COE Active Principles

Active principles
(constituents oI toxicological concern)

contained in natural sources oI Ilavourings

approved by the Committee oI Experts on Flavouring Substances
October 2005

2

The Committee oI experts appreciates the extra work oI the members oI the Ad hoc group on
active principles, Ulla Beckman Sundh and Judith Amberg-Mller, to bring the compilation oI
datasheets into the Iinal Iorm Ior publication and oIIers them special thanks.

3
Contents
Page

1. General introduction........................... 5

2. Foreword.............................. 5

3. DeIinitions................................ 6

4. Aims oI the study................................... 8

5. General principles............................ 8

6. List oI 'active principles (constituents oI toxicological concern)........ 10

Appendix 1 ReIerences......................... 13

Appendix 2 List oI 'active principles (constituents oI toxicological concern) being
evaluated by the Committee oI experts................ 15
List oI 'active principles to be evaluated............. 19
List oI 'active principles deleted aIter preliminary evaluation.... 19

Appendix 3 DeIinition oI types oI natural Ilavouring preparations......... 21

Appendix 4 Format oI datasheets Ior 'active principles
(constituents oI toxicological concern)............. 25

Appendix 5 ClassiIication system Ior natural Ilavourings sources and
preparations........................ 27

Appendix 6 List oI members oI the Committee oI experts on Ilavouring
substances........................ 29

DATA SHEETS............................ 33
Agaric acid................................. 35
beta-Asarone................................. 41
d-Camphor............................... 49
Capsaicin............................... 59
Elemicin................................ 67
Estragole.............................. . 75
Eucalyptol................................ 87
Hydrocyanic acid.............................. 97
Hypericin................................. 109
IsosaIrole................................. 119
Myristicin............................... . 125
Teucrin A................................. 137
alpha-and beta-Thujone....................... ... 143
4

5
1. General Introduction

Fields of activity of the Council of Europe

The competence oI the Council oI Europe is very wide and covers practically all aspects oI
European aIIairs, with the exception oI deIence matters. Where, however, a lesser number oI
states wish to engage in some action in which not all their European partners desire to join, they
can conclude a 'partial agreement which is binding on themselves alone.

Partial Agreement in the Social and Public Health Field

It was on this basis that the Partial Agreement in the Social and Public Health Field was
concluded in 1959 by the Council oI Europe Committee oI Ministers and revised in 1996 by the
Committee oI Ministers with eIIect Irom 1 January 1997. The Iollowing states are members oI
the Partial Agreement: Austria, Belgium, Cyprus, Denmark, Finland, France, Germany, Ireland,
Italy, Luxembourg, the Netherlands, Norway, Portugal, Slovenia, Spain, Sweden, Switzerland
and the United Kingdom oI Great Britain and Northern Ireland.

The aim oI the Partial Agreement public health activities is to protect the consumer Irom
potential health risks connected with the present-day way oI liIe. The committees oI experts
provide the scientiIic base Ior national and international regulations concerning products which
have a direct or indirect impact on the human Iood chain (control oI IoodstuIIs, nutrition, Iood
saIety, consumer health, Iood contact materials, Ilavouring substances), pesticides,
pharmaceuticals and cosmetics.

The steering committee responsible Ior the activities concerning health protection oI the
consumers is the Public Health Committee, composed oI high-ranking Ministry oI Health
oIIicials oI the member states. It supervises the committees oI experts` activities and speciIies
the general principles oI the public health policy that these committees oI experts have to Iollow
in their work.

The Partial Agreement in the Public Health Field has always closely Iollowed technical
evolution and scientiIic progress and resolutions, guidelines and reports arising Irom its work
are oIten oI a pioneering nature.

In most Iields, the approach is that oI toxicological evaluation oI chemical substance groups, the
setting oI technical and toxicological speciIications, elaboration oI maximum or guideline levels
and preparation oI inventory lists oI chemical substances used by industry.

2. Foreword

2.1 Natural sources of flavourings

Volume I oI the 4
th
edition oI the Council oI Europe`s 'Blue Book was published in 1992 and
dealt with chemically-deIined Ilavouring substances. The Committee oI Experts on Flavouring
Substances, hereaIter called Committee oI experts, is currently engaged in a major review oI the
saIety-in-use oI over 600 natural Ilavouring source materials, the conclusions oI which will
eventually be published in Volume II oI the 4
th
edition oI the 'Blue Book. This review builds
on the Committee oI experts` earlier evaluations oI natural sources, which were included in the
3
rd
edition oI the 'Blue Book which was published in 1981. In view oI the time required to
6
complete this large task, it has been agreed that interim reports should be published at regular
intervals covering the source materials so Iar evaluated. The Iirst oI these reports was published
in 2000 and provides the Committee oI experts` evaluations oI 101 source materials. The second
interim report is under publication and provides evaluations oI a Iurther 60 source materials. (1,
2, 3)

2.2 ~Active principles (constituents of toxicological concern)

The Committee oI experts is also reviewing the toxicity oI certain potentially toxic chemically-
deIined substances known as 'active principles (constituents oI toxicological concern) (see
deIinitions in section 3) which occur in natural Ilavourings. These 'active principles are being
reviewed separately to Iacilitate the evaluation oI the Ilavouring source materials and preparations
in which they occur. On the basis oI existing toxicological data, 'active principles should not be
used as Ilavourings in their own right.

Datasheets on 'active principles (constituents oI toxicological concern) summarise the
toxicological evidence Ior the classiIication oI the substance as an 'active principle, explain the
derivation oI the MDI or TMDI (see Iootnote a and deIinitions in section 3) and outline Iurther
data that may be required. They will also list the limits set Ior each oI the 'active principles in
Ioods and beverages. The conclusions oI this review will also be published as interim reports. This
current report is the Iirst oI these publications.

A list oI substances currently classiIied as 'active principles (constituents oI toxicological
concern) and their evaluation status are given in Appendix 2 to this report.

2.3 Other activities

The Committee oI experts has also considered the saIety-in-use oI smoke Ilavours, thermal
process Ilavourings, Ilavourings produced by enzymatic and microbiological processes and
Ilavourings produced using plant tissue culture and its conclusions in these areas have been
published separately (4-8).

3. Definitions

Natural sources of flavourings

Natural sources oI Ilavourings (or natural source materials) are materials oI vegetable or animal
origin, whether or not they are normally consumed as Iood, Irom which Ilavourings may be
obtained. The Committee oI experts has predominantly evaluated materials in the raw or dried
state, with the exception oI certain special products, such as vanilla, cocoa and black pepper,
which are traditionally processed (e.g. Iermented) beIore their use as source materials.

Flavouring preparations

Flavouring preparations are products (other than chemically deIined Ilavouring substances),
whether concentrated or not, with Ilavouring properties which are obtained Irom natural sources oI
Ilavourings by appropriate physical processes (including distillation and solvent extraction).
DeIinitions oI the types oI Ilavouring preparations included in this report, based on those speciIied
by the International Organization Ior Standardization (10), are given in Appendix 4.

7
~Active principles (constituents of toxicological concern)

'Active principles (constituents oI toxicological concern) are chemically deIined substances
which occur in certain natural Ilavouring source materials and preparations and which, on the
basis oI existing toxicological data, should not be used as Ilavouring substances in their own
right. They are thereIore not included in the Council oI Europe`s list oI chemically-deIined
Ilavourings (1). Maximum limits in IoodstuIIs as consumed should be set on the basis oI
existing toxicological data or with reIerence to a Maximum Daily Intake (MDI) or a Temporary
Maximum Daily Intake (TMDI), when possible. In the Iirst interim report (3) constituents oI
toxicological concern were classiIied into 'active principles or 'other chemical components.
This approach has been reIined by taking into consideration inherent properties oI the
constituents oI toxicological concern, and/or amount oI data lacking. The 'active principles
have been classiIied into three categories, and the term 'other chemical component is no longer
used.

'Active principles I are suspected to be genotoxic carcinogens and thereIore no MDI can be
set.

The maximum limits in IoodstuIIs as consumed should be set as low as possible. In IoodstuIIs
and beverages in general 'Active principles I should be non-detectable based on modern
analytical test methods. The limits oI determination should be taken into consideration as
general limits.

'Active principles II are substances which are suspected to be potent toxicants and Ior which due
to insuIIicient existing toxicological data, it was not possible to set an MDI (see section 5.2).

However a Temporary MDI (TMDI) may be set in certain circumstances.

Maximum limits in IoodstuIIs as consumed should be set based on existing toxicological data.

'Active principles III are substances which are considered to be oI toxicological concern and
Ior which an MDI has been set.

Maximum limits in IoodstuIIs as consumed should be set with reIerence to the MDI.

Exclusion as ~active principle`

Substances which have been evaluated by JECFA (Joint FAO/WHO Expert Committee on Food
Additives), SCF (EU ScientiIic Committee Ior Food), SC (EU ScientiIic Committee), EFSA
AFC-panel (European Food SaIety Authority, Panel on Iood additives, Ilavourings, processing
aids and materials in contact with Iood), Council oI Europe or other competent bodies and Ior
which an ADI
a
has been set are excluded Irom the list oI 'active principles.

a
Acceptable daily Intake (ADI) was originally deIined by the World Health Organization as an estimate
by JECFA (Joint FAO/WHO Expert committe on Food Additives) oI the amount oI a Iood additive,
expressed on a body weight basis, that can be ingested daily over a liIetime without appreciable health
risk. The deIinition oI Tolerable Daily intake (TDI) is the same as that Ior ADI but it is generally used
only Ior substances classed as contaminants. The Committee oI experts has thereIore in its second
interim report on natural sources oI Ilavourings introduced the term Maximum Daily Intake (MDI) to
cover active principles (constituents oI toxicological concern).
8
4. Aims of the study

The Committee oI experts set itselI the Iollowing aims in its study oI natural sources oI
Ilavourings and preparations thereoI and inherent 'active principles:

- to draw up a list oI natural sources oI Ilavourings and preparations, indicating their
acceptability Ior use in Iood;

- to draw attention to certain natural sources oI Ilavourings and preparations which present a
hazard to public health;

- to draw up a list oI 'active principles and set maximum limits Ior such substances in
IoodstuIIs;

- to supplement and revise the lists reIerred to above; and

- to recommend to manuIacturers oI Ilavouring source materials and Ilavouring preparations the
inIormation required by the Committee oI experts to complete its evaluations oI their saIety-
in-use.

5. General principles

5.1 Considerations

The Committee oI experts` review only covers source materials Ior which interested
manuIacturers have requested a saIety evaluation; it is thereIore not exhaustive. The Committee
oI experts` recommendations on the saIety-in-use oI natural source materials and preparations as
Ilavourings should not be interpreted as approval Ior their use in any other application.

The Committee oI experts is conscious that the level oI use oI natural Ilavouring source
materials and preparations is generally low and their use is oIten limited by their organoleptic
properties. In general, they should be used at the lowest level which is technologically eIIective.
However, these Iactors are not in themselves suIIicient to guarantee that there are no public
health risks associated with their use.

The Committee oI experts recognises that up-to-date inIormation on the current usage oI natural
source materials and preparations is an important part oI the evaluation process. As part oI this
process, the Committee oI experts receives inIormation Irom the International Organization oI
the Flavor Industry (IOFI) on the current levels oI use oI source materials and preparations in
Ioods and beverages. Where necessary, the Committee oI experts may also ask IOFI to provide
inIormation on the chemical composition oI source materials and preparations and on any
relevant toxicological studies. Since 1995, IOFI has been invited to attend part oI Committee oI
experts` meetings, making the exchange oI inIormation Iaster and more eIIicient.

The evaluation oI natural Ilavouring source materials and preparations is a large and complex
task. Several parts (e.g. Iruits, leaves, roots) oI the same source species can be used in natural
Ilavourings and, depending on the species, these can be added as such, or with very limited
processing, such as crushing, grinding or drying (e.g. herbs and spices), and/or they can be used
to derive natural Ilavouring preparations. It is also possible Ior several diIIerent types oI
Ilavouring preparations (e.g. essential oils, extracts) to be prepared Irom the same part. In
9
addition, there can be signiIicant qualitative and quantitative diIIerences in chemical
composition between diIIerent parts and between diIIerent preparations. For example, alkaloids
are Iound in the bark but not in the Iruit oI pomegranate, while anthraquinones are present in the
leaves oI rhubarb but are Iound at only trace levels in the stalks.

Natural Ilavouring source materials and preparations may contain substances deIined by the
Committee oI experts as 'active principles (constituents oI toxicological concern) (see
deIinitions in section 3). The addition oI these natural Ilavourings to Ioods and beverages will
obviously result in the presence oI these substances in the products concerned. In setting limits
Ior these substances in Ioods and beverages, the Committee oI experts considers that the general
limits should be set as low as technologically Ieasible to restrict consumer exposure. For
'active principles I where the substance concerned is a potential genotoxic carcinogen, the
general limit is set at the limit oI detection. This type oI limit is expressed as the substance
being 'non-detectable based on modern analytical methods. The Committee oI experts
recognises, however, that there may be a small number oI traditional Ioods and beverages in
which the levels oI these substances could be higher than the general limits. The Committee oI
experts thereIore sets higher limits Ior these Iew tightly deIined Iood categories.

Natural Ilavourings are prepared Irom vegetable or animal sources which may themselves be
consumed as part oI the human diet. The Committee oI experts evaluates the saIety-in-use oI the
Ilavourings derived Irom these Ioods and not the IoodstuIIs themselves.

The consumption oI 'active principles contained in natural Ilavouring source materials and
preparations listed in this report, as well as many other Iood ingredients, may sometimes
sensitise or produce hypersensitivity reactions in some people. At present, such reactions cannot
reliably be predicted Irom tests on laboratory animals or Irom in vitro studies. ThereIore, in
common with other international organisations, the Committee oI experts has not been able to
consider these issues in arriving at its decisions.

When evaluating natural Ilavouring source materials and preparations, the Committee oI experts
has oIten had to take decisions based on very limited toxicological data available Irom
published literature or other sources. Hence, it has had to adopt less rigorous criteria than those
generally applied in the saIety evaluation oI Iood additives. The value oI the available data has
been judged on a case-by-case basis rather than by adhering to rigid requirements. A general
description oI the criteria Ior evaluation is given below.

5.2 Toxicological criteria for classification

Requirements to set an MDI (Maximum Daily Intake) expressed in mg/kg bw/day:
In general, 28-day oral studies in rodents and in vitro mutagenicity studies are regarded as the
minimal requirement Ior the setting oI an MDI. However, this requirement can be reduced on a
case-by-case basis. Conversely, the requirement Ior toxicological data can be increased in
certain situations such as where the data available have given cause Ior concern and Iurther
studies are needed Ior reassurance.

Requirements to set a TMDI (Temporary Maximum Daily Intake) expressed in mg/kg bw/day:
II an MDI cannot be set, a TMDI may be set on the toxicological data available and the
additional data needed to set a Iull MDI should be indicated with the TMDI.

The requirements to set an MDI may be reduced, particularly in the Iollowing situations:
10
- The part oI the source species used in natural Ilavourings is commonly consumed as a Iood in
Europe. The division oI sources into Iood and non-Iood has been made on the basis oI the
Committee oI experts` experience oI the European Iood supply. The Committee oI experts is
aware oI a number oI plant Ioods which are saIely consumed on other continents but this may
only be because their populations have developed ways oI removing or inactivating the toxins
they contain. Without knowing exactly how each Iood consumed outside Europe is normally
handled, the amount which might normally be consumed and the likelihood that any adverse
eIIects would have been noted, it is not possible to take this history oI use into account in the
evaluation oI such source materials.

- The chemical composition oI the Ilavouring source material or preparation is closely related to
the chemical composition oI another Ilavouring source or preparation which has been more
thoroughly studied.

- The major constituents oI the source materials and/or preparations are thought unlikely to
cause adverse eIIects at the current level oI use, Ior example because they have previously
been evaluated by the Committee oI experts or by the EU`s ScientiIic Committee or another
recognised international expert group dealing with Ilavourings and Iound to be Iully
acceptable as Ilavouring substances.

- Conversely, the requirement Ior toxicological data has been increased in certain situations
such as where the data available on source material and preparations, or their constituents,
have given cause Ior concern and Iurther studies are needed Ior reassurance.

The classiIication system Ior natural Ilavouring sources and preparations is described in
Appendix 5.

6. List of ~active principles (constituents of toxicological concern)

Datasheets are prepared Ior all the 'active principles (constituents oI toxicological concern)
that are being evaluated by the Committee oI experts, with responsibility Ior their preparation
being divided between delegations. To ensure consistency oI the data, a standard Iormat has
been agreed (see Appendix 4).

However, Ior diIIerent 'active principles (constituents oI toxicological concern), the amount oI
inIormation as well as the quality oI inIormation currently available varies greatly. ThereIore
diIIerences exist in the amount oI detail provided in datasheets. These diIIerences reIlect not
only the varying amount oI published inIormation currently available on individual 'active
principles, but also the alternative approaches taken by delegations to preparing the datasheets.

The Committee oI experts has discussed whether a threshold should be used in the classiIication
oI source materials and preparations that contain 'active principles (constituents oI
toxicological concern). For the present time the Committee oI experts has agreed that all source
materials and preparations should be classiIied as containing 'active principles whatever the
concentrations oI 'active principles present.

Several key reIerence books have been consulted in preparing the datasheets. Each source
species is identiIied primarily by its systematic name based on the botanical nomenclature oI
Zander (11) or, where the source species is not listed in that reIerence book, using that oI
MansIeld (12). Source species not listed in either book are treated on a case-by-case basis, e.g.
11
based on Hager (13), Rehm (14) or Hoppe (15). Other key reIerences used include publications
by Arctander (16), Duke (17), Fenaroli (18), Leung (19), the UK Ministry oI Agriculture,
Fisheries and Food (MAFF) (20), Opdyke (21), Tanaka (22) and Usher (23). In order to save
space on the datasheets these reIerences may be simply listed by author name.

Several oI the Ilavouring sources mentioned in this report have been the subject oI national and
international evaluations, and these are noted in the datasheets. For example, the UK Food
Additives and Contaminants Committee (FAC) carried out an extensive review oI over 500
Ilavouring source materials in the 1970s, the results oI which were published in 1976 (24). In
the US, since 1958, the Food and Drug Administration (FDA), with the assistance oI FEMA,
has been compiling lists oI Ilavouring materials considered to be GRAS (Generally Recognised
As SaIe) and these are included in Chapter 21 oI the Code oI Federal Regulations (CFR). In
addition, the FEMA expert panel is continuing to publish separate lists oI materials that it
considers to be GRAS.

DeIinitions oI types oI natural Ilavouring preparations are given in Appendix 3. Further
inIormation on the preparation and Iormat oI 'active principle datasheets is given in Appendix
4. The classiIication system Ior natural Ilavouring sources and preparations is described in
Appendix 5.
12
13
Appendix 1

References

1. Council oI Europe (1992). Flavouring Substances and Natural Sources of Flavourings. 4
th

Edition. Jolume 1. Chemicallv-defined Flavouring Substances. ISBN 2-7160-0147-2.

2. Council oI Europe (1981). Flavouring Substances and Natural Sources of Flavourings. 3
rd

Edition. ISBN 2-7160-0081-6. |out oI stock|

3. Council oI Europe (2000). Natural sources of Flavourings. Report N1. ISBN 92-871-4324-
2.

4. Council oI Europe (1992). Council of Europe Guidelines concerning the transmission of
flavour of smoke to food, Health Protection oI the Consumer Series. Council oI Europe Press,
Strasbourg. ISBN 92-871-2191-5.

5. Council oI Europe (1992). Health aspects of using smoke flavours as food ingredients, Health
Protection oI the Consumer Series. Council oI Europe Press, Strasbourg. ISBN 92-871-2189-3.

