). The percentage of the
1
Methylcyclohexane: molecular weight = 98.2 g/mol; water solubility = 14 mg/L at 25 °C; and Log Kow =
3.61; vapour pressure = 5.73 kPa at 25 °C
https://pubchem.ncbi.nlm.nih.gov/compound/Methylcyclohexane
Cyclohexane: molecular weight = 84.16 g/mol, water solubility = 55 mg/L at 25 °C; and Log Kow= 3.44;
vapour pressure = 10.3 kPa at 20 °C
https://pubchem.ncbi.nlm.nih.gov/compound/Cyclohexane
308
radioactivity found in the urine ranged between 54.2 and 77.4%. The pattern of
hydroxylation suggested that steric factors influenced metabolism (for example,
hydroxylation occurred to the greatest extent at the carbon furthest away from
the methyl group). Total radioactivity accounted for in this study ranged between
65.2 and 93.9% (Elliott, Tao & Williams, 1965).
In an unpublished study summarized in an ECHA (2020f) registration
dossier, male Fisher 344 rats were administered single oral doses by gavage of
100 (n = 2), 200 (n = 25), 1000 (n = 3) or 2000 mg [14C]-cyclohexane/kg bw (n
= 3), and an additional group of three rats received a single intravenous dose
of 10 mg [14C]-cyclohexane/kg bw. Following administration of 200 mg/kg bw,
peak concentrations of total [14C] were attained by 6–12 hours post-dosing
in the whole blood and plasma and were significantly reduced after 72 hours
(approximately 0.4% of the administered 200 mg/kg bw dose). Elimination half-
lives for total [14C] from plasma and tissues were 10–15 hours. The ratio of total
[14C] in adipose tissue and blood ranged from 16:1 (low dose) to 47:1 (high dose).
Following oral administration of 100, 200 or 1000 mg/kg, unchanged cyclohexane
exhaled during the 72-hour period following dosing accounted for 59.4, 59.8
and 92.1% of the doses respectively. According to the ECHA (2020f) dossier, the
high value for cyclohexane after a dose of 1000 mg/kg may have been due to an
error in estimating the dose. The urinary metabolite profile was similar for all
test groups (both intravenous and oral exposure), with only trace quantities of
cyclohexane, cyclohexanone and cyclohexanol being present. Most of the [14C]
was present as four unidentified polar metabolites (probably glucuronic acid
conjugates of hydroxylated metabolites). A preliminary experiment showed
that only minor amounts of [14C] (<0.3% of the dose) were excreted in faeces.
Following intravenous exposure to 10 mg/kg bw, 79.5% of the dose was exhaled
unchanged during the initial 24 hours with a further 1.27% and 1.43% being
exhaled during the 24–48- and 48–72-hour periods, respectively. [14C] exhaled as
either cyclohexanone or cyclohexanol only accounted for 0.22% of the dose over
the 0–72-hour period.
In a human volunteer study, four men and four women (ages 31–
55 years) were exposed to cyclohexane in a closed exposure chamber for 8
hours at a vapour concentration of 1010 mg/m3 cyclohexane (Mráz et al., 1998).
Following exposure, the urine was collected (18 samples per person for 72 hours)
and analysed for cyclohexanol, 1,2-cyclohexanediol and 1,4-cyclohexanediol
following acidic hydrolysis of the glucuronide conjugates. The amount of each
compound determined represents the sum of its conjugated and unconjugated
forms in the urine. According to the results reported, 0.5% was excreted
in the urine as cyclohexanol, 23.4% as 1,2-cyclohexanediol and 11.3% as
1,4-cyclohexanediol. The authors estimated the elimination half-lives of the 1,2-
and 1,4-cyclohexanediol metabolites in humans as 14 to 18 hours.
309

Based on the available information, cyclohexane exhibits low acute oral toxicity
with reported LD50 values in rats and rabbits being greater than 5000 mg/kg bw
(ECB, 2004; Gad, 2014). Clinical symptoms reported following oral exposure to
high doses of cyclohexane include diarrhoea/soft faeces, weight loss, increased
respiration rate, central nervous system effects (for example, transient depression/
narcosis and convulsions) and salivation. Like many other organic solvents, the
suspected mechanism of action of central nervous system depression is the
disruption of membrane enzymes and the corresponding alterations in cell
function (Gad, 2014). Kimura, Ebert & Dodge (1971) suggested that older adult
rats are more sensitive to the acute toxicity of cyclohexane administered via the
oral route than younger adult rats.
Methylcyclohexane
In rats, mice and rabbits, LD50 values of greater than 1200 mg/kg bw have been
reported for methylcyclohexane (PubChem1). Clinical symptoms associated with
acute exposure to high doses are similar to those reported following exposure
to high doses of cyclohexane. Based on oral LD50 values in rabbits, Treon,
Crutchfield & Kitzmiller (1943) suggested that toxicity decreases from ketone (for
example, cyclohexanone) to alcohol (for example, cyclohexanol) to hydrocarbon
(cyclohexane) and that the methylated compounds (i.e. methylcyclohexane,
methylcyclohexanol and methylcyclohexanone) are more hazardous than the
corresponding non-methylated compounds (i.e. cyclohexane, cyclohexanol and
cyclohexanone). Treon, Crutchfield & Kitzmiller (1943) reported oral LD50 values
in rabbits of 5500 to 6000 mg/kg bw for cyclohexane versus 1000 to 1250 mg/kg
bw for methylcyclohexane.

No short-term or long-term oral toxicity studies on cyclohexane were identified.

The following paragraph briefly summarizes the toxicity observed following
exposure by the inhalation and intraperitoneal routes.
According to the ECB’s (2004) assessment of toxicity of cyclohexane
following exposure via inhalation, a NOAEC of 400 ppm (1400 mg/m3) was
identified in rats based on slight neurobehavioural effects observed at 2000 ppm
(6880 mg/m3) following three consecutive acute exposures. Similarly, a NOAEC
of 500 ppm (1720 mg/m3) was identified based on transient sedative effects being
observed at 2000 ppm (6880 mg/m3) in both rats and mice following 90 days of
1
https://pubchem.ncbi.nlm.nih.gov/compound/Methylcyclohexane#section=NIOSH-Toxicity-Data
310
inhalation exposure. Another NOAEC of 435 ppm (1500 mg/m3) was identified
based on an older inhalation study in rabbits showing systemic effects (slight
histopathological lesions in the liver and kidney) at 786 ppm (2700 mg/m3). In
one monkey exposed to cyclohexane at a concentration of 1243 ppm for 6 hours/
day, 5 days/week, for 10 weeks, no clinical signs of toxicity or histopathological
effects were noted (ECB, 2004). Bernard et al. (1989) observed evidence of
nephrotoxicity in female Sprague-Dawley rats following exposure to 400 mg/kg
bw per day via intraperitoneal injection, 5 days/week for 2 weeks, and suggested
that the nephrotoxic effects of cyclohexane are probably due to cyclohexanol.
Cyclohexane is a volatile organic compound and limited toxicological
information is available on exposure via the oral route. Therefore, critical
information from repeated-dose studies on oral toxicity of methylcyclohexane
and cyclohexanone is briefly summarized below.
Methylcyclohexane
In a 28-day repeated dose oral toxicity study (OECD 407; GLP), methylcyclohexane
(99.8% pure) was administered by gavage to groups of Crj:CD(SD) rats (five per
sex and group) at 0, 100, 300 and 1000 mg/kg bw per day. Animals in the recovery
group (sacrificed 14 days after termination of treatment) were also included in the
control and high-dose groups (five per sex and group). Male and female animals
that received 1000 mg/kg bw per day showed transient salivation after dosing.
Additionally, males from the high-dose groups showed increased total protein
and decreased alkaline phosphatase immediately after treatment and following
recovery. Clinical biochemistry changes in females given the highest dose
included increased total cholesterol and decreased alanine aminotransferase (not
observed following recovery). Following terminal necropsy, males and females
from the high-dose group showed increased absolute and relative liver weights
(recovery not mentioned), hepatocellular hypertrophy (not observed following
recovery) and hyaline droplet formation or degeneration in the kidneys (observed
following recovery). Males from the high-dose group also showed increased body
weight and food consumption and females showed slightly increased absolute
and relative ovary weights (only after a recovery period). Effects observed at
the intermediate dose of 300 mg/kg bw per day were restricted to the males and
consisted of transient salivation after dosing, increased body weight and food
consumption, and hyaline droplet degeneration in the kidney. Except for slight
hyaline droplet degeneration observed in one male from the low-dose group, no
other toxicologically relevant effects were noted. A NOAEL of 100 mg/kg bw per
day was identified (ECHA, 2020e).
311
Cyclohexanone
In a pair of guideline-compliant (OECD 453) 2-year studies on rats and mice,
cyclohexanone was administered via drinking water. F344 rats (52 per sex and
treatment) were given concentrations of 0, 3300 and 6500 ppm (equivalent to
~165 and 325 mg/kg bw per day1). B6C3F1 mice (controls both sexes, n = 52;
low- and middle dose females, n = 50; middle dose males, n = 52; high dose n = 47
males and n = 41 females) were given cyclohexanone at concentrations of 0, 6500
and 13 000 ppm in males (equivalent to ~585 and 1170 mg/kg bw per day) and 0,
6500, 13 000 and 25 000 ppm in females (equivalent to ~585, 1170 and 2250 mg/
kg bw per day2). Increased incidences of cancer were observed sporadically in
treated rats and mice. In male rats, the incidences of adenoma of the adrenal cortex
were 1/52, 7/52 and 1/50 in rats exposed to 0, 3300 and 6500 ppm, respectively.
In male mice, the incidences of combined benign and malignant hepatocellular
neoplasms were 16/52, 25/51 and 13/46 in mice exposed to 0, 6500 and
13 000 ppm, respectively. In female mice, the incidences of malignant lymphoma
were 8/52, 17/52, 4/50 and 0/41 in mice exposed to 0, 6500, 13 000 and 25 000 ppm,
respectively. Given the lack of a clear dose-response, the increased incidences of
cancer observed in rats and mice were not considered toxicologically significant
(Litjinsky & Kovatch, 1986).

No relevant studies of the effects of oral exposure to cyclohexane on reproductive
or developmental toxicity were identified. However, reproductive and
developmental toxicity testing of cyclohexane administered via the inhalation
route has been undertaken in rats and rabbits, and there is reproductive and
developmental toxicity information available for methylcyclohexane and cyclo-
hexanol administered via the oral route. Kreckmann et al. (2000), the US EPA
(2003b) and ECHA (2020f), extensively summarize the results of reproductive
and developmental toxicity testing in rats and rabbits following inhalation
exposure to cyclohexane. Based on the summaries available in the published

literature, the results of these inhalation studies are outlined below.
In a developmental toxicity study in Sprague-Dawley rats (25 per
concentration), dams were exposed to cyclohexane by whole-body inhalation
for 6 hours per day during GD 6 to 15 at concentrations of 0, 500, 2000 or 7000
ppm (equivalent to 0, 1721, 6886 or 24 101 mg/m3). Dams were sacrificed on GD
1
Dose conversion used the default values for chronic drinking water studies in rats proposed by EFSA (i.e.
0.05 mg/kg bw per day per mg/L).
2
Dose conversion used the default values for chronic drinking water studies in mice proposed by EFSA (i.e.
0.09 mg/kg bw per day per mg/L). (https://efsa.onlinelibrary.wiley.com/doi/pdf/10.2903/j.efsa.2012.2579
accessed 16 December 2020).
312
21 and fetuses were examined for soft tissue and skeletal abnormalities. Dams
exposed to 2000 and 7000 ppm cyclohexane showed maternal toxicity in the
form of reduced body weight and reduced response to voice stimuli. Necropsy
of the dams and offspring did not reveal any gross lesions and there were no
histopathological changes noted in the soft or skeletal tissues of the offspring.
No evidence of developmental toxicity was observed at any dose. A NOAEC for
maternal toxicity of 500 ppm (1721 mg/m3) was identified.
Pregnant New Zealand white rabbits (20 per concentration) were exposed
via whole body inhalation to 0, 500, 2000 or 7000 ppm (equivalent to 0, 1721,
6886 or 24 101 mg/m3) of cyclohexane for 6 hours/day on GD 6 to 18. This study
revealed a statistically significant decrease in the mean number of corpora lutea
of females in the groups treated with 2000 ppm and 7000 ppm. There was also a
significant trend towards an increased ratio of male pups to total number of pups
in the 2000 ppm and 7000 ppm groups. There were no significant differences
in fetal malformation between control and treatment groups. A NOAEC of
500 ppm (1721 mg/m3) was identified (Kreckmann et al., 2000; ECHA, 2020f).
In a two-generation reproduction study, male and female Crl:CD BR
rats (F0; 30 per sex and concentration) were exposed to cyclohexane (99.9%) by
inhalation, through whole-body exposure, at concentrations of 0, 500, 2000 or
7000 ppm (equivalent to 0, 1721, 6886 or 24 101 mg/m3) for 10 weeks prior to
mating for 6 hours/day and 5 days/week. F0 dams were continuously exposed
throughout GD 0–20. Exposure of the F0 dams paused on GD 21 but resumed
on PND 5 and continued until weaning. A selected group of F1 offspring (30 per
sex and group) was also exposed to the same dosing regimen as the F0 generation
beginning at least 11 weeks after weaning and continuing until parturition of
the F2 litters. Clinical signs of toxicity included transient sedation, salivation,
stained perioral area and wet chin in F0 males and females following exposure to
2000 ppm and 7000 ppm. In addition, females exposed to 2000 ppm and 7000 ppm
showed a transient diminished or absent response to a sound stimulus during each
exposure session. Although no alterations in body weight or food consumption
were observed in F0 males, F0 females exposed to 7000 ppm exhibited a significant
reduction in mean body weight during gestation and lactation. F0 dams and F1
parents (males and females) exposed to 7000 ppm showed significant reductions
in body weights and corresponding decreases in F1 and F2 pups were observed
from lactation days 7 to 25. Exposure to cyclohexane did not significantly alter
fertility or gestational indices, implantation efficiency or gestational length in
either the F0 or F1 parents. Although a significant reduction in the percentage of F1
pups born alive was observed at 7000 ppm, a normal percentage of F2 pups born
alive was observed at this dose. No toxicologically relevant effects were noted
during gross necropsy and histopathology in any of the generations. A NOAEC of
2000 ppm (6886 mg/m3) was identified for developmental effects (decreased body
313
weight in F1 and F2 pups) and a NOAEC of 500 ppm (1721 mg/m3) was identified
for maternal toxicity (decreased body weight and transient diminished or absent
response to a sound stimulus). Considering the decreased pup weights in the F1
and F2 generations, the US EPA (2003) calculated a benchmark concentration
with 95% lower confidence limit (BMCL1SD) of 1822.48 mg/m3 and proposed an
inhalation reference concentration (RfC) of 6 mg/m3 for cyclohexane, using an
overall uncertainty factor of 300.
Methylcyclohexane
ECHA (2020e) described the results of a combined repeated dose toxicity study
with the Reproduction/Developmental Toxicity Screening Test (OECD 422;
GLP) in Crj:CD(SD) rats. According to the summary provided in the ECHA
(2020e) dossier, the rats (six males and five females per group) were administered
methylcyclohexane (99.9% pure) daily (7 days/week) via oral gavage at doses of
0, 62.5, 250 and 1000 mg/kg bw per day. Satellite control and treatment groups
(six males and five females per group) were included for a 14-day recovery
period following the cessation of treatment. Treatment started 14 days prior
to mating and continued until the end of the 14-day mating period (males) or
until day 4 of lactation (females). Reproductive parameters assessed included:
estrous cycles, pairing numbers with successful copulation and conceiving days,
fertility index, length of gestation, number of corpora lutea, implantation scars
and implantation index. The developmental parameters analysed were: number
of pups born, stillbirths, sex ratio at birth, birth index, live birth index, number
of live pups on day 4 of lactation, sex ratio on day 4 of lactation, viability index,
external abnormalities and pup body weight. Treatment appeared to be well
tolerated in animals given 62.5 and 250 mg/kg bw per day. However, following
administration of 1000 mg/kg bw per day, male and female animals showed
transient salivation. Changes in haematology and clinical biochemistry were
limited to animals treated with 1000 mg/kg bw per day and consisted of increased
reticulocytes and monocytes in females following treatment but not following