6. Council oI Europe (1994). Guidelines for flavouring preparations produced bv en:vmatic of
microbiological processes, Health Protection oI the Consumer Series. Council oI Europe Press,
Strasbourg. ISBN 92-871-2586-4.

7. Council oI Europe (1995). Guidelines for safetv evaluation of thermal process flavourings,
Health Protection oI the Consumer Series. Council oI Europe Press, Strasbourg. ISBN 92-871-
2811-1.

8. Council oI Europe (1998). Council of Europe Guidelines for flavouring preparations
produced bv plant tissue culture, Health Protection oI the Consumer Series. Council oI Europe
Press, Strasbourg. ISBN 92-871-3738-2.

9. European Community (1988). Council Directive oI 22 June 1988 on the approximation oI the
laws oI Member States relating to Ilavourings Ior use in IoodstuIIs and to source materials Ior
their production (88/388/EEC). Official Journal of the European Communities No.. L 184/61-
67.

10. International Organization Ior Standardization (1997). Aromatic natural raw materials
Jocabularv. ISO 9235: 1997. International Organization Ior Standardization, Geneva.

11. Zander, R. (1984). Handwrterbuch der Pflan:ennamen, 13th Edition. R. Enche, G.
Buchheim, S. Seybold (Eds.). Verlag Eugen Ulmer, Stuttgart.

12. MansIeld, R. (1986). Jer:eichnis landwirtshaftlicher und grtnerischer kulturpflan:en
(ohne Zierpflan:en), Volumes 1-4, 2nd edition. J. Herausgegeben von Schule-Motel (Ed.).
Springer-Verlag, Berlin, Heidelberg & New York.

13. List, P.H. and Hrhammer, L. (1967-80). Hagers Handbuch der Pharma:eutischen Praxis,
Volumes 1-8, 4th Edition. Springer-Verlag, Berlin, Heidelberg & New York.

14
14. Rehm, S (Ed.) (1994). Multilingual Dictionarv of Agronomic Plants. Kluwer Academic
Publishers, Dordecht.

15. Hoppe, H.A. (1977). Drogenkunde, Vol. 1 Angiospermen, Vol. 2: Gvmnospermen,
Krvptogamen, Tierische Drogen. Walter de Gruyter, Berlin & New York.

16. Arctander, S. (1960). Perfume and Flavour Materials of Natural Origin. Arctander,
Elizabeth, New Jersey, USA.

17. Duke, J.A. (1985). Handbook of Medicinal Herbs. CRC Press, Boca Raton, Ann Arbor,
London & Tokyo.

18. Burdock, G.A. (Ed.) (1995). Fenarolis Handbook of Flavor Ingredients. Volume I and II.
3
rd
Edition. CRC Press, Boca Raton, Ann Arbor, London & Tokyo.

19. Leung, A.Y. and Foster, S. (1996). Encvclopedia of Common Natural Ingredients used in
Food, Drugs and Cosmetics. 2
nd
Edition. John Wiley & Sons, Inc., New York, Chichester,
Brisbane, Toronto & Singapore.

20. UK Ministry oI Agriculture, Fisheries and Food. (1995). Flavourings in Food. Food
Surveillance Report No 48. HMSO, London.

21. Opdyke, D.L.J. (1979). Monographs on Fragrance Raw Materials. Food and Cosmetics
Toxicologv, 17, Special Issue V. Pergamon Press, OxIord, London, New York, Paris.

22. Tanaka, T (1976). Tanakas Cvclopedia of Edible Plants of the World. S. Nakao (Ed.).
Keigaku Publishing Co, Tokyo.

23. Usher, D. (1974). Dictionarv of Plants Used bv Man. Constable, London.

24. UK Food Additives and Contaminants Committee (1976). Report on the Review of
Flavourings in Food. FAC/REP/22. HMSO, London.

15
Appendix 2

List of ~active principles (constituents of toxicological concern) being evaluated by the
Committee of experts

~Active principle I No MDI Limits (mg/kg)
beta-Asarone - Foods and beverages: ND
1

Exceptions:
Alcoholic beverages (traditionally Ilavoured
with calamus): 0.5

Elemicin - None set. The Committee considers that limits are
not required Ior elemicin as the intake (mainly
Irom nutmeg) will be restricted via a limit Ior
saIrole in nutmeg containing Ioods

Estragole -
Foods and beverages (not traditionally
Ilavoured with herbs and spices containing
estragole): ND
1

Exceptions:
Anise Ilavoured conIectionary: 100
Anise Ilavoured alcoholic beverages: 35
Alcoholic beverages traditionally Ilavoured with
tarragon, Iennel or chervil: 70
Flavoured baked goods:10
Cheese with herb:10
Flavoured preserves (with vegetable, Iish): 35
Flavoured ready meals (with Iish, meat): 35
Tarragon/basil Ilavoured oil: 175
Condiments: 200
Sauces: 100

Methyleugenol
2
- Foods and beverages: ND
1
Exceptions:
to be established

Myristicin - None set. The Committee considers that limits are
not required Ior myristicin as the intake (mainly
Irom nutmeg) will be restricted via a limit Ior
saIrole in nutmeg-containing Ioods

16
~Active principle II TMDI (mg/kg bw) Limits (mg/kg)
Agaric acid NE
3 33 3

Foods and beverages: ND
1 11 1

Exceptions:
Alcoholic beverages: 20
Camphor NE
3 33 3

Foods: 25
Beverages: 10
Exceptions:
Candy: 100
Fresh cheese: 140
Sauces and condiments: 150
Capsaicin 0.2 expressed as
total capsaicinoids
Foods and beverages: 5
Hot foods and beverages: 10
Exceptions:
Hot ketchup and similar products: 20
Tabasco, harissa, hot pimento oils and similar
preparations: 50
Eucalyptol NE
3 33 3

Exceptions:
Mint-Ilavoured syrups: 30
Candies: 1000
Micro-candies: 10500
Chewing-gums: 5000
Baked goods (salted biscuits): 50
Pork meats: 100
Sauces and condiments: 250
White cheese, Iresh cheese, cream-cheese and
Iresh spreads: 100
Oils: 60
Fish preserves: 30
Hydrocyanic acid 0.023
Foods: 0.5
Beverages: 0.05
Exceptions:
Stone Iruit juices: 0.5
Canned stone Iruit and stone Iruit preserves and
purees: 2
Marzipan, nougat and similar: 50
Almond and/or marzipan-containing
conIectionery and baked goods (or other similar
products): 10
Special` almond- and/or marzipan-containing
conIectionery and baked goods e.g. amaretti`,
Dresdner Christstollen`, `Schwarzbrtchen`,
chocolate enrobed marzipan, marzipan
novelties: 40
For every 1 alcohol by volume in alcoholic
beverages: 0.5

17
~Active principle II
(continued)
TMDI
(mg/kg bw)
Limits (mg/kg)
Hypericin 0.0025 Foods and beverages: ND
1

Exceptions:

Isosafrole NE
3 33 3
None set. According to the available data
isosaIrole occurs together with saIrole, but
at much lower concentrations. ThereIore
measures to restrict saIrole will also
restrict exposure to isosaIrole. It is not
considered necessary to set limits Ior
isosaIrole as its main intake will be
restricted via the limits to be set Ior
saIrole.

Thujone 0.01 Foods and beverages: 0.1
Exceptions:
Alcoholic beverages 25 (vermouths): 2
Alcoholic beverages ~25 (liqueurs,
bitters): 10
Herb vinegar: 5
Sage Iood and stuIIing: 10
Sweets (lozenges): 50

18
~Active principle III MDI Limits (mg/kg)
Menthofuran
2
0.1 (joint MDI
with pulegone)
Exceptions:
Mint/peppermint Ilavoured
alcoholic beverages: 100
conIectionery: 200
chewing gum: 1000

Pulegone
2
0.1 (joint MDI
with
menthofuran)
Exceptions:
Mint/peppermint Ilavoured alcoholic
beverages: 100
conIectionery: 100
Intensely strong mint/peppermint
Ilavoured conIectionary: 200
Mint/peppermint Ilavoured chewing
gum: 350

Teucrin A 0.002 Foods and beverages: ND
1

Exceptions:
Alcoholic beverages: 0.05 Ior every 1
oI alcohol by volume

1
Non-detectable based on modern analytical test methods. The limit oI determination should be taken into
consideration as general limit.
2
This substance has been evaluated by the Committee and limits have been discussed. There is no datasheet on this
substance in this compilation.
3
Not established due to insuIIicient data.
19

List of ~active principles to be evaluated

Aloin/Aloe-emodin
Berberine
CaIIeine
Carvacrol
Dill apiol
Furocoumarins
Geniposide
Glycoalkaloids
Glycyrrhizic acid
Parasorbic acid
Parsley apiol
Polyacetylene compounds
Pyrrolizidine alkaloids
Quassine
Quinine
SaIrole
Santonine
Sparteine
Xanthones

List of ~active principles deleted after preliminary evaluation

Allyl isothiocyanate (1)
trans-Anethole (1)
Cocaine (2)
Coumarin (4)
Eugenol (1)
FurIural (3)
Terpinen-4-ol (1)

Reason Ior deletion: (1) used as Ilavouring as such and included in Vol. I oI Blue Book,
(2) narcotic compound,
(3) no occurrence,
(4) evaluated by EFSA

20
21
Appendix 3

Definitions of types of natural flavouring preparations

DeIinitions oI the types oI natural Ilavouring preparations included in this report, based on those
speciIies by the International Organization Ior Standardization (10), are given below.

1. Raw materials

Exudate. natural raw material excreted by plants either spontaneously or aIter wounding.

Natural oleoresin. exudates consisting mainly oI volatile and resinous constituents (e.g. pine
oleoresin, gurjum).

Balsam. natural oleoresin characterised by the presence oI benzoic and/or cinnamic derivatives
(e.g. Peru balsam, Tolu balsam, benzoin, styrax).

Gum. exudates consisting mainly oI polysaccharides (e.g. gum Arabic, tragacanth gum).

Gum resin. exudates consisting mainly oI resinous constituents and gums (e.g. shellac gum).

Gum oleoresin. exudates consisting mainly oI resinous constituents, gums and certain amounts
oI volatile constituents (e.g. myrrh, olibanum, opoponax, galbanum).

2. Derived products. resinous materials

Resin. product obtained Irom natural oleoresins by removing, as Iar as possible, the volatile
constituents (e.g. rosin).

3. Derived products. volatile products

Essential oil. product obtained Irom vegetable raw material:
- by destillation with water or steam;
- Irom the epicarp oI Citrus Iruits by a mechanical process; or
- by dry destillation.
(NB: The essential oil is subsequently separated Irom the aqueous phase by physical means.)

Essential oil obtained bv steam distillation. essential oil obtained by distillation with or without
water in a still (e.g. pepper oil (with water), lavender oil (without water)).

Cold pressed essential oil. essential oil obtained Irom the epicarp oI Citrus Iruits by mechanical
means at room temperature.

Essence oil. essential oil obtained Irom Iruit juices during concentration or UHT (Ilash
pasteurisation) treatment.

Rectified essential oil. essential oil which has been subjected to Iractional distillation in order to
modiIy the content oI certain constituents (e.g. mint essential oils).

22
'Terpene-less` essential oil. essential oil Irom which the monoterpenic hydrocarbons have
been mainly removed.

'Terpene- and sesquiterpene-less` essential oil. essential oil Irom which the mono- and
sesquiterpenes have been mainly removed.

'X-less` essential oil. essential oil Irom which the component 'x has been partly or
completely removed (e.g. essential oil oI bergamot with partially reduced bergapten content;
essential oil oI Mentha arvensis with partially reduced menthol content).

Folded oil/concentrated oil. essential oil which has been processed to concentrate the
components oI interest by physical means.

Drv-distilled oil. essential oil obtained by dry distillation oI woods, barks or roots without
added water or steam (e.g. essential oil oI cade (Juniperus oxvcedrus), essential oil oI the bark
oI the birch tree).

Jolatile concentrate. concentrated water-soluble volatile substance recovered Irom the
evaporated water oI Iruit or vegetable juices.

Distillate. product oI condensation obtained aIter distillation oI a natural raw material.

Alcoholate. distillate which results Irom the distillation oI a natural raw material in the presence
oI ethanol at variable concentrations.

Aromatic water. aqueous distillate remaining aIter steam distillation when the essential oil has
been separated.

Terpenes. products mainly consisting oI hydrocarbons obtained as by-products Irom an
essential oil by concentration or distillation, or other isolation techniques.

4. Derived products. extraction products

Tincture. solution obtained by maceration oI a natural raw material in the presence oI ethanol at
variable concentrations (e.g. tincture oI benzoin, tincture oI ambergris).

Extract. product obtained by treating a natural raw material with a solvent then, aIter Iiltration,
removal oI the solvent by distillation, except in the case oI use oI a non-volatile solvent.

Concrete. extract with a characteristic odour, obtained Irom a Iresh vegetable raw material by
extraction with a non-aqueous solvent.

Resinoid. extract with a characteristic odour, obtained Irom a dried vegetable raw material by
extraction with a non-aqueous solvent.

Absolute. product with odour, obtained Irom a concrete or a resinoid by extraction with ethanol
at room temperature.
(NB: The ethanolic solution is generally cooled and Iiltered in order to remove the 'waxes; the
ethanol is then removed by distillation.)

23
Oleoresin. extract oI spices or aromatic herbs with a characteristic odour and/or Ilavour (e.g.
pepper oleoresin, ginger oleoresin).

Non-concentrated extract/single-fold extract. product obtained by treating a natural raw
material with a non-removed solvent (e.g. cocoa nibs in propylene glycol, asaIoetida in peanut
oil, vanilla in ethanol).

24
25
Appendix 4

Format of datasheets for ~active principles (constituents of toxicological concern)

Datasheets are prepared for all the 'active principles` (constituents of toxicological concern)
which are being evaluated bv the Committee of experts, with responsibilitv for their preparation
being divided between delegations. To ensure consistencv in the presentation of the data, a
standard format has been agreed as follows.

Chemical name of substance appears as heading on each sheet.

ACTIVE PRINCIPLE: ( I, II, III)

SYNONYMS: (CAS name, IUPAC name, etc.)

CAS No:

STRUCTURE:

REGULATORY/INTERNATIONAL STATUS: (CoE, EU, USA, FEMA, etc.)

MAIN TOXICOLOGICAL STUDIES:

Metabolism:(e.g. absorption, distribution, bioavailability, biotransIormation, excretion, etc)
studies
Animal studies
Human studies

Toxicology:
Acute toxicity
Subacute/subchronic toxicity
Chronic toxicity /carcinogenicity
Reproductive toxicity/teratogenicity
Mutagenicity/genotoxicity
Human data
Other studies (e.g. mechanism oI toxicity, enzyme induction, allergenicity, immunotoxicity,
pharmacological activity, anti-carcinogenicity studies, membrane studies, special in vitro
studies, etc.)

(Under each sub-section oI metabolism and toxicology inIormation, iI no data then 'no data
Iound)

TOXICOLOGICAL EVALUATION: (including a possible NOAEL and derivation oI an
(T)MDI in conIormity with the deIinition oI active principles)

MDI/TMDI: Iigure / not established (suspected genotoxic carcinogen / insuIIicient data)

MAIN OCCURRENCE: (natural sources, preparations and concentration oI 'constituents oI
toxicological concern; inIormation summarized as text, detailed inIormation to be added as
table(s) in Annex)
26
INTAKE ESTIMATION: (use levels oI sources and preparations in IoodstuIIs and estimated
intake; inIormation summarized as text, detailed inIormation to be added as table(s) in Annex)

CONCLUSIONS: (including evaluation oI saIety Ior consumer |comparison oI theoretical
maximum intake and MDI|)

DATA NEEDED: (toxicology, occurrence, intake etc.)

LIMITS: (general limits and exceptions)

REFERENCES:
(ReIerences should be listed as Iollows:
- Text section: author(s), year (e.g. Smith, 1999; Smith and Jones, 2000, Smith et al, 2002)
- ReIerence section:
- 1ournals: Author(s) (Year) Title oI article. International abbreviation oI scientiIic
publication, volume, pages.; (e.g. Prior, R.L., Jacobson J.J. and Smith S.B. (1981)
Metabolic pathways involved in lipogenesis Irom lactate and acetate in bovine
adipose tissue: eIIects oI metabolic inhibitors. Arch. Biochem. Biophys., 211 (1),
202-210).
- Books: Author and/or editor (Year) Title. Publisher, Place oI publication, pages iI
relevant.

DATA BASES USED: (including key words and period oI search)

APPENDIX
(including detailed inIormation on main occurrence and intake estimation given in Table(s))

Table I. Title
27
Appendix 5

Classification system for natural flavouring sources and preparations

In the 3
rd
edition oI the 'Blue Book (2), natural Ilavouring sources were listed and classiIied
into Iour categories (N1-N4). This approach has been reIined by considering individual parts
and preparations and allocating them a single classiIication or, in the case oI preparations
derived Irom the same part, a group classiIication. Flavouring source materials and preparations
have been classiIied into six numbered categories as Iollows:

Category 1
Plants, animals and other organisms, and parts oI these or products thereoI, normally consumed
as Iood items, herbs or spices in Europe Ior which it is considered that there should be no
restrictions on use.

Flavouring preparations, which are not themselves consumed as Iood but which are derived
Irom plants, animal and other organisms and parts or these or products thereoI, normally
consumed as Iood items, herbs or spices in Europe. These preparations, on the basis oI the
inIormation available, are not considered a risk to health in the quantities used.

Category 2
Plants, animals and other organisms, and parts oI these or products thereoI, and preparations
derived thereIrom, not normally consumed as Iood items, herbs or spices in Europe.

These source materials and preparations, on the basis oI the inIormation available, are not
considered to constitute a risk to health in the quantities used.

Category 3
Plants, animals and other organisms, and parts oI these or products thereoI, normally consumed
as Iood items, herbs or spices in Europe which contain deIined 'active principles requiring
limits on use levels.

Flavouring preparations, which are not themselves consumed as Iood but which are derived
Irom plants, animals and other organisms, and parts oI these or products thereoI, normally
consumed as Iood items, herbs or spices in Europe which contain deIined 'active principles
requiring limits on use levels.

These source materials and preparations are not considered to constitute a risk to health in the
quantities used provided that the limits set Ior the 'active principles are not exceeded.

Category 4
derived thereIrom, not normally consumed as Iood items, herbs or spices in Europe, which
contain deIined 'active principles requiring limits on use levels.

These source materials and preparations are not considered to constitute a risk to health in the
quantities used provided that the limits set Ior the 'active principles are not exceeded.

28
Category 5
derived thereIrom, Ior which additional toxicological and/or chemical inIormation is required.

These could temporarily be acceptable provided that any limits set Ior the 'active principles are
not exceeded.

Category 6
derived thereIrom, which are considered to be unIit Ior human consumption in any amount.

As described in Section 5.1, all preparations derived Irom each part oI a source species have
generally been given a group classiIication, but it must be stressed that this classiIication applies
only to the preparations listed on each datasheet. Other preparations may have diIIerent
compositions and these would have to be evaluated separately beIore being included in the
existing classiIication.

Source materials and preparations classiIied in categories 1 and 2, Irom Iood and non-Iood
sources respectively, are considered to be Iully acceptable, and no limits are required on their
use.

Those in categories 3 and 4 are also Iully acceptable, provided that their use does not result in
unacceptably high intakes oI 'active principles.

Where Ilavouring source materials and preparations in categories 3 and 4 contain an 'active
principle, a maximum limit has been recommended Ior the level oI the 'active principle in
IoodstuIIs in which the Ilavourings are used. The setting oI such limits does not necessarily
imply that the use oI a particular Ilavouring source material or preparation must be restricted,
since the amounts added to Iood may not be high enough to exceed the limit.

Category 5 contains source materials which have not been Iully evaluated due to a lack oI data,
but which have been categorised as temporarily acceptable on the basis oI the available
inIormation. In some cases there is insuIIicient inIormation on the composition oI the source
material or its Ilavouring preparations, while in others it has not been possible to assess the
signiIicance oI particular components which are known to occur.

Source materials placed in category 6 are considered to be unsuitable Ior use as sources oI Iood
Ilavourings due to the presence or particularly toxic substances.

Natural Ilavouring source materials whose evaluation has been postponed since there is no
evidence that they are currently in use in Europe have not been categorised.

29
Appendix 6

List of members of the Committee of experts on flavouring substances:

BELGIUM

Dr Marie-Paule DELCOUR-FIRQUET
CheI de travaux
Institut ScientiIique de Sante Publique
14 rue J. Wytsman
B-1050 BRUXELLES
Tel 32 2/642 51 28
Fax 32 2/642 52 24
E mail Marie-Paule.Delcouriph.Igov.be

DENMARK

Dr J GRY
ScientiIic Adviser, Ph. D.
Institute oI Food SaIety and Nutrition
Morkhoj Bygade 19
DK-2860 SOBORG
Tel 45 33/95 60 00
Fax 45 33/95 66 99
E-mail jgFDIR.DK

Dr H FRANDSEN
Institute oI Food SaIety and Nutrition
Danish Veterinary and Food Administration
Morkhoj Bygade 19
DK-2860 SOBORG
Tel 45 33 95 60 00
Fax 45 33 95 66 99
E-mail HFFDIR.DK

FRANCE

ProI Robert ANTON
Faculte de Pharmacie
B.P. 24
F-67401 ILLKIRCH CEDEX
Tel 33 (0)3 90 24 42 42
Fax 33(0)3 90 24 43 11
E-mail antonpharma.u-strasb.Ir

30
IRELAND

Miss Emer O`REILLY
Technical Executive
Authority oI Ireland
Abbey Court
Lower Abbey Street
DUBLIN 1
IRELAND
Tel 353-1-817 1244
Fax 353-1-817 1344
E-mail eoreillyIsai.ie
website www.Isai.ie

ITALY

Mr. Massimo DE VINCENZI
Dirigente di Ricerca
Metabolism and pathological biochemistry
Istituto Superiore di Sanita
Viale Regina Elena 299
I-00161 ROME
Tel 39 6/4990 25 26/2598
Fax 39 6/49387149
E-mail mbpsegriss.it

PORTUGAL

Dr Ana Claudia FIGUEIREDO
Pre-clinical assessor
INFARMED
Parque de Saude de Lisboa
Av. do Brasil, 53
P 1700 LISBOA
Tel 351 21 798 7381
Fax 351 21 798 7316
E-mail anaclaudia.IigueiredoinIarmed.pt

SWEDEN

Ms Ulla BECKMAN SUNDH
Toxicologist
National Food Administration
Box 622
S-75126 UPPSALA
Tel 46 18 175 755
Fax 46 18 10 5848
E-mail: USBESLV.SE
31
Mrs Carmina IONESCU
Byrdirektr
National Food Administration
Box 622
S-75126 UPPSALA
Tel 46 18 175 601
Fax 46 18 105 848
E-mail: caioslv.se

SWITZERLAND

Dr Judith AMBERG-MLLER
Toxicologist
Swiss Federal OIIice oI Public Health
Section Food Toxicology
StauIIacherstrasse 101
CH-8004 ZRICH
Tel 41 43 322 21 92
Fax 41 43 322 21 99
E mail judith.ambergbag.admin.ch

UNITED KINGDOM

Dr Rhodri EVANS
Aviation House
125 KINGSWAY
GB - LONDON WC2B 6NH
Tel 44 20 72 76 85 82
Fax 44 20 72 76 85 14
E-mail rhodri.evansIoodstandards.gsi.gov.uk

32
33
DATA SHEETS
34
35
Agaric acid

ACTIVE PRINCIPLE: II

SYNONYMS: Agaricic acid, agaricinic acid, agaricin, d-cetylcitric acid, n-hexadecylcitric acid,
2-hydroxy-1,2,3-nonadecanetricarboxylic acid, laricic acid

CAS No: 666-00-9 (Anhydrous), 6034-71-5 (Sesquihydrate)

STRUCTURE:

OH
O
HO
HO O
O
HO
CH
3

REGULATORY / INTERNATIONAL STATUS: Maximum use levels Ior agaric acid in
Ioods and beverages have been set in Annex II oI Council Directive 88/388/EEC on Ilavourings
(EEC, 1988) with maximum limits oI 20 mg/kg in IoodstuIIs and beverages and the exceptions
oI 100 mg/kg in alcoholic beverages and 100 mg/kg in Iood containing mushrooms, in
accordance with Council oI Europe, Blue Book (Council oI Europe, 1981).


Metabolism:
studies: No data Iound.
Animal studies: No data Iound.
Human studies: No data Iound.

Toxicology:
Acute toxicity: Vomiting and diarrhoea were seen in dogs and adult cats given single oral doses
oI 0.5-1.0 g agaric acid (approx. 50-100 mg/kg bw in dogs and 250-500 mg/kg bw in cats).
Lower oral doses (0.5 g per animal) had no overt adverse eIIects (Taddei et al., 1975). No
obvious toxic eIIects (apart Irom purgative eIIects) were seen when single oral doses oI 500,
1000 und 2000 mg agaric acid/kg bw were given to mice and rats; no purgative eIIects were
seen at oral doses oI 150 and 350 mg/kg, respectively (Carminati et al., 1968).
Intravenous doses oI 100 and 25 mg agaric acid/kg bw were Iatal to rabbits (LD
Lo
)
(Abdenalden, 1935) and mice respectively. Mice given i.v. 12 mg agaric acid/kg bw showed no
overt eIIects (Carminati et al., 1968). The i.p. injection oI 20 mg agaric acid/kg bw produced no
overt eIIects in mice but 30 mg/kg bw killed all the treated animals between 48 hr and 7 days
36
aIter injection (Carminati et al., 1968). In rats, i.p. injection oI 50 mg agaric acid/kg bw was
toxic and lethal in some cases (Taddei et al., 1975). In dogs, i.v. doses oI 0.5-1.0 mg/kg bw
reduced arterial pressure transiently, however, the mechanism seemed to be diIIerent Irom
atropine (Carminati et al., 1968). In young cats subcutaneous doses oI 10-30 mg agaric acid per
animal (no data on bw) inhibited transpiration, s.c. doses oI 100 mg per animal (no data on bw)
caused vomiting and diarrhea. In the Irog s.c. doses oI 25-50 mg per animal resulted in central
paralysis and death (Taddei et al., 1975).
Subacute / subchronic toxicity: In Iemale Sprague-Dawley rats (170-185 g bw) Ied diets
containing 1 and 4 agaric acid (providing about 1 and 4 g agaric acid/kg bw/day) Ior
3 weeks body weight gain and Iood intake was signiIicantly decreased (possibly due to a
palatability problem). The metabolism was aIIected including a signiIicant decrease in Iatty acid
synthesis at 1 agaric acid in the diet (which was accentuated with 4 agaric acid diet) and a
reduction oI glucose synthesis (Freedland et al., 1981).
Chronic toxicity / carcinogenicity: No data Iound.
Reproductive toxicity / teratogenicity: No data Iound.
Mutagenicity / genotoxicity: No data Iound.

Human data: The dried mushroom white agaric (containing 16.5 agaric acid) was used
therapeutically at single oral doses oI 0.05 g (Hagers Handbuch, 1998) to 0.3 g (Taddei et al.,
1975) (i.e. 8.3 to 50 mg agaric acid) and at daily oral doses oI 1 g (i.e. 165 mg agaric acid) Ior
the treatment oI hyperhydrosis. Onset oI the antihydrotic eIIect was reported to occur 5 to
6 hours aIter dosage. Doses oI 2.5-3.0 g (i.e. up to 500 mg agaric acid) were used as a purgative
(Taddei et al., 1975). Agaric acid itselI has been used as an antiperspirant at therapeutic doses oI
10-30 mg per dose and 100 mg per day (maximal daily therapeutic dose according to
Pharmacopoea Helvetica V, 1933; Taddei et al., 1975). AIter single oral doses oI 50 to 100 mg
agaric acid sudden transient nausea was reported. These adverse eIIects did not occur aIter
repeated doses oI 10 to 30 mg agaric acid (Hagers Handbuch, 1998).

Other studies: Biochemistrv. Agaric acid is an extremely eIIective inhibitor oI Iatty acid
synthesis (inhibitor oI the enzyme aconitase at 1 x 10
4
M in vitro) (Carrrano et al., 1967;
Henry et al., 1969; Thoms et al, 1907; Chavez et al., 1975; Newton et al., 1980; Liddle et al.,
1979); however, it causes many other alterations in metabolism (Freedland et al., 1981). The
physiological and biochemical properties oI agaric acid may be related to its structural similarity
to citric acid. The eIIects oI agaric acid on metabolism have been investigated in vitro and in
vivo: In isolated mitochondria agaric acid inhibited citrate translocation Irom the mitochondria
to the cytosol, and decreased Iatty acid synthesis Irom lactate by 40 and 60 at inhibitor
concentrations oI 0.2 and 1.0 mM (Stipani et al., 1976; Prior et al., 1981).
EIIects on lipogenesis (decreased Iatty acid synthesis) were observed in rats Ied diets containing
1 and 4 agaric acid (providing 1 or 4 g agaric acid/kg bw/day) Ior 3 weeks (see subacute
toxicity). However, agaric acid did not inhibit the in vitro Iormation oI Iatty acids Irom glucose
or lactate in the adipose tissue oI rats and cattle, respectively (Hood et al., 1981).
EIIects oI agaric acid on ketogenesis have been demonstrated in isolated rat hepatocytes in the
absence and in the presence oI butyrate. There was a marked increase in the production oI
ketone bodies in the presence oI 60 micromoles agaric acid per litre in the absence oI other
added substrates. However, in the presence oI 2 mmoles butyrate per litre there was a slight but
signiIicant decrease in total ketone body production. The observed increase oI ketone bodies
with agaric acid under endogenous conditions, where ketogenesis was limited by the availability
oI Iatty acids, and the observed decrease oI ketone bodies during rapid substrate utilization may
indicate a potential oI agaric acid to increase lipolysis (Freedland et al., 1981).
37
Furthermore, agaric acid aIIects cell respiration: Apart Irom eIIects on aconitase, agaric acid has
been reported to inhibit ADP-stimulated respiration without aIIecting state Iour respiration. The
eIIect oI agaric acid on the transport oI ADP and ATP was examined in rat liver mitochondria.
Respiration stimulated by ADP was progressively inhibited by agaric acid and maximal
inhibition was attained at 40 micromoles agaric acid per litre. ATPase activity was inhibited by
30 at 20 micromoles agaric acid per litre. The exchange oI adenine nucleotides was
competitively inhibited by agaric acid (Chavez et al., 1975).
Pharmacological effects. According to the observed eIIects oI agaric acid on metabolism, the
substance has been classiIied as an antihydrotic and parasympatholytic agent (Henry et al.,
1969).
Agaric acid inhibited acetylcholine-induced contractions at the isolated heart and intestine oI
guinea pigs in vitro, however activity was much lower (up to 100 times) as observed Ior atropin
(Carminati et al., 1968). Agaric acid was a nonspeciIic potentiator (with possibly nonspeciIic
musculotropic mechanisms) oI acetylcholinmimetic Iurtrethonium (at ~ 3 x 10
7
M) in isolated
rat jejunum, and when given alone, it increased both the tone and spontaneous activity oI the
jejunum. In the isolated guinea pig ileum, agaric acid (1-5 x 10
-5
M) caused a delayed onset,
slow, persistent contraction with musculotropic rather than neurotropic characteristics. The
contraction could be blocked by atropine. The observed eIIects were dose-dependent (Carrano
et al., 1967).

TOXICOLOGICAL EVALUATION: Data on acute toxicity indicate that oral doses oI agaric
acid are considerably less toxic than parenteral doses. Due to the highly irritating properties oI
agaric acid, resulting in vomiting and diarrhoea lethal doses are not achieved by acute oral
intake. Repeated single oral doses oI up to 30 mg and maximal daily doses oI 100 mg/person
were used therapeutically in humans to treat hyperhydrosis and did not result in obvious adverse
eIIects. The toxic properties oI agaric acid and its inhibitory eIIects on metabolism may be
related to its structural chemical relationships to citric acid. For the anhydrotic eIIect oI agaric
acid a parasympatholytic mechanism was suggested. The database does not allow the derivation
oI a TMDI.

(T)MDI: Not established due to insuIIicient data.

MAIN OCCURRENCE: Agaric acid has been isolated Irom the dried Iruit body oI the
mushroom White agaric (Fomes officinalis (Vill.:Fr.) Ames, Basidiomycetes; Synonyms:
Agaricum officinale (Vill.:Fr.) Donk, Boletus agaricum, B. laricis Bull., B. officinalis, B.
purgans, Fomes laricis, F. albogriseus, Fomitopsis officinalis (Vil.:Fr.) Bond. et Sing.,
Laricifomes officinalis (Vill.:Fr.) Kotl. et Pouz., Leptoporus officinalis, Polvporus officinalis
(Vill.:Fr.) Fr., Ungulina officinalis; other common names. Polypore du meleze, Agaric blanc,
Agarico, A. bianco) (Taddei et al., 1975; Freedland et al., 1981; Henry et al., 1969; Thoms et al,
1907; Liddle et al., 1979).
Resin contains 14-16 agaric acid (Hoppe, 1975). Dried white agaric contains 16.5 agaric
acid (range 15-18) (Taddei et al., 1975).

INTAKE ESTIMATION: The mushroom white agaric is used as bitter ingredient in certain
beverages (Taddei et al., 1975; Liddle et al., 1979) and in some cough drops (Usher, 1974;
Gessner et al., 1974).
Use levels oI the mushroom white agaric Ior the preparation oI alcoholic beverages have been
reported to be in the range oI 2-2.5 g/l, resulting in concentrations oI 15 mg agaric acid/l in the
Iinished product (not completely dissolved, theoretical content 412 mg agaric acid/l) (Taddei et
al., 1975). Alcoholic beverages have also been reported to be prepared with 10 g/l oI a 10
38
tincture oI the dried Iungus white agaric (no indication oI agaric acid levels) (Council oI Europe,
1999).
In alcoholic beverages containing detectable amounts oI agaric acid the concentrations
measured were in the range oI some mg per litre (Liddle et al., 1979). Higher levels (around 100
mg/l) have been cited earlier (Taddei et al., 1975). It has been demonstrated that the solubility oI
agaric acid in alcoholic beverages depends very much on the pH (low solubility at pH below 4
as in wine based alcoholic beverages) and the content oI alcohol (low solubility at
concentrations below 20) (Liddle et al., 1979).
The consumption oI 100-300 ml oI an alcoholic beverage containing 100 mg/l agaric acid (i.e.
current limit) results in a theoretical intake in the range oI the reported therapeutic single doses
oI 10-30 mg agaric acid.

CONCLUSIONS: The estimated intake based on agaric acid concentrations at the current limit
oI 100 mg/l in alcoholic beverages is in the range oI the recommended therapeutic dose to treat
hyperhydrosis (parasympatholytic eIIect). In alcoholic beverages containing white agaric
concentrations oI 15 mg agaric acid/l have been reported. There seems to be no need Ior a limit
higher than 20 mg/l.

DATA NEEDED: A 28-day oral study in rodents and in vitro mutagenicity studies in
conIormity with OECD guidelines Ior testing oI chemicals are required.

LIMITS: (mg/kg)

General limits in Ioods and beverages: ND
a

Exceptions:
Alcoholic beverages 20

REFERENCES:
Abdenalden S. (1935) Handb. Biol. Arb., General Toxicity Studies, 4, 1289.
Carminati, G.M. and Spina G. (1968) Proprieta Iarmacologiche dell'acido agarico, Boll. Chim.
Farm., 107, 249-251.
Carrano, R.A. and Malone M.H. (1967) Pharmacologic study oI norcaperatic and agaric acids. J.
Pharm. Sci., 56 (12), 1611-1614.
Chavez, E. and Klapp M. (1975) A new inhibitor oI adenine nucleotide translocase in
mitochondria: agaric acid. Biochem. Bioph. Res. Co., 67 (1), 272-278.
Council oI Europe (1981) Flavouring Substances and Natural Sources oI Flavourings. Blue
Book, 3
rd
ed., Strasbourg.
Council oI Europe (1999) Committee oI Experts on Flavouring Substances. Use levels oI Fomes
oIIicinalis RD 5/ 8-44. InIormation submitted by industry.
EEC (1988) Council Directive 88/388/EEC oI 21 June 1988 on the approximation oI the laws oI
the Member States relating to Ilavourings Ior use in IoodstuIIs and to source materials Ior
their production. OIIicial Journal oI the European Communities, 15.7.1988, L184/61-67.
Freedland, R.A. and Newton R.S. (1981) Agaric acid. Methods Enzymol., 72, 497-506.
Gessner, O. and Gerhard Orchezchowski (1974) GiItpIlanzen- und ArzneipIlanzen
Mitteleuropas. 3
rd
ed., Carl Winter Universittsverlag, Heidelberg.

a
ND non-detectable based on modern analytical test methods. The limit oI determination should be taken into

39
Hagers Handbuch der Pharmazeutischen Praxis (1977-1979) 4
th
ed. Editors List, P.H. and
Hrhammer,L., Springer Verlag Berlin.
Hagers Handbuch der Pharmazeutischen Praxis (1998) 5
th
ed., Folgeband 23: Drogen A-Z.
Editors Blaschek, W., Hnsel R., Keller K., Reichling J., Rimpler H. and Schneider G.,
Springer Verlag, Berlin.
Henry, E.D. and Sullivan G. (1969) Phytochemical evaluation oI some cantharelloid Iungi. J.
Pharm. Sci., 58 (12), 1497-1500.
Hood, R.L., Beitz D.C. and Johnson D.C. (1985) Inhibition by potential metabolic inhibitors oI
in vitro adipose tissue lipogenesis. Comp. Biochem. Physiol., 81B (3) 667-670.
Hoppe, H. (1975) Drogenkunde, Band 1 Angiospermen. 8
th
ed., Verlag De Gruyter, Berlin.
Liddle, P.A.P., De Smedt P., Cresto B. and Houllemare A. (1979) Dosage de l'acide agarique
dans les boissons alcooliques. Ann. Fals. Exp. Chim., 72 (733), 125-132.
Newton, R.S. and Freedland R. A. (1980) The eIIects pI speciIic lipogenic substrates and
metabolic inhibitors and the novo Iatty acid synthesis in isolated hepatocytes Irom
chow-Ied Iemale rats. Arch. Biochem. Biophys., 204 (1), 379-386.
Pharmacopoea Helvetica (1933) Editio Quinta, Deutsche Ausgabe, StmpIli, Bern.
Prior, R.L., Jacobson J.J. and Smith S.B. (1981) Metabolic pathways involved in lipogenesis
Irom lactate and acetate in bovine adipose tissue: eIIects oI metabolic inhibitors. Arch.
Biochem. Biophys., 211 (1), 202-210.
Stipani, I., Francia F., Flamieri F. and Quagliarello E. (1976) Inhibition by agaric acid oI
mitochondriale citrate transport. Boll. Soc. Ital. Biol. Sper., 52, 812.
Taddei, I., Giachetti D. (1975) L agarico bianco chimica, tossicologia e Iarmacologia, Riv.
Ital. Essenze - proIumi - piante oIIicinali, aromi, saponi, cosmetici, aerosol, 57, 107-112.
Thoms, H. and Vogelsang J. (1907) Ann. Chem., 357, 145.
Usher, G.A. (1974) Dictionary oI plants used by man. Constable Publisher Mac Millon.