recovery, and increased alanine aminotransferase and cholesterol in males after
treatment and recovery. Following terminal necropsy, males given 1000 mg/kg
bw per day showed increased absolute and relative liver and kidney weights (not
observed following recovery) and increased relative testes weights (after recovery
only). Females given the high dose also showed increased relative liver and
kidney weights (absolute liver weights were only measured following recovery)
and increased relative and absolute adrenal weights (not observed following
recovery). Following histopathological examination, an increased incidence of
hyaline droplets in the renal tubules was observed in males dosed with 250 and
1000 mg/kg bw per day following treatment (0/6, 0/6, 4/6 and 6/6 in the 0, 62.5,
314
250 and 1000 mg/kg bw per day dose groups, respectively; not observed following
recovery). No effects were observed in females that received 250 mg/kg bw per
day. However, males dosed with 62.5 and 250 mg/kg bw per day also showed
a slight increase in cholesterol following treatment (not statistically significant
following recovery and within historical control limits reported by Lee et al.,
2012). No significant effects on reproduction or development were noted at any
dose. Despite being exposed for longer, female rats appeared to be less sensitive
to the effects of methylcyclohexane exposure than male rats.
According to the OECD (2014) summary of this study, immuno-
histochemical examination for α-2u-globulin in the kidneys of rats in the
control and high dose (1000 mg/kg bw per day) group, revealed similar levels
of α-2u-globulin (positive controls confirmed assay function). Although α-2u-
globulin-related nephropathy in male rats is a common effect associated with
repeated exposures to hydrocarbon solvents (McKee, Adenuga & Carrillo, 2015),
the Committee concluded that the evidence of α-2u-globulin -related effects in
rats exposed to methylcyclohexane is incomplete. In the absence of definitive
evidence for α-2u-globulin-associated effects, and considering that hyaline
droplet accumulation was also observed in female rats given the high dose,
the renal effects observed in male rats are considered relevant for the current
evaluation. A NOAEL of 62.5 mg/kg bw per day was identified.
3.2.4 Genotoxicity
According to the available information, cyclohexane is not genotoxic in vitro
or in vivo. Similarly, methylcyclohexane and cyclohexanol are not genotoxic in
vitro. To add to the weight of evidence for the negative in vivo genotoxicity of
cyclohexane, it is also noted that cyclohexanone is nongenotoxic in vitro and in
vivo, and all four substances lack structural alerts for mutagenicity/genotoxicity.
The genotoxicity data that form the basis of these conclusions are summarized in
Tables 20 to 23.
3.2.5 Allergenicity
Repeated exposure to cyclohexane did not produce delayed contact hyper-
sensitivity in guinea-pigs assessed by the modified Buehler method (ECHA,
2020f). No data on the allergenic potential of methylcyclohexane were available.
3.2.6 Impurities
Some of the impurities identified in section 2.2 are associated with carcinogenicity
(for example, benzene and PAH (IARC, 2018)).
315
Table 20
Summary of in vitro and in vivo genotoxicity studies on cyclohexane
Gene mutation in Salmonella Typhimurium 0, 10, 33, 100, 333, 1000, 3333, 10 000 Negative (S9+) ECHA (2020f);
(TA98, TA100, TA1535 and TA1537) µg/plate Negative (S9−) Mortelmans et al. (1986)
Gene mutation in Salmonella Typhimurium 7 graded doses 7.1 to 5200 µg/plate Negative (not specified if US EPA (2003)
(TA98, TA100, TA1535, TA1537 and TA1538) with or without S9)
Gene mutation in Salmonella Typhimurium Up to 1000 µg/plate Negative (S9+) McCann et al. (1975)
(TA98, TA100, TA1535 and TA1537) Negative (S9−)
Gene mutation in Salmonella Typhimurium 25, 50, 100, 200, 500 µL/plate Negative Maron,
TA100 Katzenellenbogen &
Ames (1981)
Gene mutation in mouse lymphoma cells Up to 100 µg/mL Negative (S9+) US EPA (2003)
Negative (S9−)
Gene mutation in mouse lymphoma cells 313, 625, 1250, 2500, 3000, 4000, Negative (S9+) ECHA (2020f)
(L5178Y) 5000, 6000, 7000 and 8000 nL/mL Negative (S9−)
Sister chromatid exchange test in Chinese Up to 25 µg/mL Negative (S9+) US EPA (2003)
hamster ovary cells Negative (S9−)
Unscheduled DNA synthesis in human 0, 0.01, 0.001 and 0.0001 mol/L Negative (S9+) ECHA (2020f); Perocco,
lymphocytes Negative (S9−) Bolognesi & Alberghini
(1983)
DNA binding assay in Escherichia coli Q13 10 and 100 µM Equivocal Kubinski, Gutzke &
Kubinski (1981)
Bone marrow chromosome aberration 0, 100, 300, 1000 ppm via inhalation Negative ECHA (2020f)
test in male and female CRL:COBS CD(SD) route for 5 days (6 hours per day)
BR rats
Table 21
Summary of in vitro and in vivo genotoxicity studies on methylcyclohexane

Gene mutation in Salmonella Typhimurium 0.0977 to 25 µg/plate (S9−) Negative (S9−) ECHA (2020e)
(TA98, TA100, TA1535 and TA1537) 0.781 to 200 µg/plate (S9+) Negative (S9+)
Gene mutation in Escherichia coli WP2 uvrA 0.0977 to 25 µg/plate (S9−) Negative (S9−) ECHA (2020e)
0.781 to 200 µg/plate (S9+) Negative (S9+)
Chromosome aberration test in Chinese 0, 245, 490 and 980 µg/L Negative after short-term treatment ECHA (2020e)
hamster lung (CHL/IU) cells (6 hours) (S9− and S9+)
Negative after continuous treatment
(24 and 48 hours) (S9−)
316
Table 22
Summary of in vitro and in vivo genotoxicity studies on cyclohexanol
Gene mutation in Salmonella Typhimurium 0, 500, 1000, 2500, 5000, 7500, 10 000, Negative (S9+) ECHA (2020g)
(TA98, TA1535, TA1537 and TA1538) 15 000 µg/plate Negative (S9−)
Gene mutation in mouse lymphoma cells 0, 62.5, 125, 250, 500, 1000 µg/mL Negative (S9+) ECHA (2020g)
(L5178Y) Negative (S9−)
Bone marrow chromosome aberration 0, 500, 1000, 1500 mg/kg bw via Negative at all sacrifice ECHA (2020g)
test in male and female CRL:COBS CD(SD) gavage (single dose) intervals (16, 24, 48
BR rats hours)
Table 23
Summary of in vitro and in vivo genotoxicity studies on cyclohexanone
Gene mutation in Salmonella Typhimurium At least five concentrations tested (not Negative (S9−) ECHA (2020h);
(TA98, TA100, TA1535 and TA1537) specified); highest concentration was 10 Negative (S9+) Haworth et al. (1983)
mg/plate in absence of toxicity
Gene mutation in Salmonella Typhimurium 0 and 3 µmol/plate Negative (S9−) ECHA (2020h); Florin
(TA98 and TA100) Negative (S9+) et al. (1980)
Gene mutation in Salmonella Typhimurium 10 to 5000 µg/plate (standard plate test) Negative (S9−) ECHA (2020h)
(TA98, TA100, TA1535 and TA1537) 10 to 1000 µg/plate (pre-incubation test) Negative (S9+)
Gene mutation in Escherichia coli WP2 uvrA 10 to 5000 µg/plate (standard plate test) Negative (S9−) ECHA (2020h)
10 to 1000 µg/plate (pre-incubation test) Negative (S9+)
Unscheduled DNA synthesis in human Up to 9.48 mg/mL Negative (S9−) ECHA (2020h)
fibroblasts Negative (S9+)
Gene mutation in Chinese hamster ovary Experiment 1: 0, 122.5, 245, 490, 980 µg/ Negative in both ECHA (2020h)
cells mL (4-hour exposure with and without experiments (S9−)
S9) Negative in both
Experiment 2: 0, 61.3, 122.5, 245, 490, experiments (S9+)
980 µg/mL (24-hour exposure without S9)
and 0, 100, 200, 400, 980 µg/mL (4-hour
exposure with S9)
Comet assay in human epidermal skin Up to 1600 µg/cm2 Negative ECHA (2020h); Reus et
model al. (2013)
Gene mutation in mouse lymphoma cells 0, 312.5, 625, 1250, 2500, 500 µg/mL Negative (S9−) ECHA (2020h)
(L5178Y) Negative (S9+)
[3H]TdR uptake study in human 0, 0.01, 0.001, 0.0001 mol/L Negative (S9−) ECHA (2020h); Perocco,
lymphocytes Negative (S9+) Bolognesi & Alberghini
(1983)
HPRT assay in Chinese hamster ovary cells 2.5 to 12.5 µL/mL Negative (S9−) ECHA (2020h)
Negative (S9+)
317
Bone marrow chromosome aberration test 0, 50, 400 ppm via inhalation route for 1 Negative ECHA (2020h)
in male and female CD rats or 5 days (7 hours per day)
Rodent dominant lethal test in male and 0, 50, 400 ppm via inhalation route for 1 Negative ECHA (2020h)
female CD rats or 5 days (7 hours per day)
Sex-linked recessive lethal test in male and 0 or 36% saturation (~1900 ppm) via Negative ECHA (2020h)
female Drosophila melanogaster inhalation route for 1 day (4 hours per
day)
Sex-linked recessive lethal test in male and 50 ppm via inhalation route for 1 day (1, 3 Negative ECHA (2020h)
female Drosophila melanogaster or 6.75 hours per day)
400 ppm via inhalation route for 1 day
(10, 20 or 70 minutes per day)

Very limited information on exposure to cyclohexane via the oral route is available.
According to a brief entry in the Encyclopedia of toxicology (Gad, 2014), acute
ingestion of cyclohexane may cause sore throat, nausea, diarrhoea or vomiting,
whereas prolonged exposure may produce liver and kidney damage. The basis for
this statement was not provided. However, owing to its use in the occupational
environment, the effects of cyclohexane following inhalation exposure in humans
have been studied.
In the ECB (2004) assessment, the results of a double-blind, two-way
cross-over study in human volunteers (summary results later published in Hissink
et al., 2009 and Lammers et al., 2009) were used to identify an inhalation NOAEC
of 250 ppm (860 mg/m3) cyclohexane for neurobehavioural effects in humans.
Twelve male volunteers (ages ranging from 20 to 40 years; weight 66 to 95 kg) were
exposed to 0, 25 or 250 ppm (equivalent to 0, 86 or 860 mg/m3) cyclohexane for
4 hours in a specially constructed temperature and relative humidity-controlled
exposure room. Automated neurobehavioural tests and questionnaires were

administered prior to exposure, during, and approximately 60 minutes after
the exposure. Mean blood concentrations of cyclohexane, sampled near the
end of the exposure period, were 55 and 618 ng/ml, respectively, for the 25 and
250 ppm dose groups. No adverse neurobehavioural effects were reported;
however, headaches and eye and throat irritation were reported more frequently
after exposure to 250 ppm (860 mg/m3) cyclohexane.
Yasugi et al. (1994) investigated the effects of cyclohexane exposure
in female workers (n = 33 exposed; mean age ~25 years) who were in close
proximity to an automated spraying system used for glue application. The glues
used in this factory were reported to contain at least 75% cyclohexane and up
318
to 15.8% toluene. According to Yasugi et al. (1994) the workers were exposed
to mean concentrations of approximately 27 ppm cyclohexane (maximum
concentration 274 ppm). Cyclohexane exposure was correlated with “end of
shift” (~8 hours) cyclohexanol and cyclohexanone concentrations in the workers’
blood and urine. The workers did not report any significant subjective symptoms
apart from “dimmed vision” and “unusual smell”. Haematology appeared normal;
serum biochemistry was within the normal limits for liver and kidney function;
and no significant increases in sister chromatid exchange were observed. Yuasa
et al. (1996) reported that following 1.2 years of exposure to cyclohexane, no
adverse effects on the peripheral nervous system were evident in 18 women (ages
19 to 56 years) exposed to glue containing 75% cyclohexane, 12% toluene and
0.9% n-hexane. Twelve of these women were reported to have been exposed to
n-hexane for a median of 2.8 years previously.