DATA BASES USED: Medline (1966-1999), Biosis (1973-1999), Toxcas (1965-1999),
Toxline (1969-1999), Embase (1965-1999), Toxlit (1965-1993). Keywords: agaric acid, white
agaric, Fomes officinalis, Fomes laricis, Polvporus officinalis.
40
41
beta-Asarone

ACTIVE PRINCIPLE: I

SYNONYMS: 2,4,5-trimethoxy-1-propenylbenzene, cis-1-propenyl-2,4,5-trimethoxybenzene,
cis-asarone

CAS No: 5273-86-9

STRUCTURE:

O
O
O

REGULATORY / INTERNATIONAL STATUS: The Council oI Europe Committee oI
Experts on Flavouring Substances evaluated beta-asarone as an active principle in Iood
Ilavourings and recommended limits oI 0.1 mg/kg in Ioods and beverages with the exceptions oI
1 mg/kg in alcoholic beverages and 1 mg/kg in Ioods containing Acorus calamus L. or Asarum
europaeum L. (Council oI Europe, 1981). In Annex II oI Council Directive 88/388/EEC on
Ilavourings, general limits and the limit Ior alcoholic beverages are as above (EEC, 1988).
However, no separate limit Ior Ioods containing Acorus calamus L. or Asarum europaeum L.,
but additionally a limit oI 1 mg/kg has been set Ior seasonings used in snack Ioods (EEC, 1988).
In the USA, calamus and its derivatives (oil, extracts, etc.) are prohibited Irom use in human
Iood (CFR 189.110) (Federal Register, 1968). JECFA evaluated beta-asarone in 1981 and did
not establish an ADI, but recommended that calamus oil used in Iood should contain the lowest
possible level oI beta-asarone (JECFA, 1981). SCF evaluated beta-asarone in 2001 and
concluded that beta-asarone has a weak carcinogenic eIIect in rats and that a genotoxic potential
cannot be ruled out. ThereIore, SCF concluded that no threshold could be assumed and that
limitations in exposure and use levels were appropriate (SCF, 2001). Regulation oI the use oI
the various karyotypes oI Acorus calamus L. in Ioods and beverages in the EU is currently
under revision.


Metabolism:
Animal studies: The metabolic pathway oI beta-asarone has not been elucidated so Iar. It is
thought to be diIIerent to that oI saIrole/estragole where hydroxylation takes place at the 1`-
position leading to the Iormation oI sulphuric acid esters, which have been recognized as the
major ultimate electrophilic and carcinogenic metabolites oI alkenylbenzenes in vivo. Evidence
42
Ior a diIIerent metabolism was provided by the observation that pretreatment with
pentachlorphenol (a sulphotransIerase inhibitor which inhibits the esteriIication oI the 1`-
hydroxy metabolites) did not inhibit the hepatocarcinogenicity oI beta-asarone in male mice
(Wiseman et al., 1987). In preliminary investigations in male rats given 75-300 mg beta-
asarone/kg bw in saIIlower oil i.p. or orally, small amounts oI basic ninhydrin-positive
substances, believed to be substituted phenylisopropyl amines or amphetamines, have been
identiIied in the urine. No such substances were excreted by the same animals when given the
vehicle only. Similar substances were Iound in rats treated with other alkenylbenzenes such as
myristicin, saIrole or isosaIrole. Treatment with the trans-isomer (alpha-asarone) produced
much higher (10-50Iold) amounts oI ninhydrin-positive material than did either the cis-isomer
(beta-asarone), saIrole or myristicin at similar doses (Oswald et al., 1969).

Toxicology:
Acute toxicity: Beta-asarone has an oral LD
50
oI 1010 mg/kg bw in rats and an i.p. LD
50
oI
184 mg/kg bw in mice (JECFA, 1981). Body weight gain and Iood consumption were reduced
in pre-weanling Sprague-Dawley rats receiving a single i.p. injection oI 100 or 250 mg beta-
asarone/kg bw. Reduced thymus weight (20-30 wt reduction) and liver weights were recorded
at necropsy, 5 days aIter treatment (Ramos-Ocampo and Hsia, 1987).
Subacute / subchronic toxicity: Four pre-weanling Sprague-Dawley rats received 100 mg
beta-asarone/kg bw/day i.p. on 5 consecutive days. Reduced body weight gain, increased
adrenal weights, decreased heart and thymus weight, and a higher incidence oI single cell
degeneration in cortex oI thymus compared to controls was observed. There was no change in
the concentration oI microsomal protein and cytochrome P450 as well as in serum transaminase
levels observed one day aIter the last injection (Ramos-Ocampo and Hsia, 1987).
Chronic toxicity / carcinogenicity: Groups oI 25 male and 25 Iemale rats (strain not speciIied)
were Ied a diet containing 0, 0.04, 0.08 or 0.2 beta-asarone (equivalent to 0, 20, 40 and
100 mg beta-asarone/kg bw/day) Ior two years, and a positive control group received 0.25
calamus oil Irom the Indian variety oI Calamus cultivated in the Jammu area (assumed to
contain 75.8 beta-asarone (Taylor et al., 1967)) in the diet Ior two years. Mortality was
increased in the two highest dose groups. The gross pathological changes observed were serous
Iluid in the abdominal and pleural cavities, changes in the liver (hepatic angiectasis and
necrosis) and kidneys. Leiomyosarcomas (malignant, highly pleomorphic, highly anaplastic)
were observed in the small intestines oI male rats with a dose-related increase in the incidences
oI 0/25, 1/25, 6/25 and 9/25 male rats receiving 0, 0.04, 0.08 or 0.2 beta-asarone in the
diet and 1/25 males receiving 0.25 calamus oil. Leiomyosarcomas were also noted in two
Iemale controls, but in none oI the treated Iemales, and the location diIIered Irom that oI the
tumours Iound in male mice (Taylor, 1981; more details in JECFA, 1981). In another study,
groups oI 25 male and 25 Iemale rats (strain not speciIied) were Ied a diet containing 0, 0.05,
0.1, 0.25 or 0.5 oI the Indian variety oI calamus oil Irom the Jammu area (equivalent to about
0, 25, 50, 125 and 250 mg calamus oil/kg bw/day or 0, 19, 38, 95 and 190 mg beta-asarone/kg
bw/day based on the reported beta-asarone content oI 75.8 in the calamus oil) Ior two years. A
dose-related increase in the mortality was observed in all treated groups, with Iemales dying
earlier than males. Weight gain was reduced in a dose-related manner in both sexes, with
Iemales being more sensitive. Degenerative and regenerative changes were observed in the liver
on microscopic examination. Leiomyosarcomas, mainly in the duodenum, were Iound in
animals oI both sexes (none in the control group, three in Iemales on the 0.05 calamus oil diet,
Iive in males on the 0.1 diet, two in males on the 0.25 diet and none in all other groups).
Histopathological changes observed in the heart and liver were similar to those observed with
beta-asarone (Taylor et al., 1967; Taylor, 1981; more details in JECFA, 1981). Male pre-
43
weanling B6C3F
1
mice (43 animals) received beta-asarone i.p. on day 1, 8, 15 and 22 (total dose
oI approx. 1 mg/animal). At the age oI 13 months 83 oI mice had hepatomas with an average
oI 2.4 + 1.7 hepatomas per mouse. A similar response was elicited when beta-asarone was given
as a single i.p injection oI 52 mg/kg bw to male pre-weanling B6C3F
1
mice (30 animals). AIter
12 months 69 oI the mice autopsied had hepatomas with an average oI 1.1 + 1.0
hepatomas/mouse. In both instances the incidence oI hepatomas was signiIicantly diIIerent
(p0.001) Irom mice treated with solvent only (Wiseman et al., 1987).
Reproductive toxicity / teratogenicity: Survival oI chicken embryos was 43 and 0 when
eggs were inoculated with a solution containing 0.04 or 4.0 mg beta-asarone/egg, respectively
(Yabiku 1980).
Mutagenicity / genotoxicity: Several in vitro studies on genotoxicity have been carried out.
Beta-asarone was not mutagenic in the Ames test with Salmonella tvphimurium strains TA98,
TA100, TA1525, TA1537 and TA1538 with and without metabolic activation at 0.2-1000
micrograms/plate (Hsia et al., 1979; Goggelmann and Schimmer, 1983; Ramos-Ocampo and
Hsia, 1988), except Ior one study in which a positive response was Iound in strain TA100 with
metabolic activation, however, at two oI the tested concentrations only and without dose-
relation (Goggelmann and Schimmer, 1983).
No unscheduled DNA synthesis (UDS) was observed in isolated rat hepatocytes at
concentrations oI up to 1 mmole/l (equivalent to 208 micrograms/ml) in one study (Ramos-
Ocampo and Hsia, 1988), but in a more recent study, UDS in rat hepatocytes was increased at
0.5 mmol/l oI beta-asarone (equivalent to 104 micrograms/ml). As UDS response was less
pronounced in the presence oI the cytochrome P450 inhibitor cimetidine, it has been suggested
that genotoxicity oI beta-asarone is mediated by metabolite(s) Iormed by cytochrome P450-
mediated oxidation (Hasheminejad and Caldwell, 1994). Clastogenic eIIects were produced in
human lymphocytes by a 70:30 mixture oI alpha- and beta-asarone at a concentration oI
107 micrograms/ml in vitro with and without metabolic activation. Chromosome breaks and a
small increase in sister chromatid exchanges (SCE), which was not signiIicant, were recorded
(less than twoIold compared to control), and the author suggested not to consider the increase oI
SCE as relevant (Abel, 1987).

Human data: No data Iound.

Other studies: Pharmacologv studies: In heart perIusion studies, beta-asarone was Iound to
cause diastolic stoppage at a dose oI 300 micrograms in the Irog and rabbit heart. Beta-asarone
produced a Iall in blood pressure in anaesthetised dogs by about 50 mm Hg at a i.v. dose oI 3
mg/kg bw. Beta-asarone has been reported to have anti-acetyl cholinesterase activity in guinea-
pig ileum, rat uterus, Irog rectus and dog tracheal muscle preparations (Sharma and Dandiya,
1962). An i.p. dose oI 20 mg beta-asarone/kg bw potentiated convulsions induced by metrazol
and picrotoxin (Sharma et al., 1961).

TOXICOLOGICAL EVALUATION: Beta-asarone is not acutely toxic in rats and mice.
There are only Iew data available on subchronic and chronic toxicity. Beta-asarone was
carcinogenic in rodents, inducing leimyosarcomas in the small intestine oI male rats and
hepatomas in the liver oI male mice. The genotoxic potential oI beta-asarone is unclear. The Iew
available data, e.g. mutagenicity studies in Salmonella tvphimurium and UDS tests in rat
hepatocytes gave equivocal results.

(T)MDI: Not established (suspected genotoxic carcinogen).

44
MAIN OCCURENCE: Beta-asarone is an active principle with Ilavouring properties Iound in
the rhizomes oI calamus (Acorus calamus L., CE No. 13) and in the whole plant oI hazelwort
(Asarum europaeum L., CE No. 76). Asarum europaeum L. contains up to 50 beta-asarone in
the essential oil (Wren, 1988). The beta-asarone content in the essential oils oI Acorus calamus
L. ranges Irom 0 to up to 95, depending on the karyotype and the origin oI the plant (SCF,
2001). The Indian tetraploid variety oI Acorus calamus L. contains up to 95 beta-asarone in
the essential oil, the European triploid variety up to 10 (SCF, 2001), and the diploid Iorm only
traces (Liddle and Bossard, 1985).
Industry endeavours to use the European variety oI calamus with the lower beta-asarone content
in the essential oil. The diploid Iorm oI calamus is currently not commercially available, though
it is cultivated in Mongolia and used in the USA and Canada as a source oI aromatherapy
products (Council oI Europe, 1998).

INTAKE ESTIMATION: Rhizomes oI calamus (Acorus calamus L.) are used in the
preparation oI bitters and in the Iormulation oI vermouths. The essential oil, obtained Irom
steam distillation oI rhizomes, is used Ior the preparation oI compounded oils Ior liqueurs,
conIectionery, cakes, dairy products, syrups and sauces (Fenaroli, 1975; IOFI, 1998). Intake
Irom Ioods Ilavoured with calamus oil is relatively low. Cakes, desserts, conIectionary and dairy
products contribute to about the same extent (about 0.5-2.3 micrograms beta-asarone/day) to the
total daily intake (see Table I). Highest intakes are most likely to arise Irom the consumption oI
alcoholic beverages Ilavoured with calamus rhizome and preparations thereoI. Levels oI beta-
asarone in alcoholic beverages have been reported to range Irom 0.002-4.9 mg/l (Council oI
Europe, 1998). Assuming a daily consumption oI 40-100 microlitres alcoholic beverages
containing the maximum limit oI 1 mg beta-asarone/kg as set in Council Directive 88/388/EEC
(EEC, 1988), the intake would be in the range oI 20-100 micrograms/day. No reIerences could
be Iound on the use oI Asarum europaeum L. as a Ilavouring.
The total intake oI beta-asarone Irom Ioods and alcoholic beverages has been estimated to be in
the range oI 8.5-49 micrograms/day (0.12-0.7 micrograms/kg bw/day) Ior mean and extreme
consumers, respectively, assuming that beta-asarone levels in alcoholic beverages do not exceed
1 mg/kg. This estimation was based on the reported use levels oI essential oils as indicated in
Table I and Iood intake data Irom the dietary and nutritional survey oI British adults (British
adult study) (Gregory et al., 1990).

CONCLUSIONS: Beta-asarone is clearly carcinogenic in rodents and potentially genotoxic.
Comparison with other alkenylbenzenes is diIIicult because there is no DNA-binding study
available, and as there is indication that the activation oI beta-asarone does not involve a
sulphation step, as known Ior other alkenylbenzenes, the mechanism oI carcinogenicity may be
diIIerent Irom the saIrole-type. It is prudent to reduce the level oI beta-asarone in Ioods as Iar as
possible, but there may be a need Ior speciIic exceptions Ior alcoholic beverages. Due to the
high beta-asarone content oI up to 95 in essential oils oI the tetraploid Iorm and up to 10 in
the triploid Iorm oI Acorus calamus L., their use should be restricted by classiIying them as not
suitable Ior human consumption (category 6
a
). Concerted eIIorts should be made to investigate
the commercial availability oI the diploid Iorm (containing no beta-asarone or traces) with the
aim oI ensuring that no natural sources containing beta-asarone are used in Ioods or beverages
or as sources oI natural Ilavourings or other Iood or beverage components.

DATA NEEDED: To clariIy whether beta-asarone is genotoxic or not, in vivo mutagenicity
studies, especially DNA binding studies are needed.

a
Category 6: Plants, animals and other organisms, and parts oI these or products thereoI, and preparations derived
thereIrom, which are considered to be unIit Ior human consumption in any amount (Council oI Europe, 2000)
45
LIMITS: (mg/kg)

General limit in Ioods and beverages: ND
a

Exceptions:
Alcoholic beverages traditionally Ilavoured with calamus: 0.5

REFERENCES:
Abel, G. (1987) Chromosome-damaging eIIect oI beta-asarone on human lymphocytes. Planta
Medica, 53 (3), 251-253.
Adam, L. and Postel W. (1992) Bestimmung von alpha- und beta-Thujon, SaIrol, IsosaIrol,
beta-Asaron und Cumarin in weinhaltigen Getrnken und Spirituosen. Die
BranntweinwirtschaIt, 132 (12), 202-206.
Book, 3
rd
ed., Strasbourg.
Council oI Europe (1998) Committee oI Experts on Flavouring Substances. Review oI the
toxicity oI the active principles Ior the 4
th
edition part II oI 'Ilavouring substances and
natural sources oI Ilavourings, Annex 2 'beta-asarone and calamus- RD 4.2/16-42.
Document submitted by IOFI.
Council oI Europe (2000) Natural sources oI Ilavourings, Report No. 1, p. 11, ClassiIication
system Ior natural Ilavouring sources and preparation, Strasbourg.
Federal Register (1968) May 9.
Fenaroli (1975) Fenaroli`s Handbook oI Flavor Ingredients, Second edition, CRC Press, Boca
Raton.
Fenaroli (1995) Fenaroli`s Handbook oI Flavor Ingredients, Volume I, Ed. G.A. Burdock, Third
edition, CRC Press, Boca Raton.
Goggelmann, W. and Schimmer O. (1983) Mutagenicity testing oI beta-asarone and commercial
calamus drugs with Salmonella typhimurium. Mutat. Res., 121 (3-4), 191-194.
Gregory, J., Foster K., Tyler H. and Wiseman M. (1990). The dietary and nutritional survey oI
British Adults. HMSO, London.
Hasheminejad, G and Caldwell J. (1994) Genotoxicity oI the alkenylbenzenes alpha- and beta-
asarone, myristicin and elemicin as determined by the UDS assay in cultured rat
hepatocytes. Fd. Chem. Toxic., 32(3), 223231.
Hsia, M.T S., Adamovics J.A.A. and Kreamer L.W. (1979) Microbial mutagenicity studies oI
insect growth regulators and other potential insecticidal compounds in Salmonella
typhimurium. Chemosphere, 8 (9), 521-529.
IOFI (1998) Personal communication to Council oI Europe, Committee oI Experts on
Flavouring Substances, 42
nd
meeting, Bruxelles, April 1998.
JECFA (1981) Joint FAO/WHO Expert Committee on Food Additives. Monograph on beta-
asarone. WHO Food Additive Series No. 16.
Lander, V., Wrner M., Kirchenmayer C., Wintoch H. and Schreier P. (1990) Anwendung der
Festphasenextraktion zur raschen Probenvorbereitung bei der Bestimmung von
LebensmittelinhaltsstoIIen. II. Asaron, Chinin, Cumarin und Quassin in Trinkbranntwein.
Z. Lebensm. Unters. Forsch., 190, 410-413.

a
ND Non-detectable based on modern analytical test methods. The limit oI determination should be taken into
46
Lawrence, B.M. (1997) Progress in Essential Oils: Calamus oil. PerIumer & Flavourist, 22 (2),
65-67.
Liddle, P.A.P. and Bossard A. (1985) Volatile naturally-occuring restricted compounds derived
Irom Ilavourings, and their determination in Ioods and beverages. In: Progress in Flavour
Research. Proceedings oI the 4th Weurman Flavour Research Symposium, Dourdan,
France, 9-11 May 1984. Adda, J. (Ed.), Elsevier Science Publishers B.V., Amsterdam, p.
467-476.
Merat, E., Martin E., Duret M. and Vogel J. (1976) Extraction et dosage pour chromatographie
en phase gazeuse de beta-asarone et beta-thuyone dans les aperitiIs. Trav. chim. aliment.
hyg., 67, 521-526.
Micali, G., Curro P. and Calabro G. (1980) Reversed-phase high-perIormance liquid
chromatography Ior the determination oI beta-asarone. J. Chromatography, 194 (2), 245-
250.
Morales-Ramirez, P., Madrigal-Bujaidar E., Mercader-Martinez J., Cassini M., Gonzalez G.,
Chamorro-Cevallos G. and Salazar-Jacobo M. (1992) Sister-chromatid exchange induction
produced by in vivo and in vitro exposure to alpha-asarone. Mutat. Res., 279(4), 269-273.
Oswald, E.O., Fishbein L. and Corbett B.J. (1969) Metabolism oI naturally occurring propenyl
benzene derivatives. I. Chromatographic separation oI ninhydrin positive materials in rat
urine. J. Chromatography, 45, 437-445.
Przyborski, H. and Bandion F. (1992) Zur Bestimmung von beta-Asaron, Pulegon, SaIrol,
Santonin und Thujon in Spirituosen und Wein. Mitteilungen Klosterneuburg, 42, 171-178.
Ramos-Ocampo, V.E. and Hsia M.T.S. (1987) EIIects oI acute treatments oI calamus oil, beta-
asarone and dimethoxypropenylbenzenes in laboratory rats. Phillip. Ent, 7 (2), 129-158.
Ramos-Ocampo, V. E. and Hsia M.T.S. (1988) Mutagenic and DNA-damaging activity oI
calamus oil, asarone isomers and dimethoxypropenylbenzenes analogues. Phillip. Ent., 7
(3), 275-291.
ScientiIic Committee on Food (SCF) (2001) Opinion oI the ScientiIic Committee on Food on
the presence oI beta-asarone in Ilavourings and other Iood ingredients with Ilavouring
properties.
Sharma, J.D, Dandiya P.C., Baxter R.M. and Kandel S.I. (1961) Pharmacodynamical eIIects oI
asarone and beta-asarone. Nature, 192 (4809), 1299-1300.
Sharma, J.D. and Dandiya P.C. (1962) Studies on Acorus calamus Part VI. Pharmacological
actions oI asarone and beta-asarone on cardiovascular system and smooth muscles. Ind. J.
Med. Res., 50 (1), 61-65.
Taylor, J.M. Jones W.I., Hagan E.C., Gross M.A., Davis D.A. and Cook E.L. (1967) Toxicity oI
oil oI calamus (Jammus variety). Toxicol. Appl. Pharmacol., 10, 405 (abstract).
Taylor, J. M. (1981) (Food and Drug Administration) Personal communication to the World
Health Organization concerning unpublished studies on beta-asarone and calamus oils.
(cited in JECFA, 1981)
Todorova, M.N. and Ognyanov I.V. (1995) Chemical composition oI essential oil Irom
mongolian Acorus calamus L. rhizomes. J. Ess. Oil Res., 7, 191-193.
Vashist, V.N. and Handa K.L. (1964) A chromatographic inestigation oI indian calamus oils.
Soap, PerIumery & Cosmetics, 37, 135-139.
Wiseman, R.W., Miller E.C., Miller J.A. and Liem A. (1987) Structure-activity studies oI the
hepatocarcinogenicities oI alkenylbenzene derivatives related to estragole and saIrole on
administration to preweanling male C57BL/6J x C3H/HeJ F1 mice. Cancer Res., 47 (9),
2275-2283.
Wren, R.C. (1988) Potters New Cyclopedia oI Botanical Drugs and Preparations. CW Daniel
Company Ltd, SaIIron Walden.
47
Yabiku, H. Y. (1980) Calamus oil Toxicological aspects and their control in alcoholic
beverages. M. S. thesis, Sao Paulo, Brazil, submitted to FAO/WHO (cited in JECFA,
1981).

DATA BASES USED: Biosis (1973-1998), Medline (1966-1998), Analytical Abstracts (1980-
1998), FSTA (1990-1998), Leatherhead Food Science Database (FROSTI) (1975-1998).
Keywords: beta-asarone, cis-asarone, Calamus.
48
49
d-Camphor
a


SYNONYMS: D-2-bornanone; D-2-camphanone; (1R)-1,7,7-trimethyl-bicyclo|2.2.1|-heptane-
2-one; 2-keto-1,7,7-trimethyl-nor-camphane; bicyclo |2.2.1|-heptan-2-one-1,7,7-trimethyl (1R):
d-camphor (CAS name), ()-camphor

CAS No: 464-49-3 (natural d-camphor)

STRUCTURE:

O

REGULATORY / INTERNATIONAL STATUS: d-Camphor has been classiIied as a
chemically-deIined Ilavouring substance Ior provisional use in IoodstuIIs (Category B) in the
Blue Book, Volume I (Council oI Europe, 1992). d-Camphor is also listed in the EU-Register oI
chemically-deIined Ilavouring substances (EEC, 1999). In the USA, leaves oI the camphor tree
(Cinnamomum camphora ssp.) and d-camphor were given GRAS status by FEMA and are
approved by the FDA Ior Iood use (CFR 172.510, saIrole-Iree; 21 CFR 172.515). Camphor oil
(Japanese, white) has also GRAS status (FEMA No. 2231) (FEMA, 1995).


Metabolism:
studies: In rat and rabbit liver preparations (microsomes) camphor was oxidized to 5-
endo- and 5-exo-hydroxycamphor as main metabolites and to ()-2,5-bornanedione as minor
metabolite. In liver cytosol reduction oI the 5-keto group occurred, and in the presence oI both
microsomes and cytosol interconversion between the endo- and exo-isomers oI 5-
hydroxycamphor was observed (Leibmann and Ortiz, 1973).
In vivo studies: The major pathways in dogs and other mammals were Iound to be oxidation at
the C-3 and especially at the C-5 position to hydroxycamphors, which were mainly excreted in
urine as glucuronides. A small amount was Iound to be present in the expired air (Leibmann and
Ortiz, 1973; Williams, 1959; Robertson and Hussain, 1969). Conversion oI the
hydroxycamphors to ketocamphors (oxocamphors) with Iurther metabolization may also occur
(review in Scheline, 1991). A minor metabolic pathway is the reduction oI the keto group to
yield ()-borneol (2-endo-hydroxybornane) (Williams, 1959).

a
Camphor occurs in nature as the d- or l-Iorm, the optical inactive Iorm (dl-) is rarely encountered. In the present evaluation
only the d-Iorm is considered. II camphor is written without indication oI the isomer, inIormation on the isomer has not
been reported.
50
Human studies: The major metabolic reactions observed in humans were hydroxylation at the
3-, 5-, 8- and 9-position. The resulting 5- and 8- (or 9-) hydroxycamphors were Iurther oxidized
to a ketone and carboxylic acid. The Iormed metabolites were excreted unchanged, except Ior
the carboxylic acid which was conjugated with glucuronic acid (Kppel et al., 1982).
Stereoselective reduction to borneol and consecutive excretion in urine as glucuronide has been
reported as a minor pathway (Wagreich et al., 1941).

Toxicology:
Acute toxicity: An oral LD
50
oI 5 g/kg bw in rats and 1.31 g/kg bw in mice has been reported
(Opdyke, 1978).
Subacute / subchronic toxicity: In a poorly reported study, groups oI Iive rats were given 0,
250, 500, 1000 or 1200 mg sage oil/kg bw/day (equivalent to about 0, 75, 150, 300 and 360 mg
camphor/kg bw/day; no inIormation given on the content oI thujone in the sage oil used) orally
Ior 8 weeks. The lowest dose caused no eIIects on mortality, body weight gain and behaviour.
At 500 mg/kg bw/day, reduced body weight gain, cramps and one case oI mortality was
observed. At the highest dose, these eIIects occurred in most animals (Von Skramlik, 1959).
Chronic toxicity / Carcinogenicity: No carcinomas were seen in mice aIter a period oI 4.5-
6 months during which skin was painted twice a week with a mixture oI cyclic terpenes
including camphor (Benko et al., 1963). No increase in the incidence oI primary lung tumour
was seen in strain A mice when the maximum tolerated dose oI 750 mg/kg bw/day (MTD;
maximum dose tolerated by Iive mice when six i.p. injections were administered over a 2-week
period) or 150 mg/kg bw/day (20 MTD) were injected i.p. 3 times/week Ior 8 weeks and
animals were killed aIter a total oI 24 weeks (Stoner et al., 1973).
Reproductive toxicity / teratogenicity: Female rats were given 0, 100, 400 or 800 mg
camphor/kg bw/day, Irom day 6 to 15 oI gestation. There was evidence oI maternal toxicity and
some clinical signs at the two highest doses; but no adverse eIIects on Ietal growth, viability or
morphological development (NTP, 1992). No evidence oI teratogenicity was seen when
pregnant rats were administered orally 1000 mg camphor/kg bw/day during the Ietal period oI
organogenesis, or when oral doses up to 681 mg camphor/kg bw/day were given to pregnant
rabbits (Leuschner, 1997).
Mutagenicity / Genotoxicity: Camphor was negative in the Ames test with Salmonella
tvphimurium strains TA1535, TA1538, TA98 and TA100 at 4, 20, 100, 500 and
2500 micrograms/plate with and without metabolic activation (Anderson and Styles, 1978).

Human data: Many cases oI accidental non-Iatal and Iatal camphor poisoning, especially in
children, have been reported aIter oral doses oI camphor (Kppel et al., 1982; Smith and
Margolis, 1954; Riggs et al., 1965; Aronow and Spigiel, 1976; Phelan, 1976; Dupeyron et al.,
1976; Geller et al., 1984). Signs oI camphor intoxication include central nervous stimulation
with mouth, throat and gastric irritation, nausea and vomiting, excitement, hallucinations,
delirium, muscular excitability, tremors and convulsions. Coma and respiratory Iailure Iollowed
by death have been reported to occur aIter large exposures. An evaluation oI 64 cases Irom
literature and 182 cases Irom poison centers was carried out to detect the dose-eIIect relation Ior
the management oI suspected camphor ingestion. OI the 102 poison centre cases, 81 cases
ingested a dose estimated to exceed 2 mg/kg bw. The reported 101 cases with ingestion oI less
than 2 mg/kg bw remained asymptomatic. Seventy-threee patients ingesting over 2 mg/kg bw
remained asymptomatic (90), 3 (4, mean dose 15 mg/kg bw, median dose 13 mg/kg bw,
dose range 5-27 mg/kg bw) developed minor symptoms, and 5 (6, mean dose 152 mg/kg bw,
median dose 80 mg/kg bw, dose range 59-475 mg/kg bw) developed symptoms deemed major
(syncope, cyanosis, hypotension, arrhythmias, mental status changes). There were no deaths
among poison centre cases. Based on the observed dose-related eIIects, patients without
51
symptoms or signs who ingested less than 10 mg/kg bw were considered not to be at risk Ior
clinical complications, and patients ingesting between 10 and 30 mg/kg bw appeared to be at
risk Ior the development oI minor symptoms (sleepy but arousable, gagging, crying), but still at
(low) risk Ior seizures. Major symptoms were also reported aIter oral doses oI 31-430 mg/kg bw
(median dose oI 86 mg/kg bw) in 23 oI the 64 literature cases. Six Iatalities were reported aIter
ingestion oI 60-570 mg camphor/kg bw (median dose 113 mg/kg bw) in the literature cases (no
Iurther details given). Overall, it was concluded that clinically signiIicant camphor toxicity does
not maniIest below oral doses oI 30 mg/kg bw, and is uncommon below 50 mg/kg bw (Geller et
al., 1984). No dermal irritation and no sensitization reactions were seen in humans when 4
camphor in petrolatum was administered in a closed-patch test Ior 48 hours (Opdyke, 1987).

Other studies:
Pharmacologv. Camphor has a CNS stimulating activity in laboratory animals and humans,
producing convulsions iI given at doses close to the lethal dose. Numerous studies on the
cardiovascular action have produced inconclusive results. Camphor has a slight expectorating
activity and acts as a local irritant (Opdyke, 1987).
Cvtotoxicitv. No cytotoxic eIIects were seen in HeLa cells at concentrations oI 1, 10 and
100 micrograms camphor/ml (Zolotovich et al., 1967).
Effects on en:vme activities. In vitro. Cyclooxygenase activity oI sheep vesicular gland was
inhibited at concentrations oI more than 76 micrograms camphor/ml (Dewhirst, 1980). In vivo.
Female Swiss albino mice (groups oI 8 animals) were treated with oral doses oI 0, 50, 150 or
300 mg camphor/kg bw/day Ior 20 days. Animals in the highest dose group had increased
hepatic cytochrome P450 and b5 levels, increased aryl hydroxylase activity, and a reduced
glutathione and glutathione-S-transIerase activity, compared to controls treated with olive oil
(p0.05). No signiIicant eIIects on enzyme activity were observed in the lower dose groups
(Bananerjee et al., 1995).

TOXICOLOGICAL EVALUATION: There are only limited data available on the toxicology
oI camphor in laboratory animals and humans. The quality oI some oI the studies is insuIIicient.
Acute oral toxicity oI camphor in rats and mice is low. In a limited oral 8-week study with sage
oil, no adverse eIIects were noted at doses oI 250 mg sage oil/kg bw/day (equivalent to 75 mg
camphor/kg bw/day) in rats. Reduced body weight gain, cramps and increased mortality
occurred at higher doses. It has to be considered that sage oil generally contains high amounts oI
thujone (in average approx. 50) Ior which a NOAEL Ior convulsive eIIects oI 5 mg/kg bw/day
was derived Irom a 90-day oral study in rats. Thus, it may be assumed that the eIIects observed
in the above study in rats are caused by thujone. Oral chronic toxicity and carcinogenicity
studies on camphor in animals are lacking. d-Camphor has been recognised as a neurotoxic
compound. The very limited toxicity data available gave no evidence Ior carcinogenic,
genotoxic or teratogenic activity oI camphor in animals. Adverse eIIects Iollowing accidental
exposure oI humans, especially oI children, to camphor have been described in a large number
oI case reports. Evaluation oI 245 cases with camphor ingestion indicated that in humans
clinically signiIicant camphor toxicity is unlikely to occur aIter oral doses oI up to 50 mg
camphor/kg bw/day. Fatal cases have been reported aIter oral doses oI 60 to 570 mg
camphor/kg bw. Due to the insuIIicient data available a (T)MDI could not be established.


MAIN OCCURRENCE: Highest concentrations oI d-camphor occur in the camphor tree
(Cinnamomum camphora (L.) Nees. et Eber. var. camphora, CE No. 130a and a number oI
related varieties). Camphor is present in the essential oil oI the wood, stumpwood, branches and
52
leaves, oI which camphor oil is obtained by steam distillation. Considerable amounts oI 10 to
70 camphor are also Iound in essential oils oI tansy (Chrvsanthemum vulgare (L.) Bernh., CE
No. 446), sage (Salvia officinalis L., CE No. 414), Spanish sage (Salvia lavandulifolia Vahl, CE
No. 413), rosemary (Rosmarinus officinalis L., CE No. 406), spike lavender (Lavandula
latifolia Medik., CE No. 256), IeverIew (Chrvsanthemum parthenium (L.) Bernh., CE No. 382),
and white mugwort (Artemisia herba-alba Asso, CE No. 2011). Low concentrations oI camphor
have been reported in essential oils oI other natural sources oI Ilavourings (see Table I). The
ratio oI d/l-camphor is not speciIied in most cases.
Some oI the camphor-containing natural sources contain high amounts oI thujone in their
essential oils, i.e. 12 to 65 alpha-thujone and 1.2 to 36 beta-thujone in sage, nearly 80 in
tansy (mainly beta-thujone) and 2.5 to 82 oI alpha-thujone and 0.5 to 94 oI beta-thujone in
white mugwort depending on the chemotype. Considerable amounts oI eucalyptol are also
Iound in essential oils oI the camphor tree (7 in bark oil), sage (8 to 22) and Spanish sage
(12 to 41) (Council oI Europe, 2001; Council oI Europe, 2004).

INTAKE ESTIMATION: The main sources oI camphor in the human diet derive Irom the
sage plant and the essential oils oI coriander, basil and rosemary used in meat products,
alcoholic beverages, condiments and sauces and Irom cheese (Table II, III and IV). The mean
daily intake oI camphor Irom Ioods and beverages is estimated to be 1.5 mg/person (0.025
mg/kg bw/day) (Council oI Europe, 2000a). Since the ratio oI d/l-camphor is mostly not
speciIied it is assumed that the estimated camphor intake comprises both isomers.

CONCLUSIONS: Due to the insuIIicient toxicological data a (T)MDI could not be allocated
and camphor was considered as a class II active principle. However, the limits set Ior camphor
in IoodstuIIs ensure a satisIactory margin oI saIety Ior the intake oI camphor. The estimated
daily intake oI camphor Irom Ioods and beverages oI approximately 0.025 mg camphor/kg
bw/day (or 1.5 mg/person) is a Iactor oI 80 below the dose oI 2 mg/kg bw that generally does
not exert clinical symptoms and a Iactor oI 1200-2400 below the lowest doses reported to result
in major symptoms (30 mg/kg bw) or death (60 mg/kg bw).

DATA NEEDED: In order to reconsider the classiIication oI camphor a 28-day oral study in
rodents and two in vitro mutagenicity studies with d-camphor in conIormity with OECD
guidelines Ior testing oI chemicals should be carried out. Furthermore, adequate studies to
disclose the neurotoxic eIIects oI camphor are needed.

LIMITS: (mg/kg)

General limits in Ioods and beverages:
Foods 25
Beverages (including alcoholic drinks) 10

Exceptions:
Candy 100
Fresh cheese 140
Sauces and condiments 150

REFERENCES:
Anderson, D. and Styles J.A. (1978) An evaluation oI 6 short-term tests Ior detecting organic
chemical carcinogens. Appendix 2. The bacterial mutation test. Br. J. Cancer, 37, 924-930.
53
Aronow, R. and Spigiel R.W. (1976) Implications oI camphor poisoning: therapeutic and
administrative. Drug Intell. Clin. Pharm., 10, 631-634.
Banerjee, S., Welsch C.W. and Rao A.R. (1995) Modulatory inIluence oI camphor on the
activities oI hepatic carcinogen metabolizing enzymes and the levels oI hepatic and
extrahepatic reduced glutathione in mice. Cancer Lett., 88 (2), 163-169.
Benko, A., Tiboldi T. and Bardos J. (1963) The eIIect oI painting with clinical terpenes on the
skin oI white mice and on the skin carcinoma developed by benzpyrene painting. Acta
Unio Int. Contra Cancrum, 19, 786-788.
Council oI Europe (1992) Flavouring substances and natural sources oI Ilavourings, Volume I:
Chemically deIined Ilavouring substances, 4
th
edition (Blue Book).
Council oI Europe (2000a) Committee oI Experts on Flavouring Substances, Presentation de
calculs dìngestion concernant le camphre (Intake calculations on camphor) - RD 4.3/1-47.
Council oI Europe (2000b) Natural sources oI Ilavourings, Report No. 1, Strasbourg.
Council oI Europe (2001) Committee oI Experts on Flavouring Substances, New datasheets on
natural sources - RD 5.2/6-49.
Council oI Europe (2004) Natural sources oI Ilavourings, Report No. 2, Strasbourg (in press).
Dewhirst, F.E. (1980) Structure-activity relationships Ior inhibition oI prostaglandin
cyclooxygenase by phenolic compounds. Prostaglandins, 20 (2), 209-222.
Dupeyron, J.P., Quattrocchi F., Castaing H. and Fabiani P. (1976) Intoxication aigue du
nourrisson par application cutanee dùne pommade revulsive locale et antiseptique
pulmonaire. Eur. J. Toxicol., 9 (5), 313-320.
EEC (1999) Commission decision oI 23 February 1999 adopting a register oI Ilavouring
substances used in or on IoodstuIIs drawn up in application oI Regulation (EC) No 2232/96
oI the European Parliament and oI the Council oI 28 October 1996.
Flavor and Extract ManuIacturers' Association (FEMA) (1995) The FEMA GRAS Assessment
Program Ior Flavor Ingredients. The GRAS Status oI Alicyclic Substances Used as Flavor
Ingredients 3, p. 1.
Fenaroli (1995) Fenaroli`s Handbook oI Flavor Ingredients. Volume I, 3rd edition, G.A.
Burdock (Ed.), CRC Press, Boca Raton.
Geller, R.J., Spyker D.A., Garrettson L.K. and Rogol A.D. (1984) Camphor toxicity:
development oI a triage strategy. Vet. Hum. Toxicol., 26 (suppl. 2), 8-10.
Kppel, C., Tenezer J., Schirop Th. and Ibe K. (1982) Camphor poisoning. Arch. Toxicol., 51,
101-106.
Leibmann, K.C. and Ortiz E. (1973) Mammalian metabolism oI terpenoids. I. Reduction and
hydroxylation oI camphor and related compounds. Drug Metab. Dispos., 1 (2), 543-551.
Leuschner, J. (1997) Reproductive toxicity studies oI d-camphor in rats and rabbits. Arzneim.
Forsch., 47, 124-128.
National Toxicology Program (NTP) (1992) NTP Report PB92-170034.
Opdyke, D.L.J. (1978) Camphor USP. Food Cosmet. Toxicol., 16 (S1), 665-671.
Phelan, W.J. (1976) Camphor poisoning: over-the-counter dangers. Pediatrics, 57 (3), 428-431.
Ravid, U., Putievsky E. and Katzir I. (1993) Determination oI the enantiomeric composition oI
(1R) ()- and (1S) (-)-camphor in essential oils oI some lamiaceae and compositae herbs.
Flav. Frag. J., 8, 225-228.
Riggs, J., Hamilton R., Homel S. and McCabe J. (1965) Camphorated oil intoxication in
pregnancy. Report oI a case. Obstet. Gynecol. 25, 225-258.
Robertson, J.S. and Hussain M. (1969) Metabolism oI camphors and related compounds.
Biochem. J., 113 (1), 57-65.
Scheline, R.R. (1991) CRC Handbook oI mammalian metabolism oI plant compounds, CRC
Press Inc.
54
Smith, A. G. and Margolis G. (1954) Camphor poisoning; anatomical and pharmacologic study;
report oI a Iatal case; experimental investigation oI protective action oI barbiturate. Am. J.
Pathol., 30 (5), 857-869.
Stoner, G.D., Shimkin M.B., KniazeII A.J., Weisburger J.H., Weisburger E.K. and Gori G.B.
(1973) Test Ior carcinogenicity oI Iood additives and chemotherapeutic agents by the
pulmonary tumor response in strain A mice. Cancer Res., 33 (12), 3069-3085.
Von Skramlik, E. (1959) Aetherische le. Die Pharmazie, 14 (8), 435-445.
Wagreich, H., Bernstein A., Pader M. and Harrow B. (1941) Detoxication oI borneol by
glucuronic acid in humans. Proc. Soc. Exp. Biol. Med., 46, 582-586.
Williams, R.T. (1959) Detoxication mechanisms. The metabolism and detoxication oI drugs,
toxic substances and other organic compounds, Chapman & Hall Ltd, New York
Zolotovich, G., Nachev K., Silyanovska K. and Stoichev S. (1967) Cytotoxic eIIect oI carbonyl
compounds isolated Irom essential oils. C.R. Acad. Bulg. Sci., 20 (11), 1213-1216.