Cyclohexane may be used as an extraction solvent in the preparation
of flavourings from natural flavouring materials, at levels up to 1 mg/kg in food
in the EU (2009/32/EC) or as a diluent in colour additive mixtures in the United
States (21 CFR 73.1). However, no estimates of cyclohexane concentrations in
foods or of exposure from these sources were identified.
No data were found on concentrations of cyclohexane in food oils due
day, based on a worst-case concentration of 100 mg/kg and an oil intake of
3 g/kg bw per day by infants and young children who are high consumers (see
section A4.3).  
5. Comments

The chemical and technical considerations for cyclohexane are summarized in
Table 24.
319
Table 24
Chemical and technical considerations for cyclohexane
Name: Cyclohexane
110-82-7 None
Chemical details Cyclohexane; hexamethylene; hexanaphthene
Colourless, clear, flammable liquid, with characteristic odour

Melting point: 4–7 °C
Insoluble in water; soluble or miscible with ethanol, methanol, diethyl ether, acetone, benzene and
carbon tetrachloride.
Route(s) of synthesis Produced by hydrogenation of benzene in either the liquid or the vapour phase in the presence of
hydrogen or by fractional distillation of petroleum.
Composition Cyclohexane produced by hydrogenation of benzene may contain residues of benzene, whereas
cyclohexane from petroleum contains residues of hydrocarbons of similar volatility, mainly C5–C7,
possibly including benzene. Cyclohexane may contain residues of sulfur and polycyclic aromatic
hydrocarbons.
Uses Used as a solvent for lacquers and resins, as a paint and varnish remover, as an extraction solvent in
the preparation of flavouring agents, in industrial recrystallization of steroids, and in the manufacture
of adipic acid and caprolactam that is used in the production of polyamide (nylon) for food packaging
materials.
Analytical methods None identified for previous cargoes. Cyclohexane can be analysed by GC-FID or GC-MS.
Potential reaction(s) with a It is not expected to react with edible fats and oils.
subsequent cargo of fat or oil

Following oral exposure, cyclohexane undergoes hydroxylation to cyclohexanol

and is excreted in the urine as glucuronide conjugates or expired as carbon dioxide
or unchanged parent compound (Elliott, Parke & Williams, 1959). Cyclohexane
administered orally to rats is eliminated from the plasma and tissues with a
measured elimination half-life of 10–15 hours (ECHA, 2020f). Toxicokinetic
information on methylcyclohexane, a structural analogue of cyclohexane,
indicates that methylcyclohexane also undergoes hydroxylation and excretion as
a glucuronide or is expired as carbon dioxide or unchanged parent compound
(Elliott, Tao & Williams, 1965). However, due to its lower volatility, it is estimated
that significantly less unchanged methylcyclohexane is excreted in the expired
air following ingestion compared to cyclohexane (Elliott, Tao & Williams, 1965)
320
and greater systemic exposure to methylcyclohexane may be expected compared

to cyclohexane. Both cyclohexane and methylcyclohexane have a high affinity for
adipose tissues.

Cyclohexane exhibits low acute oral toxicity in rodents, with an estimated LD50 of
> 5000 mg/kg bw (ECB, 2004). Methylcyclohexane exhibits a slightly higher acute
oral toxicity with reported oral LD50 values of ≥1200 mg/kg bw (PubChem1).
Based on oral LD50 values in rabbits Treon, Crutchfield & Kitzmiller (1943)
suggested that toxicity decreases from ketone (for example, cyclohexanone)
to alcohol (for example, cyclohexanol) to hydrocarbon (cyclohexane) and
that the methylated compounds (i.e. methylcyclohexane, methylcyclohexanol
and methylcyclohexanone) are more hazardous than the corresponding non-
methylated compounds (i.e. cyclohexane, cyclohexanol and cyclohexanone).
No information on the short-term or long-term oral toxicity of cyclohexane
via the oral route was identified. Following short-term exposure via inhalation,
mice and rats showed increased liver weights and centrilobular hypertrophy at
concentrations of between 6000 and 7000 ppm (US EPA, 2003). Bernard et al.
(1989) observed evidence of nephrotoxicity in female rats following exposure
to 400 mg/kg bw per day via intraperitoneal injection, 5 days/week for 2 weeks,
and suggested that the nephrotoxic effects of cyclohexane are likely to be due to
cyclohexanol.
In a guideline-compliant 28-day repeated dose oral toxicity study
(OECD 407; GLP) methylcyclohexane was administered via gavage to groups
of rats at doses of 0, 100, 300 and 1000 mg/kg bw per day (ECHA, 2020e). At
1000 mg/kg bw per day, male and female animals showed transient salivation
after dosing, changes in clinical chemistry parameters and histopathological
changes in the liver (hepatocellular hypertrophy) and kidney (hyaline droplet
degeneration). Effects observed at the intermediate dose of 300 mg/kg bw per
day were restricted to the males and consisted of transient salivation after dosing,
increased body weight and food consumption, and hyaline droplet degeneration
in the kidney. With the exception of slight hyaline droplet degeneration observed
in one male, no other toxicologically relevant effects were observed in animals
dosed at 100 mg/kg bw per day.
In a combined repeated dose toxicity study with the Reproduction/
Developmental Toxicity Screening Test (OECD 422; GLP), rats were administered
methylcyclohexane daily via oral gavage at doses of 0, 62.5, 250 and 1000 mg/kg
bw per day (ECHA, 2020e). Once again, male and female animals exposed to
1
https://pubchem.ncbi.nlm.nih.gov/compound/Methylcyclohexane#section=NIOSH-Toxicity-Data
321
1000 mg/kg bw per day showed transient salivation after dosing and evidence of
liver (increased absolute and relative organ weights) and kidney effects (increased
relative organ weight). Toxicologically significant effects observed at 250 mg/kg
bw per day (i.e. hyaline droplets in the renal tubules) were limited to male rats.
No significant effects on reproduction or development were noted at any dose.
According to the OECD (2014) summary of this study, immunohistochemical
examination for α2u-globulin in the kidneys of rats in the control and high-dose
(1000 mg/kg bw per day) groups, revealed similar levels of α2u-globulin (positive
controls confirmed assay function). Although α2u-globulin-related nephropathy
in male rats is a common effect associated with repeated exposures to hydrocarbon
solvents (McKee, Adenuga & Carrillo, 2015), the Committee concluded that the
evidence on α2u-globulin-related effects in rats exposed to methylcyclohexane
is incomplete. In the absence of definitive evidence for α2u-globulin-associated
effects, and considering that hyaline droplet accumulation was also observed in
female rats in the high-dose group, the renal effects observed in male rats were
considered relevant for the current evaluation.
The Committee also concluded that cyclohexane is non-genotoxic in
vitro and in vivo.
5.4 Allergenicity
The Committee did not identify any reports that indicated that cyclohexane or
methylcyclohexane elicits an allergenic response upon oral exposure. There are
also no data available that indicate that cyclohexane would contain a known food
allergen.
5.5 Impurities
Cyclohexane transported as a previous cargo may contain benzene, but is unlikely
to contain more than 0.1% benzene (EFSA, 2012a). The estimated exposure to
benzene present at a concentration of 0.1% in cyclohexane carried as a previous
cargo is 0.3 µg/kg bw per day. The major route of exposure to benzene is inhalation,
rather than diet (WHO, 2000; Duarte-Davidson et al., 2001). Estimated exposures
to benzene in the United Kingdom range from 0.71 µg/kg bw per day for children
in rural areas to 14.12 µg/kg bw per day for adult smokers in urban areas who
work adjacent to busy roads (Duarte-Davidson et al., 2001). Estimates of dietary
exposure range from 1.4 to 2.8 µg/day (WHO, 2000), equivalent to 0.02–
0.05 µg/kg bw per day for adults weighing 60 kg. The estimated maximum dietary
exposure to benzene present in fats and oils when cyclohexane is carried as a
322
previous cargo, 0.3 µg/kg bw per day, is below the estimated range of inhalation
exposures, although it is high compared to estimated total dietary exposures.
Cyclohexane may also contain PAH residues, but these residues have
neither been characterized nor quantified, and exposures to PAH substances in
cyclohexane carried as a previous cargo therefore cannot be estimated.