DATA BASES USED: Chemical Abstracts (1967-1998), Toxline (1969-1998). Keywords:
camphor, 2-bornanone

55
APPENDIX:

Table I. Main occurrence oI camphor in natural sources oI Ilavourings

Botanical name Common name Camphor content in essential oil ()
Cinnamomum camphora Nees.
et Eber. var. camphora
(Irom China, Japan and the East
Indies)
Camphor tree up to 60 in bark essential oil
b

up to 84 in leaves essential oil
b

Lavandula latifolia Medik.
(syn. Lavandula spica auct. non
L.)
Spike lavender 12.4-25.5 in herb essential oil
Salvia officinalis L. Sage 20.3-31.3 in leaI essential oil
(d-Iorm with high enantiomeric purity detected in 2
types, ratio d/l 89/11 and 75/25)
a

Salvia lavandulifolia Vahl Spanish sage 36 in leaI essential oil
c

Rosmarinus officinalis L. Rosemary 13-31 in herb essential oil
(ratio d/l-camphor 66/34)
a,e

Chrvsanthemum vulgare (L.)
Bernh.
(syn. Tanacetum vulgare L.)
Tansy 21.9 in herb essential oil
(ratio d/l-camphor 75/25)
a

Sassafras officinale Nees. et
Ebern.
(syn. Sassafras albidum (Nutt.)
Nees var. molle (RaI.) Fern.
SassaIras up to 5 in bark essential oil
d

Coriandrum sativum L. Coriander 6 in Iruit essential oil
3-9 in herb essential oil
c

Origanum maforana L. Spanish
marjoram
2 in herb essential oil
c

Ocimum basilicum L. Sweet basil 0.8-1.5 in herb essential oil
c

Chrvsanthemum parthenium (L.)
Bernh.
(syn. Tanacetum parthenium
Schultz-Bip)
Common
pellitory,
FeverIew,
Flirwort,
FeatherIew
42-62 in herb essential oil
(100 l-camphor)
a

Artemisia herba-alba Asso White mugwort 2.5-70 in herb essential oil depending on
chemotype
d

According to
a
Ravid et al., 1993;
b
Fenaroli, 1995;
c
Council oI Europe, 2000a;
d
Council oI
Europe, 2000b;

e
Council oI Europe, 2001.
56
Table II. Use levels (mg/kg) oI camphor in various Iood categories based on camphor content in
essential oils as listed in Table I

Foodstuffs Basil

plant
Basil

EO
Coriander

EO
Marjoram

EO
Rosemary

EO
Sage

plant
Sage

EO
Spanis
h sage
EO
d-
camphor
Non-alcoholic
beverages
a

10 13 7.1 8 1 6
Alcoholic
beverages
a

48 8 30 16 3 10
Ice creams
a
20 11 - 6 - 24
Candies
a
37 70 44 6 200 25
Baked Ioods
a
58 56 50 103 1 2 15
Meat products
a

57 70 79 27 11
Prepared
dishes
b

10000 30 25
Condiments
and sauces
a

50000
b
1143 104 50 271 279 53
Cheese
b
40000 200 10 40000
Cheese spread
b

40000 200 40000
Oils
b
500
Fish preserves
b

15000 10
Vegetables
preserves
b

10000

Use levels according to
a
EFFA/IOFI;
b
ANIA/SNIAA

Abbreviations:
EO Essential oil
EFFA European Flavour and Fragrance Association
IOFI International Organization oI the Flavor Industry
ANIA Association nationale de lìndustrie agro-alimentaire (French Iood and Ilavour
industry)
SNIAA Syndicat national des industries aromatiques alimentaires

57
Table III. Consumption Iigures based on a survey carried out by the French Iood saIety agency
a

in the year 2000

Foodstuffs Consumption
(g/day)
Portion of foodstuffs flavoured
()
Non-alcoholic beverages
b
14.19 100
Alcoholic beverages 10.88 100
Ice creams 6.16 100
Candies 0.87 100
Baked Ioods 1.05 5
Meat products 37.37 10 (Rosemary and sage)
Prepared dishes 74.65 80
Condiments and sauces 4.34 5 (Rosemary and sage)
Cheese 16.67 10 (Basil and sage)
Cheese spread 1.24 10 (Basil and sage)
Oils 2.14 0.1
Fish preserves 11.32 30
Vegetables preserves 16.01 20

a
Observatoire des Consommations Alimentaires (OCA), Agence Franaise de Securite
Sanitaire des Aliments (AFFSA)
b
Without Iruit juices and soIt drinks
58
Table IV. Intake oI camphor Irom essential oils, herbs and use oI d-camphor as Ilavouring
(mg/person/day)

Foods /
Beverages
Basil

plant
Basil

EO
Coriander

EO
Marjoram

EO
Rosemary

EO
Sage

plant
Sage

EO
Spanishsa
ge
EO
d-camphor Total
Non-alcoholic
beverages
0 0.002 0.011 0 0.019 0 0.028 0.005 0.085 0.15
Alcoholic
beverages
0 0.008 0.005 0 0.06 0 0.043 0.012 0.109 0.236
Ice creams 0 0.002 0.004 0 0 0 0.009 0 0.146 0.161
Candies 0 5E-04 0.004 0 0.007 0 0.001 0.063 0.022 0.097
Baked Ioods 0 5E-05 2E-04 0.002 0.001 0 1E-05 4E-05 0.016 0.017
Meat products 0 0.032 0.157 0 0.055 0 0.025 0.015 0 0.283
Prepared dishes 0.024 0.027 0 0 0 0 0 0 0 0.051
Condiments and
sauces
0.009 0.074 0.027 0.007 0.011 0 0.015 0.004 0.109 0.249
Cheese 0.003 0.005 0 0.011 0 0.227 0 0 0 0.234
Cheese spread 2E-04 4E-04 0 0 0 0.017 0 0 0 0.017
Oils 0 2E-05 0 0 0 0 0 0 0 2E-05
Fish preserves 0.002 5E-04 0 0 0 0 0 0 0 0.003
Vegetable
preserves
0.001 0 0 0 0 0 0 0 0 0.001
Total 0.039 0.151 0.208 0.02 0.153 0.244 0.12 0.098 0.487 1.5

Abbreviations :
EO essential oil
59
Capsaicin


SYNONYMS: N-|(4-Hydroxy-3-methoxybenzyl|-8-methyl-trans-6-nonenamide; N-|(4-
Hydroxy-3-methoxy-phenyl) methyl|-8-methyl-trans-6-nonenamide; N-(3-Methoxy-4-
hydroxybenzyl)-8-methyl-non-trans-6-enamide; trans-8-methyl-N-vanillyl-6-nonenamide;
Isodecenoic acid vanillylamide; 8-Methylnon-6-enoyl-4-hydroxy-3-methoxybenzylamide.

CAS No: 404-86-4

STRUCTURE:

NH
O
O
OH

REGULATORY / INTERNATIONAL STATUS: SCF considered the available toxicological
data as inadequate to establish a saIe exposure level Ior capsaicinoids in Iood. It stated that high
consumption oI chillies (25-200 mg capsaicinoids/day) in Mexico, Thailand and India was
reported to be associated with cancer oI the upper digestive tract, whereas no increase in the
incidence oI gastric cancer was Iound in association with occasional and lower intakes oI
chillies (1.5 mg capsaicinoids/day) in Europe (SCF, 2002). Capsaicin has been deleted Irom the
register oI chemically-deIined Ilavouring substances used in or on IoodstuIIs in the EC due to
observed genotoxic activity in vitro and in vivo (EC, 2004). In the USA, Capsicum (C. annuum
L. or C. frutescens L.) plant and preparations are generally recognised as saIe (GRAS) Ior their
intended use (CFR 182.10, CFR 182.20).


Metabolism:
studies: In vitro intestinal absorption oI capsaicin in rats and hamsters was shown to be
proportional to its concentration in the mucosal medium (Monsereenusorn, 1980). Capsaicin
impaired the intestinal glucose and Iluid absorption, possibly by a secondary inhibitory eIIect on
the Na
-K
-ATPase dependent sodium pump. Inhibition oI intestinal Na
-K
-ATPase, as
demonstrated in everted sac preparations and crude mucosal homogenate, was much more
pronounced in hamsters than in rats (Monsereenusorn and Glinsukon, 1979).
DiIIerent biotransIormation pathways have been described: (a) oxidation by hepatic mixed-
Iunction oxidase systems Iorming a catechol metabolite via hydroxylation oI the vanillyl ring
moiety as shown with phenobarbital-induced rat liver microsomes (Lee and Kumar, 1980), or
60
Iorming omega-hydroxycapsaicin by oxidation at the terminal carbon oI the side chain (Surh et
al., 1995); (b) one electron oxidation oI the ring hydroxyl Iorming phenoxy radicals and 5,5`-
bis-capsaicin (capsaicinoid dimer) by enzymatic or non-enzymatic reactions (Lawson and
Gannett P., 1989; Boersch et al., 1991); (c) hydrolysis at the acid-amide bond to Iorm
vanillylamine and a Iatty acyl moiety and oxidative deamination oI vanillylamine to vanillin
which, in turn, undergoes oxidation to vanillic acid or reduction to vanillyl alcohol (Kawada and
Iwai, 1985). Similar metabolic pathways have been observed with dihydrocapsaicin, a
commercial mixture oI capsaicin and dihydrocapsaicin (Kawada and Iwai, 1985) and with
olvanil |N-(3-methoxy-4-hydroxybenzyl)oleamide|, which has a longer side chain than
capsaicin (Wehmeyer et al., 1990).
Animal studies: Capsaicin is rapidly and extensively absorbed Irom the gastro-intestinal tract in
rats: when capsaicin, dihydrocapsaicin or a commercial capsaicin mixture containing 85
capsaicin and 15 dihydrocapsaicin was administered in vivo, about 85 oI the dose was
absorbed within 3 hours. AIter in situ administration into stomach, jejunum, and ileum, about
50-80 oI the dose was absorbed within 60 minutes (Kawada and Iwai, 1985). Rapid
accumulation oI capsaicin was Iound in brain, spinal cord and liver 3 min aIter i.v.
administration oI 2 mg/kg bw to rats, with 3- to 5-Iold higher concentrations than in blood.
AIter ten minutes concentrations in blood and liver had greatly decreased, but were still highly
elevated in brain and spinal cord (Saria et al., 1982). Within 48 hrs aIter oral administration oI
dihydrocapsaicin to male rats, 8.7 oI the dose was excreted unchanged in urine and 10 in
Ieces. Metabolites Iound in urine were vanillylamine (4.7); vanillin (4.6), vanillyl alcohol
(37.6) and vanillic acid (19.2) in Iree Iorm or as glucuronic acid conjugate (Kawada and
Iwai, 1985). The activity oI capsaicin hydrolyzing enzyme activity has been Iound to be
inducible by continuous oral administration oI capsaicin (Oi et al., 1992).

Toxicology:
Acute toxicity: In male mice, an oral LD
50
oI 60-75 mg/kg bw (in ethanol) and 190 (122-294)
mg/kg bw (in dimethylsulIoxide) was reported, Iollowing intragastric intubation; the i.v. LD
50
was 0.56 mg/kg

bw (Glinsukon et al., 1980). Intraduodenal and intragastric administration oI
either a preparation oI 10 dried Iruit oI Capsicum minimum L. in saline solution or 0.014
capsaicin in saline solution to male rats produced identical cellular alterations in duodenal
mucosa cells such as swollen mitochondria, shrunken nuclei and an increase oI Iree ribosomes;
other organs were not examined histopathologically (Nopanitaya and Nye, 1974). Capsaicinoids
are powerIul irritants, causing burn and pain on the skin and mucous membranes.
Subacute / subchronic toxicity: Mice. A 4-week oral Ieeding study with groups oI 5 male
B6C3F
1
mice receiving 0, 0.5, 1, 2.5, 5, 7.5 or 10 ground dried Iruit oI Capsicum annuum L.
in the diet (equivalent to 0, 750, 1500, 3800, 11300 and 15000 mg capsaicin/kg bw/day did not
apparently aIIect general health, body weight and Iood intake. The only observed adverse eIIect
was a slight glycogen depletion and anisocytosis oI hepatocytes in the 10 group (Jang et al.,
1992).
Groups oI 10 male and 10 Iemale B6C3F
1
mice were orally administered a mixture oI
capsaicinoids (64.5 capsaicin and 32.6 dihydrocapsaicin) at dose levels oI 0, 0.06, 0.13,
0.25, 0.5 and 1 in the diet (equivalent to 0, 90, 190, 380, 750 or 1500 mg/kg bw/day Ior 13
weeks. Food intake and body weight gain were signiIicantly decreased in all treated groups,
especially in Iemales. Renal toxicity (Iocal tubular dilatation) was observed in males
administered 1 capsaicinoids (Akagi et al., 1998).
Groups oI 4 male and 4 Iemale Swiss albino mice were given a mixture oI capsaicinoids in the
diet (65 capsaicin, 31 dihydrocapsaicin, 0.9 nordihydrocapsaicin, 1 homocapsaicin,
0.6 homodihydrocapsaicin, 0.5 norcapsaicin and 0.3 nornorcapsaicin) Ior 35 days at 10
61
dose levels ranging Irom 0.03 to 16 in the diet (equivalent to 45-24000 mg capsaicinoids/kg
bw/day based on 20 g body weight and 3 g Iood consumption per day). Animals died at an age
oI 62-126 weeks and one adenocarcinomas oI the duodenum had developed at each dose level,
except Ior the highest dose, while no such tumours occurred in a historical control group oI 100
males and 100 Iemales. Since there was no concurrent control group and the observed tumours
were not dose-related, the appearance oI the duodenal tumours could not be attributed to
capsaicin treatment (Toth and Gannett, 1984).
Balb/c mice were given an alcoholic chilli extract in drinking water 5 days a week until 16
month oI age (27 males, 25 micrograms capsaicin/week, equivalent to about 0.125 mg/kg
bw/day) or on the tongue 2 days a week Ior 14 months (22 males without and 19 males with 1
atropine solution prior to application, 50 micrograms capsaicin/week, equivalent to about 0.25
mg/kg bw/day). The treated animals showed an increased mortality and histopathological
changes in liver, kidneys, stomach and tongue compared to 40 untreated mice. The lesions in the
liver observed in all treated mice were in the Iorm oI Iocal necrosis with inIlammatory cells
around, Iatty changes and Iibrosis (Agrawal and Bhide, 1987).
Rats. Groups oI 10-14 rats were Ied by stomach tube with 50 mg capsaicin/kg bw/day or
500 mg capsicum extract/kg bw/day Ior 60 days: There was a signiIicant reduction in body
weight gain, plasma urea nitrogen, glucose, phospholipids, triglycerides, total cholesterol, Iree
Iatty acids, glutamic pyruvic transaminase and alkaline phosphatase aIter more than one month
oI treatment with capsaicin or capsicum extract. No gross pathological changes and no
diIIerences in organ weights Irom control values were observed at autopsy, except a slight
hyperemia in the livers and reddening with increasing mucous material in the gastric mucosa.
However, the organs were not examined histopathologically (Monsereenusorn, 1983).
Hamsters. Thirty-six male Syrian golden hamsters (8-10 weeks old, 95-100 g bw) received
20 micrograms oI capsaicin (equivalent to about 0.2 mg capsaicin/kg bw/day; applied as an
alcoholic chilli extract containing 2.5 mg oI capsaicin/ml) in the right cheek pouch 5 days a
week Ior 14 months. Thirty untreated animals and 17 hamsters treated with 20 microlitres
alcohol were used as controls. Increased mortality and histopathological lesions in liver,
kidneys, stomach and cheek pouch occurred in animals treated with chilli extract. The main
lesions were liver cirrhosis (49 oI examined livers compared to 8 and 17 in the control
groups) and degeneration (50 oI examined kidneys oI exposed animals compared to 8 and 0
in the control groups), respectively (Agrawal and Bhide, 1988).