Cyclohexane may be used as an extraction solvent in the preparation of flavouring
agents from natural flavouring materials, at levels up to 1 mg/kg in food in
the EU (2009/32/EC) or as a diluent in colour additive mixtures in the United
States (21 CFR 73.1). However, no estimates of cyclohexane concentrations in
foods or of exposure from these sources were identified.
No data were found on concentrations of cyclohexane in food oils due
A4.3).  
6. Evaluation
No information regarding the short-term and long-term toxicity of cyclohexane
was identified; however, cyclohexane exhibits relatively low systemic toxicity
following short-term exposure via inhalation. The Committee identified a
NOAEL of 62.5 mg/kg bw per day from two short-term oral toxicity studies
with the structural analogue methylcyclohexane. Cyclohexane may be used as
an extraction solvent for flavouring agents or as a diluent in colour additive
mixtures. However, no estimates of cyclohexane concentrations in foods or
of exposure from these sources were identified. A comparison of the NOAEL
of 62.5 mg/kg bw per day with the estimated generic human dietary exposure
value for previous cargoes of 0.3 mg/kg bw per day yields an MOE of 208. The
Committee noted that this MOE is based on a potentially more toxic compound
(Treon, Crutchfield & Kitzmiller, 1943) and a sensitive critical effect (hyaline
droplets in the renal tubules of male rats). In consideration of the conservative
nature of both the exposure and hazard metrics used, the Committee concluded
that this MOE is sufficient to address the uncertainties in the database.
323
There are no data on allergenicity upon oral exposure to cyclohexane

Cyclohexane as a previous cargo is not expected to react with edible fats
and oils.
Although exposure to cyclohexane as a result of transporting cyclohexane
as a previous cargo does not appear to be a health concern, there is uncertainty
concerning the purity or “grade” of cyclohexane that will be transported as
a previous cargo. Since cyclohexane may contain carcinogenic impurities in
amounts that could significantly increase dietary exposure, the Committee could
not reach a conclusion on the safety of transporting cyclohexane as a previous
cargo for edible fats and oils until the nature and the quantities of these impurities
in cyclohexane have been clarified.
7. Recommendations
The Committee recommended that sufficient chemical information that allows
the evaluation of acetic anhydride and cyclohexane transported as previous
cargoes be made available prior to the next evaluation. At a minimum this
information should address the following:
■■ product grade(s) and composition, including characterization and
levels of impurities arising from all methods of manufacture.
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335
ANNEX 1
Reports and other documents resulting from previous

meetings of the Joint FAO/WHO Expert Committee on
Food Additives
1. General principles governing the use of food additives (First report of the Joint FAO/WHO Expert Committee
on Food Additives). FAO Nutrition Meetings Report Series, No. 15, 1957; WHO Technical Report Series, No. 129,
1957 (out of print).
2. Procedures for the testing of intentional food additives to establish their safety for use (Second report of the
Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings Report Series, No. 17, 1958; WHO
Technical Report Series, No. 144, 1958 (out of print).
3. Specifications for identity and purity of food additives (antimicrobial preservatives and antioxidants) (Third
report of the Joint FAO/WHO Expert Committee on Food Additives). These specifications were subsequently
revised and published as Specifications for identity and purity of food additives, Vol. I. Antimicrobial preservatives
and antioxidants, Rome, Food and Agriculture Organization of the United Nations, 1962 (out of print).
4. Specifications for identity and purity of food additives (food colours) (Fourth report of the Joint FAO/WHO Expert
Committee on Food Additives). These specifications were subsequently revised and published as Specifications
for identity and purity of food additives, Vol. II. Food colours, Rome, Food and Agriculture Organization of the
United Nations, 1963 (out of print).
5. Evaluation of the carcinogenic hazards of food additives (Fifth report of the Joint FAO/WHO Expert Committee
on Food Additives). FAO Nutrition Meetings Report Series, No. 29, 1961; WHO Technical Report Series, No. 220,
1961 (out of print).
6. Evaluation of the toxicity of a number of antimicrobials and antioxidants (Sixth report of the Joint FAO/WHO
Expert Committee on Food Additives). FAO Nutrition Meetings Report Series, No. 31, 1962; WHO Technical
Report Series, No. 228, 1962 (out of print).
7. Specifications for the identity and purity of food additives and their toxicological evaluation: emulsifiers,
stabilizers, bleaching and maturing agents (Seventh report of the Joint FAO/WHO Expert Committee on Food
Additives). FAO Nutrition Meetings Series, No. 35, 1964; WHO Technical Report Series, No. 281, 1964 (out of
print).
8. Specifications for the identity and purity of food additives and their toxicological evaluation: food colours
and some antimicrobials and antioxidants (Eighth report of the Joint FAO/WHO Expert Committee on Food
Additives). FAO Nutrition Meetings Series, No. 38, 1965; WHO Technical Report Series, No. 309, 1965 (out of
print).
9. Specifications for identity and purity and toxicological evaluation of some antimicrobials and antioxidants. FAO
Nutrition Meetings Report Series, No. 38A, 1965; WHO/Food Add/24.65 (out of print).
10. Specifications for identity and purity and toxicological evaluation of food colours. FAO Nutrition Meetings
Report Series, No. 38B, 1966; WHO/Food Add/66.25.
337
11. Specifications for the identity and purity of food additives and their toxicological evaluation: some antimicrobials,
antioxidants, emulsifiers, stabilizers, flour treatment agents, acids, and bases (Ninth report of the Joint FAO/
WHO Expert Committee on Food Additives). FAO Nutrition Meetings Series, No. 40, 1966; WHO Technical Report
Series, No. 339, 1966 (out of print).
12. Toxicological evaluation of some antimicrobials, antioxidants, emulsifiers, stabilizers, flour treatment agents,
acids, and bases. FAO Nutrition Meetings Report Series, No. 40A, B, C; WHO/Food Add/67.29.
13. Specifications for the identity and purity of food additives and their toxicological evaluation: some emulsifiers
and stabilizers and certain other substances (Tenth report of the Joint FAO/WHO Expert Committee on Food
Additives). FAO Nutrition Meetings Series, No. 43, 1967; WHO Technical Report Series, No. 373, 1967.
14. Specifications for the identity and purity of food additives and their toxicological evaluation: some flavouring
substances and non nutritive sweetening agents (Eleventh report of the Joint FAO/WHO Expert Committee on
Food Additives). FAO Nutrition Meetings Series, No. 44, 1968; WHO Technical Report Series, No. 383, 1968.
15. Toxicological evaluation of some flavouring substances and non nutritive sweetening agents. FAO Nutrition
Meetings Report Series, No. 44A, 1968; WHO/Food Add/68.33.
16. Specifications and criteria for identity and purity of some flavouring substances and non-nutritive sweetening
agents. FAO Nutrition Meetings Report Series, No. 44B, 1969; WHO/Food Add/69.31.
17. Specifications for the identity and purity of food additives and their toxicological evaluation: some antibiotics
(Twelfth report of the Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings Series, No.
45, 1969; WHO Technical Report Series, No. 430, 1969.
18. Specifications for the identity and purity of some antibiotics. FAO Nutrition Meetings Series, No. 45A, 1969;
WHO/Food Add/69.34.
19. Specifications for the identity and purity of food additives and their toxicological evaluation: some food colours,
emulsifiers, stabilizers, anticaking agents, and certain other substances (Thirteenth report of the Joint FAO/
WHO Expert Committee on Food Additives). FAO Nutrition Meetings Series, No. 46, 1970; WHO Technical Report
Series, No. 445, 1970.
20. Toxicological evaluation of some food colours, emulsifiers, stabilizers, anticaking agents, and certain other
substances. FAO Nutrition Meetings Report Series, No. 46A, 1970; WHO/Food Add/70.36.
21. Specifications for the identity and purity of some food colours, emulsifiers, stabilizers, anticaking agents, and
certain other food additives. FAO Nutrition Meetings Report Series, No. 46B, 1970; WHO/Food Add/70.37.
22. Evaluation of food additives: specifications for the identity and purity of food additives and their toxicological
evaluation: some extraction solvents and certain other substances; and a review of the technological efficacy of
some antimicrobial agents (Fourteenth report of the Joint FAO/WHO Expert Committee on Food Additives). FAO
Nutrition Meetings Series, No. 48, 1971; WHO Technical Report Series, No. 462, 1971.
23. Toxicological evaluation of some extraction solvents and certain other substances. FAO Nutrition Meetings
Report Series, No. 48A, 1971; WHO/Food Add/70.39.
24. Specifications for the identity and purity of some extraction solvents and certain other substances. FAO Nutrition
Meetings Report Series, No. 48B, 1971; WHO/Food Add/70.40.
25. A review of the technological efficacy of some antimicrobial agents. FAO Nutrition Meetings Report Series, No.
48C, 1971; WHO/Food Add/70.41.
26. Evaluation of food additives: some enzymes, modified starches, and certain other substances: Toxicological
evaluations and specifications and a review of the technological efficacy of some antioxidants (Fifteenth report
338
Annex 1
of the Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings Series, No. 50, 1972; WHO
Technical Report Series, No. 488, 1972.
27. Toxicological evaluation of some enzymes, modified starches, and certain other substances. FAO Nutrition
Meetings Report Series, No. 50A, 1972; WHO Food Additives Series, No. 1, 1972.
28. Specifications for the identity and purity of some enzymes and certain other substances. FAO Nutrition Meetings
Report Series, No. 50B, 1972; WHO Food Additives Series, No. 2, 1972.
29. A review of the technological efficacy of some antioxidants and synergists. FAO Nutrition Meetings Report
Series, No. 50C, 1972; WHO Food Additives Series, No. 3, 1972.
30. Evaluation of certain food additives and the contaminants mercury, lead, and cadmium (Sixteenth report of
the Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings Series, No. 51, 1972; WHO
Technical Report Series, No. 505, 1972, and corrigendum.
31. Evaluation of mercury, lead, cadmium and the food additives amaranth, diethylpyrocarbamate, and octyl
gallate. FAO Nutrition Meetings Report Series, No. 51A, 1972; WHO Food Additives Series, No. 4, 1972.
32. Toxicological evaluation of certain food additives with a review of general principles and of specifications
(Seventeenth report of the Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings Series,
No. 53, 1974; WHO Technical Report Series, No. 539, 1974, and corrigendum (out of print).
33. Toxicological evaluation of some food additives including anticaking agents, antimicrobials, antioxidants,
emulsifiers, and thickening agents. FAO Nutrition Meetings Report Series, No. 53A, 1974; WHO Food Additives
Series, No. 5, 1974.
34. Specifications for identity and purity of thickening agents, anticaking agents, antimicrobials, antioxidants and
emulsifiers. FAO Food and Nutrition Paper, No. 4, 1978.
35. Evaluation of certain food additives (Eighteenth report of the Joint FAO/WHO Expert Committee on Food
Additives). FAO Nutrition Meetings Series, No. 54, 1974; WHO Technical Report Series, No. 557, 1974, and
corrigendum.
36. Toxicological evaluation of some food colours, enzymes, flavour enhancers, thickening agents, and certain other
food additives. FAO Nutrition Meetings Report Series, No. 54A, 1975; WHO Food Additives Series, No. 6, 1975.
37. Specifications for the identity and purity of some food colours, enhancers, thickening agents, and certain food
additives. FAO Nutrition Meetings Report Series, No. 54B, 1975; WHO Food Additives Series, No. 7, 1975.
38. Evaluation of certain food additives: some food colours, thickening agents, smoke condensates, and certain
other substances. (Nineteenth report of the Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition
Meetings Series, No. 55, 1975; WHO Technical Report Series, No. 576, 1975.
39. Toxicological evaluation of some food colours, thickening agents, and certain other substances. FAO Nutrition
Meetings Report Series, No. 55A, 1975; WHO Food Additives Series, No. 8, 1975.
40. Specifications for the identity and purity of certain food additives. FAO Nutrition Meetings Report Series, No.
55B, 1976; WHO Food Additives Series, No. 9, 1976.
41. Evaluation of certain food additives (Twentieth report of the Joint FAO/WHO Expert Committee on Food
Additives). FAO Food and Nutrition Meetings Series, No. 1, 1976; WHO Technical Report Series, No. 599, 1976.
42. Toxicological evaluation of certain food additives. WHO Food Additives Series, No. 10, 1976.
43. Specifications for the identity and purity of some food additives. FAO Food and Nutrition Series, No. 1B, 1977;
WHO Food Additives Series, No. 11, 1977.
339
44. Evaluation of certain food additives (Twenty-first report of the Joint FAO/WHO Expert Committee on Food
Additives). WHO Technical Report Series, No. 617, 1978.
45. Summary of toxicological data of certain food additives. WHO Food Additives Series, No. 12, 1977.
46. Specifications for identity and purity of some food additives, including antioxidant, food colours, thickeners, and
others. FAO Nutrition Meetings Report Series, No. 57, 1977.
47. Evaluation of certain food additives and contaminants (Twenty-second report of the Joint FAO/WHO Expert
Committee on Food Additives). WHO Technical Report Series, No. 631, 1978.
48. Summary of toxicological data of certain food additives and contaminants. WHO Food Additives Series, No. 13,
1978.
49. Specifications for the identity and purity of certain food additives. FAO Food and Nutrition Paper, No. 7, 1978.
50. Evaluation of certain food additives (Twenty-third report of the Joint FAO/WHO Expert Committee on Food
Additives). WHO Technical Report Series, No. 648, 1980, and corrigenda.
52. Specifications for identity and purity of food colours, flavouring agents, and other food additives. FAO Food and
Nutrition Paper, No. 12, 1979.
53. Evaluation of certain food additives (Twenty-fourth report of the Joint FAO/WHO Expert Committee on Food
55. Specifications for identity and purity of food additives (sweetening agents, emulsifying agents, and other food
additives). FAO Food and Nutrition Paper, No. 17, 1980.
56. Evaluation of certain food additives (Twenty-fifth report of the Joint FAO/WHO Expert Committee on Food
58. Specifications for identity and purity of food additives (carrier solvents, emulsifiers and stabilizers, enzyme
preparations, flavouring agents, food colours, sweetening agents, and other food additives). FAO Food and
Nutrition Paper, No. 19, 1981.
59. Evaluation of certain food additives and contaminants (Twenty-sixth report of the Joint FAO/WHO Expert
62. Evaluation of certain food additives and contaminants (Twenty-seventh report of the Joint FAO/WHO Expert
Committee on Food Additives). WHO Technical Report Series, No. 696, 1983, and corrigenda.
63. Toxicological evaluation of certain food additives and contaminants. WHO Food Additives Series, No. 18, 1983.
65. Guide to specifications – General notices, general methods, identification tests, test solutions, and other
reference materials. FAO Food and Nutrition Paper, No. 5, Rev. 1, 1983.
66. Evaluation of certain food additives and contaminants (Twenty-eighth report of the Joint FAO/WHO Expert
Committee on Food Additives). WHO Technical Report Series, No. 710, 1984, and corrigendum.
340
Annex 1
68. Specifications for the identity and purity of food colours. FAO Food and Nutrition Paper, No. 31/1, 1984.
69. Specifications for the identity and purity of food additives. FAO Food and Nutrition Paper, No. 31/2, 1984.
70. Evaluation of certain food additives and contaminants (Twenty-ninth report of the Joint FAO/WHO Expert
72. Toxicological evaluation of certain food additives and contaminants. WHO Food Additives Series, No. 20.
Cambridge University Press, 1987.
73. Evaluation of certain food additives and contaminants (Thirtieth report of the Joint FAO/WHO Expert Committee
on Food Additives). WHO Technical Report Series, No. 751, 1987.
76. Principles for the safety assessment of food additives and contaminants in food. WHO Environmental Health
Criteria, No. 70. Geneva, World Health Organization, 1987 (out of print). The full text is available electronically
at www.who.int/pcs.
77. Evaluation of certain food additives and contaminants (Thirty-first report of the Joint FAO/WHO Expert
78. Toxicological evaluation of certain food additives. WHO Food Additives Series, No. 22. Cambridge University
Press, 1988.
80. Evaluation of certain veterinary drug residues in food (Thirty-second report of the Joint FAO/WHO Expert
81. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 23.
82. Residues of some veterinary drugs in animals and foods. FAO Food and Nutrition Paper, No. 41, 1988.
83. Evaluation of certain food additives and contaminants (Thirty-third report of the Joint FAO/WHO Expert
85. Evaluation of certain veterinary drug residues in food (Thirty-fourth report of the Joint FAO/WHO Expert
86. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 25, 1990.
87. Residues of some veterinary drugs in animals and foods. FAO Food and Nutrition Paper, No. 41/2, 1990.
88. Evaluation of certain food additives and contaminants (Thirty-fifth report of the Joint FAO/WHO Expert
341
90. Specifications for identity and purity of certain food additives. FAO Food and Nutrition Paper, No. 49, 1990.
91. Evaluation of certain veterinary drug residues in food (Thirty-sixth report of the Joint FAO/WHO Expert
94. Evaluation of certain food additives and contaminants (Thirty-seventh report of the Joint FAO/WHO Expert
96. Compendium of food additive specifications (Joint FAO/WHO Expert Committee on Food Additives (JECFA)).
Combined specifications from 1st through the 37th meetings, 1956–1990. Rome, Food and Agriculture
Organization of the United Nations, 1992 (2 volumes).
97. Evaluation of certain veterinary drug residues in food (Thirty-eighth report of the Joint FAO/WHO Expert
98. Toxicological evaluation of certain veterinary residues in food. WHO Food Additives Series, No. 29, 1991.
100. Guide to specifications – General notices, general analytical techniques, identification tests, test solutions, and
other reference materials. FAO Food and Nutrition Paper, No. 5, Ref. 2, 1991.
101. Evaluation of certain food additives and naturally occurring toxicants (Thirty-ninth report of the Joint FAO/WHO
Expert Committee on Food Additives). WHO Technical Report Series, No. 828, 1992.
102. Toxicological evaluation of certain food additives and naturally occurring toxicants. WHO Food Additives Series,
No. 30, 1993.
103. Compendium of food additive specifications: addendum 1. FAO Food and Nutrition Paper, No. 52, 1992.
104. Evaluation of certain veterinary drug residues in food (Fortieth report of the Joint FAO/WHO Expert Committee
106. Residues of some veterinary drugs in animals and food. FAO Food and Nutrition Paper, No. 41/5, 1993.
107. Evaluation of certain food additives and contaminants (Forty-first report of the Joint FAO/WHO Expert
109. Compendium of food additive specifications: addendum 2. FAO Food and Nutrition Paper, No. 52, Add. 2, 1993.
110. Evaluation of certain veterinary drug residues in food (Forty-second report of the Joint FAO/WHO Expert
113. Evaluation of certain veterinary drug residues in food (Forty-third report of the Joint FAO/WHO Expert Committee
on Food Additives). WHO Technical Report Series, No. 855, 1995, and corrigendum.
342
Annex 1
116. Evaluation of certain food additives and contaminants (Forty-fourth report of the Joint FAO/WHO Expert
119. Evaluation of certain veterinary drug residues in food (Forty-fifth report of the Joint FAO/WHO Expert Committee
122. Evaluation of certain food additives and contaminants (Forty-sixth report of the Joint FAO/WHO Expert
124. Compendium of food additive specifications, addendum 4. FAO Food and Nutrition Paper, No. 52, Add. 4, 1996.
125. Evaluation of certain veterinary drug residues in food (Forty-seventh report of the Joint FAO/WHO Expert
128. Evaluation of certain veterinary drug residues in food (Forty-eighth report of the Joint FAO/WHO Expert
131. Evaluation of certain food additives and contaminants (Forty-ninth report of the Joint FAO/WHO Expert
132. Safety evaluation of certain food additives and contaminants. WHO Food Additives Series, No. 40, 1998.
134. Evaluation of certain veterinary drug residues in food (Fiftieth report of the Joint FAO/WHO Expert Committee
137. Evaluation of certain food additives (Fifty-first report of the Joint FAO/WHO Expert Committee on Food
138. Safety evaluation of certain food additives. WHO Food Additives Series, No. 42, 1999.
343
140. Evaluation of certain veterinary drug residues in food (Fifty-second report of the Joint FAO/WHO Expert
143. Evaluation of certain food additives and contaminants (Fifty-third report of the Joint FAO/WHO Expert
146. Evaluation of certain veterinary drug residues in food (Fifty-fourth report of the Joint FAO/WHO Expert
149. Evaluation of certain food additives and contaminants (Fifty-fifth report of the Joint FAO/WHO Expert Committee
152. Evaluation of certain mycotoxins in food (Fifty-sixth report of the Joint FAO/WHO Expert Committee on Food
153. Safety evaluation of certain mycotoxins in food. WHO Food Additives Series, No. 47; FAO Food and Nutrition
Paper, No. 74, 2001.
154. Evaluation of certain food additives and contaminants (Fifty-seventh report of the Joint FAO/WHO Expert
157. Evaluation of certain veterinary drug residues in food (Fifty-eighth report of the Joint FAO/WHO Expert
160. Evaluation of certain food additives and contaminants (Fifty-ninth report of the Joint FAO/WHO Expert
162. Compendium of food additive specifications: addendum 10. FAO Food and Nutrition Paper, No. 52, Add. 10,
2002.
163. Evaluation of certain veterinary drug residues in food (Sixtieth report of the Joint FAO/WHO Expert Committee
344
Annex 1
166. Evaluation of certain food additives and contaminants (Sixty-first report of the Joint FAO/WHO Expert Committee
2003.
169. Evaluation of certain veterinary drug residues in food (Sixty-second report of the Joint FAO/WHO Expert
2004.
173. Evaluation of certain food additives (Sixty-third report of the Joint FAO/WHO Expert Committee on Food
175. Compendium of food additive specifications: addendum 13. FAO Food and Nutrition Paper, No. 52, Add. 13 (with
Errata), 2005.
176. Evaluation of certain food contaminants (Sixty-fourth report of the Joint FAO/WHO Expert Committee on Food
177. Safety evaluation of certain contaminants in food. WHO Food Additives Series, No. 55; FAO Food and Nutrition
Paper, No. 82, 2006.
178. Evaluation of certain food additives (Sixty-fifth report of the Joint FAO/WHO Expert Committee on Food
180. Combined compendium of food additive specifications. FAO JECFA Monographs 1, Volumes 1–4, 2005, 2006.
181. Evaluation of certain veterinary drug residues in food (Sixty-sixth report of the Joint FAO/WHO Expert Committee
182. Residue evaluation of certain veterinary drugs. FAO JECFA Monographs 2, 2006.
184. Evaluation of certain food additives and contaminants (Sixty-seventh report of the Joint FAO/WHO Expert
185. Compendium of food additive specifications. FAO JECFA Monographs 3, 2006.
187. Evaluation of certain food additives and contaminants (Sixty-eighth report of the Joint FAO/WHO Expert
345