Chronic toxicity / carcinogenicity: Mice. A mixture oI capsaicinoids (65 capsaicin, 31
dihydrocapsaicin, 0.9 nordihydrocapsaicin, 1 homocapsaicin, 0.6 homodihydrocapsaicin,
0.5 norcapsaicin and 0.3 nornorcapsaicin), was orally administered at 0.03 in a
semisynthetic diet (equivalent to 45 mg capsaicinoids/kg bw/day) to groups oI 50 male and 50
Iemale Swiss albino mice during Iull liIetime. Benign polypoid adenomas oI the caecum
occurred in 22 oI treated Iemales and 14 oI treated males compared to 8 in controls. The
increase in tumour incidence was statistically signiIicant in Iemales (p0.05). The survival rate
oI the treated animals was not substantially altered (Toth and Gannett, 1992).
Groups oI 50 male and 50 Iemale B6C3F
1
mice were given a mixture oI capsaicinoids (64.5
capsaicin and 32.6 dihydrocapsaicin) at 0, 0.025, 0.083 and 0.25 in the diet (equivalent to 0,
38, 125 and 375 mg/kg bw/day) Ior 79 weeks, Iollowed by a basal diet Ior 4 weeks. Food intake
was signiIicantly reduced in all dose groups, especially in Iemales which showed a signiIicantly
reduced body weight gain. There was a high background tumour incidence in control mice
(31/49 males and 16/50 Iemales). The number oI tumour-bearing animals was signiIicantly
reduced in Iemales treated with the highest dose (5/50), possibly due to the reduced Iood intake.
In general, tumor incidences were similar in the treated and control groups. Renal carcinomas
62
developed only in one animal each oI 0.025 and 0.25 capsaicinoid-treated males compared to
none in treated Iemales and in control groups (Akagi et al., 1998).
Rats. Rats (26 animals) were given a semi-synthetic diet containing 10 chilli pepper and
ardein (peanut protein) Ior seven months. FiIteen oI the treated animals developed neoplastic
changes in the liver (hepatomas, multiple cystic cholangiomas, adenomas or adenocarcinomas
oI the bile duct), whereas no tumours were Iound in control animals. The authors stress that the
tumour incidence could be inIluenced by the composition oI the diet (Hoch-Ligeti, 1951). There
is no two-year oral carcinogenicity study in rats available.
Reproductive toxicity / teratogenicity: Mice. Capsaicin was administered intraperitoneally to
adult male Swiss albino mice at dose levels oI 0, 0.4, 0.8 or 1.6 mg/kg bw/day on 5 consecutive
days and animals were killed 35 days aIter the end oI treatment. No clinical signs oI toxicity and
no signiIicant alterations were observed in epididymal weights, caudal sperm counts, testicular
weights or testicular histology. In the sperm morphology assay, at 1, 3, 5, 7 weeks aIter the end
oI treatment there was no treatment-related increase in the incidence oI sperm-head
abnormalities observed. In another experiment, 15 male Swiss albino mice were administered
1.6 mg/kg bw/day i.p. on Iive consecutive days, and were then mated sequentially once a week
Ior 8 weeks (2 Iemales per male). The uterus content oI pregnant Iemales was examined and no
changes in total implantations, early and late resorptions, and live and dead Ietuses were Iound
(Muralidhara and Narasimhamurthy, 1988).
Mutagenicity / genotoxicity: In vitro. Mutagenicity studies with capsaicin and chilli extract in
bacteria and mammalian cell cultures gave conIlicting results (review in Surh and Lee 1995):
capsaicin exhibited a low level oI mutagenicity in the Ames test with Salmonella tvphimurium
TA98 with S9, while negative results were observed with TA97, TA100, TA102 in the same
study (Toth et al., 1984). The positive result with TA98 could not be repeated subsequently
using identical exxperimental conditions (Toth and Gannet, 1992). Capsaicin containing 20
dihydrocapsaicin was Iound to be mutagenic in several strains oI Salmonella tvphimurium but
did not induce gene mutations in 8-azaguanine resistant mutants oI Chinese hamster V79 cells.
These Iindings, however, could not be reproduced when synthetic capsaicin oI higher purity was
tested in both assay systems. In another study, no mutagenic activity was Iound Ior capsaicin
and dihydrocapsaicin in Salmonella tvphimurium TA98 and TA1535, whereas in Chinese
hamster V79 cells both compounds as well as red pepper extract were active. In a preliminary
study capsaicin caused chromosomal aberrations in cultured human lymphocytes (Surh and Lee,
1995).
In vivo: Capsaicin Iailed to induce dominant-lethal mutations in mice (15 males administered
1.6 mg/kg bw/day i.p. on Iive consecutive days were mated with untreated Iemales)
(Muralidhara and Narasimhamurthy, 1988). When injected i.p. into mice, capsaicin produced
micronuclei in polychromatic erythrocytes in the mouse bone marrow assay at 7.5 mg/kg bw,
but not at 1.8 mg/kg bw. Inhibition oI DNA synthesis in the testes was observed at these dose
levels (Nagabhushan and Bhide, 1986).
Intraperitoneal administration oI 1.46 or 1.94 mg/kg bw/day oI capsaicin Ior 32 days to male
mice induced sister chromatid exchanges and micronucleated normochromatic erythrocytes in
mouse bone marrow at the higher dose only (Diaz Barriga Arceo et al., 1995). A Iraction oI an
alcoholic extract oI Iruits oI Capsicum frutescens was clastogenic as determined by induction oI
micronuclei in mouse bone marrow cells (Villasenor and De Ocampo, 1994).
A number oI studies have shown that capsaicin and chilli extract can act as tumour promoters
(Surh and Lee, 1995).
On the other side, capsaicin has been suggested to exert chemoprotective eIIects through
modulation oI metabolism oI carcinogens and their interaction with DNA (Surh and Lee, 1995).

63
Human data: In a case-control study carried out in India, red chilli powder was Iound to be a
risk Iactor Ior cancer oI the oral cavity, pharynx, esophagus and larynx (2- to 3-Iold higher risk
with a dose-response relationship) compared to population controls, but not compared to
hospital controls (Notani and Jayant, 1987). In a population-based case-control study in Mexico
City which included 220 cases oI gastric cancer and 752 controls, chilli pepper consumers were
at a 5.5-Iold greater risk Ior gastric cancer compared with non-consumers, selI-rated heavy
consumers at a 17-Iold risk. However, there was no signiIicant dose response relationship when
daily chilli pepper consumption was measured (Lopez-Carillo et al., 1994). There is no
indication oI an increase in the incidence oI gastric cancer with occasional and lower intakes oI
chillies in Europe (Buiatti et al., 1989; SCF, 2002).

Other studies: No relevant data Iound.

TOXICOLOGICAL EVALUATION: Capsaicin, capsaicinoid mixtures and chilli extracts
have been reported to cause histopathological and biochemical changes, including erosion oI the
gastric mucosa and lesions oI liver and kidneys, aIter oral ingestion. Experimental data on
subchronic and chronic toxicity oI orally applied capsaicin and Capsicum sp. preparations are
mainly reIlecting the locally irritative activity oI capsaicin and other capsaicinoids. However,
the studies are considered to be oI limited quality and no conclusions on eIIective doses could
be drawn. Data on the genotoxicity and carcinogenicity oI capsaicin are contradictory. Some oI
the available studies, however regarded as limited, indicated a carcinogenic potential oI
capsaicin. Yet, the most recent carcinogenicity study did not show carcinogenic eIIects in mice
at doses up to 375 mg capsaicinoids/kg bw/day. In studies in humans high consumption oI
chillies in India and Mexico was associated with cancer oI the upper digestive tract, whereas
there is no indication oI an increase in the incidence oI gastric cancer with occasional and lower
intakes oI chillies in Europe. The increased risk Ior gastric cancer observed in chilli pepper
consumers in Mexico, as compared to non-consumers, is considered to be associated with a
daily intake oI 200 mg capsaicinoids/person (equivalent to 3.3 mg capsaicinoids/kg bw Ior an
adult oI 60 kg bw) as estimated Ior average Mexican consumers (see intake estimation below).
Based on the rough assumption oI low toxicity Ior oral doses oI 4 mg capsaicinoids/kg bw in
humans and using a saIety Iactor oI 20, a TMDI expressed as total capsaicinoids was set at 0.2
mg/kg bw.

TMDI: 0.2 mg/kg bw, expressed as total capsaicinoids.

MAIN OCCURENCE: Capsaicin is the pungent principle in the Iruits oI the natural sources
Capsicum annuum L. (red pepper, Spanish pepper, paprika; CE No. 107) and Capsicum
frutescens L. (cayenne pepper, chilli pepper; CE No. 108) and other Capsicum sp. which are
widely used as spices. Only Capsicum sp. preparations and no pure capsaicin are used as
Ilavouring agents (Fenaroli, 1995). In the capsicum Iruits and capsicum preparations, capsaicin
is always accompanied by other capsaicinoids, mainly dihydrocapsaicin and small amounts oI
nordihydro-, homo-, homodihydro-, nor- and nornorcapsaicin; dihydro- and capsaicin making
about 80-90 oI the capsaicinoids. The ratio oI dihydrocapsaicin to capsaicin is generally
around 1:1 to 1:2 (Govindarajan and Sathyanarayana, 1991). The capsaicinoid content in the
whole dried powdered Iruit can vary Irom 0.11-1.5 (Evans, 1989). The capsaicin content
typically ranges Irom 0.1 mg/g dry wt in chilli pepper to 2 mg/g dry wt in red pepper and
60 mg/g in oleoresin red pepper (Parrish, 1996).

INTAKE ESTIMATION: The maximum daily intake oI capsaicin in the USA and in Europe
Irom mild chillies and paprika was roughly estimated to be 0.025 mg/kg bw, equivalent to
64
1.5 mg/person (Govindarajan and Sathyanarayana, 1991). According to consumption data Irom
CREDOC (Observatoire des Consommations Alimentaires), France, the estimated mean, 95th
percentile and maximum intake oI capsaicin Irom industrially prepared Ioods and beverages are
0.77, 1.39 and 2.64 mg/person/day (equivalent to 0.01, 0.02 and 0.04 mg/kg bw/day Ior an adult
oI 60 kg bw) (CREDOC, 1998; Council oI Europe, 1998). The estimation is based on a daily
consumption oI 150 g oI Ioods and beverages containing capsaicinoids at the recommended
general limit oI 5 mg/kg and 99.9 oI the population being consumers. In some countries,
capsicum spices are consumed in large quantities: the likely consumption is about 2.5
g/person/day in India, 5 g/person/day in Thailand (Monsereenusorn, 1983) and 20 g/person/day
in Mexico (Lopez-Carillo, 1994). Assuming that the average content oI capsaicinoids in
capsicum Iruits is about 1, the daily intake oI capsaicinoids Irom spices would be 25-200
mg/person/day, equivalent to 0.4-3.3 mg/kg bw Ior an adult oI 60 kg bw.

CONCLUSIONS: The estimated mean, 95th percentile and maximum capsaicin intakes oI
consumers oI a rather mild average European diet (0.01 to 0.04 mg capsaicinoids/kg bw/day)
are well within the TMDI oI 0.2 mg/kg bw. Nevertheless, the daily intake oI heavy consumers
oI spicy dishes (0.4-3.3 mg/kg bw/day) can exceed the TMDI by a Iactor oI up to 15. Data about
carcinogenicity in animals and humans are limited and contradictory.

DATA NEEDED: Further data are needed to clariIy the genotoxic potential oI capsaicin.
An epidemiological study designed in a way that a saIe exposure level oI capsaicin in Iood can
be established is needed.

LIMITS: (expressed as total capsaicinoids in mg/kg)

Foods and beverages 5
Hot Ioods and beverages 10

Exceptions (condiments and sauces):
Hot ketchup and similar products 20
Tabasco, harissa, hot pimento oils and similar preparations 50

REFERENCES:
Agrawal, R.C. and Bhide S.V. (1987) Biological studies on carcinogenicity oI chillies in balb/c
mice. Indian J. Med. Res., 86, 391-396.
Agrawal, R.C. and Bhide S.V. (1988) Histopathological studies on toxicity oI chilli (capsaicin)
in Syrian golden hamsters. Indian J. Experim. Biol., 26, 377-382.
Akagi, A., Sano N., Uehara H., Minami T., Otsuka H. and Izumi K. (1998) Non-carcinogenicity
oI capsaicinoids in B6C3F1 Mice. Food Chem. Toxicol., 36 (12), 1065-1071.
Boersch, A., Callingham B.A., Lembeck F. and Sharman D.F. (1991) Enzymic oxidation oI
capsaicin. Biochem. Pharmacol., 41 (12), 1863-1869.
Buiatti, E., Palli D., Decarli A., Amadori D., Avelini C., Bianchi S., Biserni R., Cipriani F.,
Cocco P., Giacosa A., Marubini E., Puntoni R., Vindigni C., Fraumeni J., Jr. and Blot W.,
(1989) A case control study oI gastric cancer and diet in Italy. Int. J. Cancer, 44, 611-616.
Council oI Europe (1998) Committee oI Experts on Flavouring Substances. Exposure
calculations Ior saIrole, estragole, coumarin and capsaicin RD 6/3-42.
65
CREDOC/OCA (Observatoire des Consommations Alimentaires) (1998). Estimation des
niveaux dìngestion de substances aromatisantes SaIrole, Estragole, Coumarine et
Capsaicine. Note technique No. 98.25.
Diaz Barriga Arceo, S., Madrigal-Bujaidar E., Calderon Montellano E., Ramirez Herrera L. and
Diaz Garcia B.D. (1995) Genotoxic eIIects produced by capsaicin in mouse during
subchronic treatment. Mutat. Res., 345 (3-4):105-109.
EC (2004) Commission decision 2004/357/EC amending decision 1999/217/EC as regards the
register oI Ilavouring substances. OIIicial Journal oI the European Union, 20.4.2004, L
113/28.
Evans, W.C. (1989) Trease and Evans Pharmacognosy London: Bailliere Tindall.
Fenaroli (1995) Fenaroli`s Handbook oI Flavor Ingredients. Volume I, 3rd ed. Editor G.A.
Burdock, CRC Press, Boca Raton.
Glinsukon, T., Stitmunnaithum V., Toskulkao C., Buranawuti T. and Tangkrisanavi-nont V.
(1980) Acute toxicity oI capsaicin in several animal species. Toxicon, 18, 215-220.
Govindarajan, V.S. and Sathyanarayana M.N. (1991) Capsicum-production, technology,
chemistry, and quality. Part V. Impact on physiology, pharmacology, nutrition, and
metabolism; structure, pungency, pain, and desensitization sequences. Crit. Rev. Food.
Sci. Nutr. 29 (6), 435-473.
Hoch-Ligeti, H. (1951) Production oI liver tumours by dietary means: EIIect oI Ieeding chillies
to rats. Acta Unio Int. contra cancrum, 7, 606-611.
Kawada, T. and Iwai K. (1985) In vivo and in vitro metabolism oI dihydrocapsaicin, a pungent
principle oI hot pepper, in rats. Agric. Biol. Chem., 49 (2), 441-448.
Jang, J.J., Devor D.E., Logsdon D.L. and Ward J.M. (1992) A 4-week Ieeding study oI ground
red chilli (Capsicum annuum in male B6C3F1 mice. Food Chem. Toxicol., 30 (9), 783-
787.
Lawson, T. and Gannett P. (1989) The mutagenicity oI capsaicin and dihydro-capsaicin in V79
cells. Cancer Lett., 48, 109-113.
Lee, S.S. and Kumar S. (1980) Metabolism in vivo oI capsaicin, a pungent principle oI red
pepper with rat liver homogenates. In: Microsomes, Drug Oxidations and Chemical
Carcinogenesis (Coon M.J., Conney A.H., Estabrook R.W., Gelboin H.V., Gillette J.R.
and O'Brien P.J., eds.) Vol. II, 1009-1012, Academic Press, New York, NY.
Lopez-Carillo, L., Avila M.H. and Dubrow R. (1994) Chili pepper consumption and gastric
cancer in Mexico: A case-control study. Am. J. Epidem., 139 (3), 263-271.
Monsereenusorn, Y. and Glinsukon T. (1979) The inhibitory eIIect oI capsaicin on intestinal
glucose absorption in vitro: II. EIIect oI capsaicin upon intestinal Na
-K
-ATPase
activities. Toxicol. Lett., 4 (5), 399-406.
Monsereenusorn, Y. (1980) In vitro intestinal absorption oI capsaicin. Toxicol. Appl.
Pharmacol., 53, 134-139.
Monsereenusorn, Y. (1983) Subchronic toxicity studies oI capsaicin and capsicum in rats. Res.
Commun. Chem. Pathol. Pharmacol., 41 (1), 95-110.
Muralidhara and Narasimhamurthy K. (1988) Non-mutagenicity oI capsaicin in albino mice.
Food Chem. Toxicol., 26 (11-12), 955-958.
Nagabhushan, M. and Bhide S.V. (1986) Mutagenicity oI chilli extract and capsaicin in short-
term tests. Environ. Mutagen., 7, 881-888.
Nopanitaya, W. and Nye S. W. (1974) Duodenal mucosal response to the pungent principle oI
hot pepper (capsaicin) in the rat: Light and electron microscopic study. Toxicol. Appl.
Pharmacol., 30, 149-161.
Notani, P.N. and Jayant K. (1987) Role oI diet in upper aerodigestive tract cancers. Nutr.
Cancer, 10 (1/2), 103-113.
66
Oi, Y., Kawada T., Watanabe T. and Iwai K. (1992) Induction oI capsaicin-hydrolyzing enzyme
activity in rat liver by continuous oral administration oI capsaicin. J. Agric. Food Chem.,
40 (3), 467-470.
Parrish, M. (1996) Liquid chromatographic method oI determining capsaicinoids in capsicums
and their extractives: collaborative study. J. AOAC Intern., 79 (3), 738-745.
Saria, A., SkoIitsch G. and Lembeck F. (1982) Distribution oI capsaicin in rat tissues aIter
systemic administration. J. Pharm. Pharmacol., 34, 273-275.
SCF (2002) Opinion oI the ScientiIic Committee on Food on Capsaicin adopted on 26 February
2002.
Surh, Y.-J., Ahn S.H., Kim C.-K., Park J.-B., Sohn Y.W. and Lee S.S. (1995) Metabolism oI
capsaicinoids: Evidence Ior aliphatic hydroxylation and its pharmacological implications.
LiIe Sci., 56 (16), PL305-PL311.
Surh, Y.-J. and Lee S.S. (1995) Capsaicin, a double-edged sword: Toxicity, metabolism, and
chemopreventive potential. LiIe Sci., 56 (22), 1845-1855.
Thomas, B.V., Schreiber A.A. and WeisskopI C.P. (1998) Simple method Ior quantitation oI
capsaicinoids in peppers using Capillary gas chromatography. J. Agric. Food Chem., 46
(7), 2655-2663.
Todd, P.H., Jr., Bensinger M.G. and BiItu T. (1977) Determination oI pungency due to
capsicum by gas-liquid chromatography. J. Food Sci., 42 (3), 660-665.
Toth, B., Rogan E. and Walker B. (1984) Tumorigenicity and mutagenicity studies with
capsaicin oI hot peppers. Anticancer Res. 4(3):117-119.
Toth, B. and Gannett P. (1992) Carcinogenicity oI liIelong administration oI capsaicin oI hot
pepper in mice. In vivo, 6, 59-64.
Villasenor, I.M. and De Ocampo E.J. (1994) Clastogenicity oI red pepper (Capsicum frutescens
L.) extracts. Mutat. Res., 312, 151-155.
Wehmeyer, K.R., Kasting G.B., Powell J.H,. Kuhlenbeck D.L., Underwood R.A. and Bowman
L.A. (1990) Application oI liquid chromatography with on-line radiochemical detection
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183.

DATA BASES USED: Chemical Abstracts (1967-2002), Pascal (1973-2002). Keywords:
capsaicin, capsicum, cayenne pepper

67
Elemicin

ACTIVE PRINCIPLE: I

SYNONYMS: 5-Allyl-1,2,3-trimethoxybenzene

CAS No: 487-11-6

STRUCTURE:

O
O
O

REGULATORY / INTERNATIONAL STATUS: In the USA, nutmeg (the seed oI Mvristica
fragrans Houtt.), which is the main source oI elemicin intake, is generally recognized as saIe
(GRAS) Ior its intended use (CFR 182.10, CFR 182.20, CFR 582.20).


Metabolism:
Animal studies: Urinary and biliary metabolites were identiIied in rats Iollowing oral doses oI
400 mg/kg bw. The major metabolic reactions Iollow the cinnamoyl pathway or the epoxide-diol
pathway. The Iormer routes give 3-(3,4,5-trimethoxyphenyl)propionic acid and its glycine
conjugate as major urinary metabolites, whereas 3-(3,4,5-trimethoxyphenyl)propane-1,2-diol is
the most prominent metabolite oI the latter route. Small amounts oI the epoxide oI the 3-O-
demethylated derivative oI elemicin were identiIied in the urine (Solheim et al., 1980).