190. Evaluation of certain food additives (Sixty-ninth report of the Joint FAO/WHO Expert Committee on Food
193. Evaluation of certain veterinary drug residues in food (Seventieth report of the Joint FAO/WHO Expert Committee
196. Evaluation of certain food additives (Seventy-first report of the Joint FAO/WHO Expert Committee on Food
199. Evaluation of certain contaminants in food (Seventy-second report of the Joint FAO/WHO Expert Committee on
Food Additives). WHO Technical Report Series, No. 959, 2011.
200. Safety evaluation of certain contaminants in food. WHO Food Additives Series, No. 63; FAO JECFA Monographs
8, 2011.
202. Evaluation of certain food additives and contaminants (Seventy-third report of the Joint FAO/WHO Expert
205. Evaluation of certain food additives and contaminants (Seventy-fourth report of the Joint FAO/WHO Expert
208. Evaluation of certain veterinary drug residues in food (Seventy-fifth report of the Joint FAO/WHO Expert
211. Evaluation of certain food additives (Seventy-sixth report of the Joint FAO/WHO Expert Committee on Food
214. Evaluation of certain food additives and contaminants (Seventy-seventh report of the Joint FAO/WHO Expert
346
Annex 1
217. Evaluation of certain veterinary drug residues in food (Seventy-eighth report of the Joint FAO/WHO Expert
220. Evaluation of certain food additives (Seventy-ninth report of the Joint FAO/WHO Expert Committee on Food
223. Evaluation of certain food additives and contaminants (Eightieth report of the Joint FAO/WHO Expert Committee
226. Evaluation of certain veterinary drug residues in food (Eighty-first report of the Joint FAO/WHO Expert
229. Safety evaluation of certain food additives and contaminants. Supplement 1: Non-dioxin-like polychlorinated
biphenyls. WHO Food Additives Series, No. 71-1, 2016.
230. Evaluation of certain food additives (Eighty-second report of the Joint FAO/WHO Expert Committee on Food
233. Evaluation of certain contaminants in food (Eighty-third report of the Joint FAO/WHO Expert Committee on Food
Additives) WHO Technical Report Series, No.1002, 2017.
234. Evaluation of certain food additives (Eighty-fourth report of the Joint FAO/WHO Expert Committee on Food
Additives), WHO Technical Report Series, No. 1007, 2017.
235. Safety evaluation of certain contaminants in food. WHO Food Additives Series, No. 74, FAO JECFA Monographs
19 bis, 2018.
238. Evaluation of certain veterinary drug residues in food (Eighty-fifth report of the Joint FAO/WHO Expert
347
242. Evaluation of certain food additives (Eighty-sixth report of the Joint FAO/WHO Expert Committee on Food
243. Evaluation of certain food additives (Eighty-seventh report of the Joint FAO/WHO Expert Committee on Food
Additives) WHO Technical Report Series, No. 1020, 2019.
244. Evaluation of veterinary drug residues in food (Eighty-eighth report of the Joint FAO/WHO Expert Committee on
Food Additives) WHO Technical Report Series, No. 1023, 2020.
245. Evaluation of certain food additives (Eighty-ninth report of the Joint FAO/WHO Expert Committee on Food
Additives) WHO Technical Report Series, No. 1027, 2021.
246. Evaluation of certain contaminants in food (Ninetieth report of the Joint FAO/WHO Expert Committee on Food
Additives) WHO Technical Teport Series, No. 1032, 2022.
348
ANNEX 2
Abbreviations and acronyms used in the monographs
24HDR 24-hour dietary recall

ADI acceptable daily intake
bw body weight
CAC Codex Alimentarius Commission
CAS Chemical Abstracts Service
CCCF Codex Committee on Contaminants in Foods
CCFO Codex Committee on Fats and Oils
CIFOCOss Chronic Individual Food Consumption Database – Summary
statistics
CHO Chinese hamster ovary
Cmax maximum concentration
CONTAM European Food Safety Authority (EFSA) Panel on Contaminants in
the Food Chain
CPSC Consumer Product Safety Commission
DMBA 7,12-dimethybenz[a]anthracene
EAT ergotamine tartrate
ECHA European Chemicals Agency
EFSA European Food Safety Authority
ELSO epoxidized linseed oil
EPA (United States) Environmental Protection Agency
ESBO epoxidized soybean oil
ETBE ethyl tertiary butyl ether
EU European Union
FAO Food and Agriculture Organization of the United Nations
GC-FID gas chromatography with flame ionization detection
GC-MS gas chromatography–mass spectrometry
FCID Food Commodity Intake Database (US Environmental Protection
Agency)
FFQ food frequency questionnaire
GEMS/Food Global Environment Monitoring System, Food Contamination
Monitoring and Assessment Programme
GF-AAS graphite furnace atomic absorption spectrometry
HBGV health-based guidance value
HPLC high-performance liquid chromatography
IMO International Maritime Organization
349
JECFA Joint FAO/WHO Expert Committee on Food Additives

LB lower bound
LC-GC-FID on-line coupled liquid chromatography-gas chromatography-flame
ionization detection
LC-HRMS liquid chromatography-high resolution mass spectrometry
LC-MS/MS liquid chromatography with tandem mass spectrometry
LD50 median lethal dose
LOD limit of detection
LOQ limit of quantification
LOR limits of reporting
MTBE methyl tertiary butyl ether
MOAH mineral oil aromatic hydrocarbons
MOE margin of exposure
MOH mineral oil hydrocarbons
MOSH mineral oil saturated hydrocarbons
MTDI maximum tolerable daily intake
NOAEL no-observed-adverse-effect level
OECD Organisation for Economic Co-operation and Development
P95 95th percentile
PBTK physiologically based toxicokinetic
PCB polychlorinated biphenyl
PTMI provisional tolerable monthly intake
QSAR quantitative structure-activity relationship
RP reference point
SCF EU Scientific Committee on Food
SIDS Screening Information Dataset
SPE solid phase extraction
TBA tertiary butyl alcohol
TDI tolerable daily intake
TLC thin layer chromatography
Tmax time to maximum concentration

TRS Technical Report Series
UB upper bound
UL upper intake level
USA United States of America
WHO World Health Organization
350
ANNEX 3
Participants in the ninetieth meeting of the Joint FAO/

WHO Expert Committee on Food Additives
Virtual meeting 1 to 12 February 2021
Members
Dr A. Agudo, Unit of Cancer and Nutrition, Catalan Institute of Oncology, Barcelona, Spain
Dr S. Barlow, Brighton, East Sussex, United Kingdom
Dr D.J. Benford, Cheddington (Bucks), United Kingdom (Vice-Chairperson)
Dr R.C. Cantrill, Halifax, Nova Scotia, Canada (Chairperson)
Mr P.J. Cressey, Institute of Environmental Science and Research Limited (ESR),
Christchurch, New Zealand
Mr M. Feeley, Ottawa, Canada (Joint Rapporteur)
Ms K.B. Laurvick, Food Standards, United States Pharmacopeia, Rockville (MD), USA (Joint
Rapporteur)
Dr U. Mueller, Perth, Western Australia, Australia (Joint Rapporteur)
Dr J. Schlatter, Zurich, Switzerland
Dr G.S. Shephard, Cape Town, South Africa
Professor I. Stankovic, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia
Secretariat
DMr A. Afghan, Health Products and Foods Branch, Health Canada, Ottawa, Canada (WHO
Temporary Adviser)
Dr N. Arnich, Risk Assessment Department, French Agency for Food, Environmental
and Occupational Health and Safety (ANSES), Maisons-Alfort Cedex, France (WHO
Temporary Adviser)
Dr P.E. Boon, Department of Food Safety, Centre for Nutrition, Prevention and Health,
National Institute for Public Health and the Environment (RIVM), Bilthoven, the
Netherlands (WHO Temporary Adviser)
Dr G.J.B. Gnonlonfin, Department of Industry and Private Sector Promotion & Directorate
of Agriculture and Rural Development, ECOWAS Commission, Abuja FCT, Nigeria (FAO
Expert)
351
Dr L. Edler, Dudenhofen, Germany (WHO Temporary Adviser)

Dr V. Fattori, Food Systems and Food Safety Division, Food and Agriculture Organization of
the United Nations, Rome, Italy (FAO Joint Secretariat)
Ms N.Y. Ho, Department of Nutrition and Food Safety, World Health Organization, Geneva,
Switzerland (WHO Joint Secretariat)
Dr E. Kirrane, US Environmental Protection Agency’s Center for Public Health and
Environmental Assessment, Research Triangle Park (NC), United States of America
(WHO Temporary Adviser)
Dr J-C. Leblanc, Laboratory for Food Safety, French Agency for Food, Environmental
and Occupational Health and Safety (ANSES), Maisons-Alfort Cedex, France (WHO
Temporary Adviser)
Dr M. Lipp, Food Systems and Food Safety Division, Food and Agriculture Organization of
the United Nations, Rome, Italy (FAO Joint Secretariat)
Mr P. Loeven, Health Products and Foods Branch, Health Canada, Ottawa, Canada (WHO
Temporary Adviser)
Dr D.P. Lovell, Population Health Research Institute, St. George's Medical School, University
of London, London, United Kingdom (WHO Temporary Adviser)
Dr K. Mukherjee, Food Systems and Food Safety Division, Food and Agriculture
Organization of the United Nations, Rome, Italy (FAO Joint Secretariat)
Dr I. P. Oswald, Toxalim (Research Center in Food Toxicology), Université de Toulouse, INRA,
ENVT, INP-Purpan, Toulouse, France (FAO Expert)
Mr K. Petersen, Department of Nutrition and Food Safety, World Health Organization,
Geneva, Switzerland (WHO Joint Secretary)
Ms J.H. Spungen, US Food and Drug Administration (FDA), Center for Food Safety and
Applied Nutrition (CFSAN), College Park (MD), United States of America (WHO
Temporary Adviser)
Dr S.G. Walch, Chemisches und Veterinäruntersuchungsamt (CVUA) Karlsruhe, Karlsruhe,

Germany (FAO Expert)
Dr Y. Kiparissis, Health Products and Foods Branch, Health Canada, Ottawa, Canada (WHO
Temporary Adviser)
Ms S. Kaplan, Bern, Switzerland (FAO Technical Editor)
352
This volume contains monographs prepared at the ninety-
first meeting of the Joint FAO/WHO Expert Committee
on Food Additives (JECFA), which met virtually online
from 1 to 12 February 2021.
The detailed monographs in this volume summarize data

on specific contaminants in food. Individual monographs
present the assessment of exposure to cadmium from
all food sources, the technical, analytical, dietary
exposure and toxicological data on ergot alkaloids,
an assessment of five substances that may occur as
previous cargoes, and a revision of the specifications for
steviol glycosides.
This volume and others in the WHO Food Additives

series contain information that is useful to those who
produce and use food additives and veterinary drugs and
those involved with controlling contaminants in food,
government and food regulatory officers, industrial
testing laboratories, toxicological laboratories and
universities.

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WHO FOOD ADDITIVES SERIES: 82

Prepared by the ninety-first meeting of the

World Health Organization, Geneva, 2023

ISBN (WHO) 978-92-4-006076-0 (electronic version)

(ff) Viet Nam 26

25 μg/kg bw, reflecting the long half-life of cadmium in humans. Reported

2. Food consumption and dietary exposure assessment

2.1 Concentrations in food used in the dietary exposure estimates

2.2 Food consumption data used in the dietary exposure estimates

2.3 National estimates of chronic dietary exposure from the literature

As part of a multicountry total diet study in sub-Saharan Africa, foods

laboratories in the various areas. Although no information was provided on the

information on the contribution from cocoa products was reported.

(l) French Polynesia

(n) Hong Kong Special Administrative Region (SAR), China

(o) Iran, Islamic Republic of

(s) Korea, Republic of

rice, peanuts and millet. No information on the contribution of cocoa products

(u) The Netherlands

Consumption Survey. Based on LB, MB and UB mean concentrations, median

(v) New Zealand

cadmium by GF-AAS, following nitric acid/hydrogen peroxide digestion. The

(aa) Sri Lanka

(dd) United Kingdom

(ee) United States of America

data were obtained from a 24HDR administered on two non-consecutive days.

(ff) Viet Nam

2.4 International estimates of chronic dietary exposure

bread products, other

Estimated dietary Contribution from

Owing to the widespread nature of cadmium contamination across the

P95 187 – 2810 753 – – – – – 1000 2592

Based on the international estimates of dietary cadmium exposure, the

2.4.1 Temporal trends in dietary cadmium exposure

2.4.2 Impact of cocoa product source on dietary cadmium exposure

limited number of records (n = 30) were submitted by Indonesia (G09). Due to

cluster with the highest concentrations of cadmium in cocoa products (G05)

2.4.3 Impact of proposed maximum limits for cadmium on cocoa product

■■ chocolates containing or declaring < 30% total cocoa solids on a dry

2.4% (cluster G07). The contributions to dietary cadmium exposure without

data to allow application of the MLs.

country grouping as reported in the literature. The mean dietary exposure to

decrease to 0.9% or increase to almost 10% if cocoa products were sourced

of young children to inorganic trace elements. Environ Int. 97:28–36.

Global mean cadmium concentrations used for international

Food N N<LOD N<LOD (%) MB mean

Confectionery 554 279 50 71.9

Food N N<LOD N<LOD (%) MB mean

Mandarin and mandarin-like hybrid 275 256 93 2.9

Food N N<LOD N<LOD (%) MB mean

Rice flour 230 17 7 38.3

Food N N<LOD N<LOD (%) MB mean

2.4.4 Epidemiological studies 132

8.4 Assessments of acute dietary exposure 171

9.2.4 Selection of mathematical model 178

2.1 Biochemical aspects

After oral administration of 0.2 to 2 mg of [3H]-labelled ergotamine (in a

ergotamine tartrate intravenously and, at least 14 days later, 2 mg of ergotamine

radioimmunoassay (LOD: 2–14 pg/mL). The maximum plasma levels were