Toxicology:
Acute toxicity: No data Iound.
Subacute / subchronic toxicity: No data Iound.
Chronic toxicity / carcinogenicity: Elemicin and 1`-hydroxyelemicin had no detectable
hepatocarcinogenic activity when administered i.p. to preweanling male B6C3F
1
mice at 4 doses
totaling 4.75 micromoles/mouse (equivalent to 49.5 mg elemicin/kg bw assuming a bw oI 20 g).
Doses were in the ratio 1:2:4:12 on days 1 ( 24 hrs aIter birth), 8, 15 and 22 aIter birth. Livers
were examined by laparatomy at 13 months and when animals were killed at 18 months (Miller et
al., 1983). In another study, the administration oI a single dose oI 0.1 or 0.25 mmoles 1`-
hydroxyelemicin/kg bw (equivalent to 20.8 and 52.1 mg/kg bw, respectively) to B6C3F
1
mice at
the age oI 12 days, similarly did not result in a signiIicant hepatocarcinogenic response (incidence
oI 0.30.6 and 0.40.6 hepatomas/mouse, respectively, by 12 months), while 0.1 mmoles oI 1`-
hydroxysaIrole/kg bw or oI 1`-hydroxy-2`,3`-dehydrosaIrole induced 1.41.2 hepatomas/mouse
68
and 10.72.8 hepatomas/mouse (p0.001), respectively. In solvent-treated controls
0.20.5 hepatomas/mouse were Iound (Wiseman et al., 1987). However, preweanling male
B6C3F
1
mice given a total i.p. dose oI 9.5 micromoles/mouse oI 1`-hydroxyelemicin or 1`-
acetoxyelemicin (equivalent to 107 and 127 mg/kg bw, respectively) in 4 doses on days 1, 8, 15
and 22 aIter birth, had an average oI 0.81.0 hepatomas/mouse by 13 months, which was
signiIicantly diIIerent (p0.001) Irom the observed 0.10.3 hepatomas/mouse in the solvent-
treated controls. Based on Iurther results oI this study, these two elemicin derivatives are
considered to be approximately 30-Iold less active, on a molar basis, than 1`-hydroxyestragole and
200-Iold less active than 1`-hydroxy-2`,3`-dehydroestragole, Ior the induction oI hepatomas in
preweanling male B6C3F
1
mice. For comparison, 1`-hydroxyestragole induced twice as much
hepatomas in C3H/HeJ F
1
mice (3.02.3 hepatomas/mouse) by 14 months than did 1`-
hydroxysaIrole (1.61.6 hepatomas/mouse) at the same total molar dose oI approx. 1.5 mmoles/kg
bw given in 3 doses on days 8, 15 and 22 aIter birth (Wiseman et al., 1987).
Mutagenicity / genotoxicity: Elemicin induced unscheduled DNS synthesis (UDS) in a dose-
dependent manner in hepatocytes derived Irom male Fischer 344 rats (Hasheminejad et al.,
1994).


Other studies: DNA-binding capacitv. The binding oI a series oI alkenylbenzenes to liver DNA
Irom adult Iemale CD-1 mice was investigated. The test compounds saIrole, estragole,
methyleugenol, allylbenzene, anethole, myristicin, parsley apiol, dill apiol, eugenol and
elemicin were administered i.p. at 2 or 10 mg/mouse. The known hepatocarcinogens saIrole,
estragole and methyleugenol exhibited the strongest binding, with 200-300 pmol adduct/mg
DNA aIter a 10 mg dose, while all the other investigated compounds, except Ior Eugenol,
showed intermediate to low binding levels (approx. level in pmol adduct/mg DNA aIter 10 mg:
myristicin 50, elemicin 17, dill apiol 14, isosaIrole 5, others less) in this
32
P-postlabelling assay.
The DNA-binding could also be expressed by the covalent binding index (CBI). The calculated
CBI value (mean oI CBI values measured aIter 2 and 10 mg/mouse) was 2.2 Ior elemicin, 10.7
Ior myristicin, 7.5 Ior dill apiol and 1.3 Ior isosaIrole, whereas the CBI values Ior saIrole,
estragole and methyleugenol were 29.2, 30.0 and 31.1, respectively (Randerath et al., 1984).
In another study, the binding oI various alkenylbenzenes to mouse liver DNA Irom male C57Bl
x C3H/He F
1
-mice was investigated when the mice were administered the compounds i.p. on
days 1, 8, 15 and 22 aIter birth at doses oI 0.25, 0.5, 1.0 and 3.0 micromoles (equivalent to
approx. 2.6-31.2 mg/kg bw). A total dose oI 4.75 micromoles (equivalent to 49.4 mg/kg bw) was
given to each animal. The amounts oI DNA adducts present in the livers were quantiIied in mice
killed 23, 29 and 43 days aIter birth. The highest levels oI adducts were detected with
methyleugenol, estragole and saIrole (72.7, 30 and 17.5 pmoles/mg DNA, respectively) at 23 or
29 days. The highest level oI DNA-binding oI elemicin was Iound to be 3.7 pmol/mg DNA at
29 days. As with the three Iirst-mentioned (carcinogenic) compounds, it was apparent that the
majority oI the DNA adducts oI elemicin seen at 23 and 29 days persisted up to 43 days
(Phillips et al., 1984).

TOXICOLOGICAL EVALUATION: Toxicological inIormation on elemicin is very limited.
There are no data available on short-term or chronic toxicity. Elemicin was Iound to be genotoxic
in a UDS assay in rat hepatocytes. However, no mutagenicity studies and no Iurther studies on
genotoxicity are available. From the short-term carcinogenicity studies in mice there is no
indication that elemicin may exert carcinogenic activity. A weak hepatocarcinogenic response
was noted Ior the possibly Iormed metabolites 1`-hydroxyelemicin and 1`-acetoxyelemicin in one
69
oI two studies in preweanling mice. The activity oI 1`-hydroxyelemicin to induce hepatomas in
mice was 5 to 15 Iold lower than that oI 1`-hydroxysaIrole. Low but signiIicant DNA-binding oI
elemicin was Iound in mouse liver in
32
P-postlabelling assays oI some alkylbenzenes. However,
the DNA-binding oI elemicin was considerably weaker (CBI 2.2) than that oI known
hepatocarcinogens such as saIrole, estragole and methyleugenol (CBI 30). The DNA adducts oI
elemicin were as persistent as the ones oI saIrole, estragole and methyleugenol. Overall, Irom
the carcinogenicity studies in mice as well as the DNA-binding studies, it can be concluded that
elemicin has an approx. 10-Iold lower hepatocarcinogenic activity than saIrole. Due to the lack oI
essential toxicological data and the uncertain genotoxic potential, no (T)MDI was set.


MAIN OCCURENCE: The highest concentrations oI elemicin are Iound in essential oils oI the
Elemi-tree (Canarium commune L., CE No. 104; up to 10.6), oI parsley (Petroselinum
crispum (Mill.) Nym. ex A.W. Hill, syn. Petroselinum sativum HoIIm., CE No. 326; 4.6) and
nutmeg (Mvristica fragrans Houtt., CE No. 296; up to 7.5). In bitterwort (Gentiana lutea L.,
CE No. 214), sassaIras (Sassafras albidum (Nutt.) Nees var. molle (RaI.) Fern., CE No. 424) and
sweetIlag (Acorus calamus Pav., CE No. 13) levels oI 0.6-1.5 elemicin in essential oils have
been reported (Table I).
The essential oil oI nutmeg (seed oI Mvristica fragrans Houtt.) also contains considerable
amounts oI saIrole (2.0 in the East Indian variety and 0.3 in the West Indian variety)
(Council oI Europe, 1997a). Mace and mace essential oil contain similar constituents as nutmeg
and nutmeg oil, except Ior a higher myristicin content (Leung, 1996).

INTAKE ESTIMATION: The main sources oI intake oI elemicin Irom Ilavoured Ioods and
beverages appear to be nutmeg and mace, with small contribution Irom parsley and essential
oils oI the Elemi-tree. The highest levels oI elemicin in Ioods and beverages Irom essential oils
have been reported to occur in meats and baked goods Ilavoured with nutmeg essential oil
(Table II). Elemicin intake Irom selected IoodstuIIs has been estimated based on reported use
levels oI essential oils used Ior Ilavouring purposes and Iood intake data Irom the dietary and
nutritional survey oI British adults (British adult study) (Gregory et al., 1990) (Table II). The
Iood groups listed in Table II are considered to contribute most to the intake oI elemicin, with
meats and baked goods Ilavoured with nutmeg being the most important single sources oI
intake.
The daily intake oI elemicin Irom essential oils seems to be limited. An approximate estimate oI
the elemicin intake Irom all sources, based on the largely overestimating assumption that all
Ioods consumed in the given amounts are Ilavoured with the three main elemicin containing
essential oils, results in less than 1 mg per person/day (i.e. less than 0.017 mg/kg bw/day) Ior
mean consumers and in the order oI a Iew milligrams per person/day Ior extreme consumers.
The intake oI elemicin Irom the use oI nutmeg as a spice has been estimated by industry to be
0.51 mg per person/day (0.0084 mg/kg bw/day) Ior average consumers. Contributions Irom
other sources were considered negligibly low (Council oI Europe, 1997a). Thus, a total daily
elemicin intake oI about 1.5 mg/person/day can be assumed.
As Ior elemicin, the greatest proportion oI saIrole intake is provided by nutmeg, mace and their
essential oils. The intake oI saIrole Irom the use oI nutmeg as a spicy component in Ioods has
been calculated by industry to be 0.82 mg per person/day (0.0137 mg/kg bw/day) Ior average
consumers (Council oI Europe, 1997a). The Committee oI Experts roughly estimated the intake
oI saIrole Irom Ioods and spices to be about 1 mg/person/day (equivalent to about 0.017 mg/kg
bw/day). The intake Irom the use oI essential oils as Iood Ilavourings has not been calculated
70
but a similar intake to that Irom Iood has been assumed, thus amounting the daily saIrole intake
to about 2 mg per person (equivalent to about 0.033 mg/kg bw) (Council oI Europe, 1997b).
In conclusion, the daily intake oI elemicin Irom Ioods by the use oI nutmeg, mace and their
essential oils is in the same order oI magnitude as the concurrent intake oI saIrole.

CONCLUSIONS: As essential toxicological data are lacking, it is not possible to assess
whether the present intake oI elemicin may represent a health risk. Further studies are needed to
clariIy the uncertainty on the genotoxic and carcinogenic potential oI elemicin. Nevertheless, by
the consumption oI nutmeg and mace, which are the major Iood sources oI elemicin, saIrole is
ingested at similar levels. Due to the about 10-Iold lower carcinogenic activity oI elemicin
compared to saIrole, the Committee oI Experts considers that limits Ior elimicin are not required
as its intake will be restricted via limits Ior saIrol. ThereIore the Committee does not ask Ior
Iurther data.

DATA NEEDED: -

LIMITS: It is not considered necessary to set limits Ior elemicin as its main intake Irom
nutmeg would be restricted via the limits set Ior saIrole.

REFERENCES:
Council oI Europe (1997a) Committee oI Experts on Flavouring Substances. Overview on toxicity
and occurrence oI alkenylbenzenes: allylbenzenes and propenylbenzenes - RD 4.1/1-40.
Council oI Europe (1997b) Committee oI Experts on Flavouring Substances. Revised datasheet on
saIrole RD 4.2/10-41.
Council oI Europe (2000) Natural sources oI Ilavourings, Report No. 1, Strasbourg.
Duke, J.A. (1985) Handbook oI Medicinal Herbs, CRC Press, Boca Raton.
Fenaroli (1995) Fenaroli`s Handbook oI Flavor Ingredients, Volume I, Ed. G.A. Burdock, Third
edition, CRC Press, Boca Raton.
British Adults, HMSO, London.
Hasheminejad, G. and Caldwell J. (1994) Genotoxicity oI the alkenylbenzenes alpha- and beta-
asarone, myristicin and elemicin as determined by the UDS assay in cultured rat
hepatocytes. Fd. Chem. Toxic. 32 (3), 223-231.
Leung A.Y. (1996) Encyclopedia oI Common Natural Ingredients Used in Food, Drugs, and
Cosmetics. John Wiley & Sons, New York.
Miller, E.C., Swanson A.B., Phillips D.H., Fletcher T.L., Liem A. and Miller J.A. (1983)
Structure-activity studies oI the carcinogenicities in the mouse and rat oI some naturally
occurring and synthetic alkenylbenzene derivatives related to saIrole and estragole. Cancer
Res., 43 (3), 1124-1134.
Phillips, D.H., Reddy M.V., and Randerath K. (1984)
32
P-post-labelling analysis oI DNA
adducts Iormed in the livers oI animals treated with saIrole, estragole and other naturally-
occurring alkenylbenzenes. II. Newborn male B6C3F
1
mice. Carcinogenesis, 5 (12), 1623-
1628.
Randerath, K., Haglund R.E., Phillips D.H. and Reddy M.V. (1984)
32
P-post-labelling analysis
oI DNA adducts Iormed in the livers oI animals treated with saIrole, estragole and other
naturally-occurring alkenylbenzenes. I. Adult Iemale CD-1 mice. Carcinogenesis, 5 (12),
1613-1622.
Reineccius G. (ed.) (1994) Source book oI Ilavors, second edition, Chapman and Hall, New
York.
71
Solheim, E. and Scheline R.R. (1980) Metabolism oI alkenebenzene derivatives in the rat. III.
Elemicin and isoelemicin. Xenobiotica, 10 (5), 371-380.
Wichtl M. (ed.) (1989) Teedrogen. Ein Handbuch Ir die Praxis auI wissenschaItlicher
Grundlage, 2. AuIlage, Wiss. Verl.-Ges., Stuttgart.
administration to preweanling male C57BL/6J x C3H/HeJ F1 mice. Cancer Res., 47 (9),
2275-2283.

DATA BASES USED: FSTA (1969-2002), Chemical Abstracts (1967-1990), Biosis (1973-
1990), Pascal (1973-1990), Toxline (1969-2002), Medline (1966-2002). Keywords: elemicin.
72
APPENDIX:

Table I. Main occurrence oI elemicin in natural sources oI Ilavourings

Botanical name Common name Essential oil in plant
()
a
Elemicin in essential oil
()
a
Canarium commune L. Elemi-tree 20-30 in the crude
resin (Elemi gum)
2.5-10.6
Petroselinum crispum
(Mill.) Nym. ex A.W. Hill
(syn. Petroselinum sativum
HoIIm.)
Parsley 1.5-3.5 in seed,
0.05-0.3 in herb
4.6

Mvristica fragrans Houtt. Nutmeg 2-16 in the seed
depending on the
source
(usually approx. 10)
0.3-4.6
Gentiana lutea L. Bitterwort 0.002-0.007 in dried
roots
1.5
Sassafras albidum (Nutt.)
Nees var. molle (RaI.) Fern
SassaIras 1-3 in dried root bark

1.0
Acorus calamus Pav. SweetIlag 0.5-10 in dried roots
depending on source,
usually 1.5-3.5
0.58

a
According to Leung, 1996; Wichtl, 1989; Reineccius, 1994; Council oI Europe, 2000; and Duke, 1985
73
Table II. Use levels, and elemicin intake Irom Ioods and beverages

Intake of food
(g/day)
b

Elemicin intake
(mg/day)

Foodstuffs Level of use
of ess. oil
(mg/kg)
Elemicin in
food
(mg/kg)
a

Mean Extreme Mean Extreme
Petroselinum crispum (Mill.) Nym. ex A.W. Hill (parsley)
Baked goods 24
c
1.1 51 153 0.06 0.17
Condiments 64
c
2.9
Meats 62
c
2.8 25 86 0.07 0.24
Soups 66
c
3.0
Mvristica fragrans Houtt. (nutmeg)
Non-alcoholic
beverages
14 0.98 130 529 0.13 0.52
Ice cream 13 0.91 18 62 0.02 0.06
Condiments 21 1.47
Meats 150 10.5 25 86 0.26 0.90
Baked goods 75 5.25 51 153 0.26 0.80
Candy 19 1.33
Canarium commune L. (Elemi-tree)
Baked goods 17
c
1.7 51 153 0.08 0.26
SoIt candy 17
c
1.7
Soups 10
c
1.0

Total 0.88 2.95

a Calculation based on 4.6 in parsley essential oil; 7 in nutmeg essential oil and 10 in Elemi-tree oil.
b Data Irom the dietary and nutritional survey oI British adults (British adult study) (Gregory et al., 1990).
c Use levels Irom Fenaroli (1995)
74
75
Estragole

ACTIVE PRINCIPLE: I
SYNONYMS: 1-allyl-4-methoxybenzene, methylchavicol, p-allylanisole, chavicol methylether;
4-methoxyallylbenzene.

CAS No: 140-67-0

STRUCTURE:

O

REGULATORY / INTERNATIONAL STATUS: In the USA, estragole was given GRAS
status by FEMA (Hall and Oser, 1965) and is approved by the FDA Ior Iood use (21 CFR
121.1164). In 1981 JECFA evaluated estragole, and no ADI was allocated. The European
Regulation No. 1576/89 requires that anise Ilavoured spirit drinks be derived exclusively Irom
anise, star anise and/or Iennel. These drinks may thereIore contain up to 100 mg/kg estragole.
SCF considered estragole as genotoxic and carcinogenic. ThereIore, a saIe exposure limit could
not be established and SCF concluded that reductions in exposure and restrictions in use levels
are to be achieved (SCF, 2001).


Metabolism:
Animal studies: There are three major pathways oI metabolism: (I) O-demethylation, (II) 1`-
hydroxylation and (III) side chain expoxidation. Pathways I and III are detoxiIication reactions.
Pathway II is involved in the Iormation oI reactive electrophilic compounds. High dose levels as
used in toxicity studies lead to a change in the metabolic pattern Iavouring the Iormation oI the
1`-hydroxy compound (Anthony et al., 1987). In mice and rats, the metabolite 1`-
hydroxyestragole excreted in the urine accounted Ior 1.3-5.4 oI the dose at the dose-range oI
0.05 to 50 mg/kg bw and Ior 7.8-13.7 at the dose-range oI 500-1000 mg/kg bw (Caldwell et
al., 1990). Single doses oI estragole in the range oI 0.05 to 50 mg/kg bw, administered to Iemale
wistar albino rats by oral intubation, were largely (52-58) excreted as CO
2
. At higher doses
(500 and 1000 mg/kg bw), CO
2
excretion only accounted Ior 28-29 oI the administered dose.
These data indicated that side-chain cleavage and O-demethylation, oI which CO
2
is the
terminal metabolite, were more important than 1`-hydroxylation in the low dose range (Anthony
et al., 1987; Zangouras et al., 1981). More recently, it has been demonstrated that the Iormation
oI 2`,3`-epoxide metabolites accounting Ior up to 30 oI the total metabolic clearance oI
76
estragole, are very rapidly detoxiIied by epoxide hydrolase and glutathione transIerase to non-
toxic metabolites (Guenthner et al., 2001).
Human studies: AIter oral administration oI estragole (100 micrograms/person/day Ior 6
months) to two volunteers the urinary excretion oI 1`-hydroxyestragole amounted to 0.2 and
0.4 oI the administered dose. In the urine, Iive metabolites (1'-hydroxyestragole 0.3, 4-
methoxyhippuric acid 12, 4-methoxyphenyllactic acid 4, 4-methoxycinnamoylglycine 0.8
and 4-methoxyphenylacetic acid 0.5) were identiIied (Sangster et al., 1987).

Toxicology:
Subacute / subchronic toxicity: Rats given Iour daily doses oI 605 mg estragole/kg bw
displayed liver injury observed on gross examination (Taylor et al., 1964).
Chronic toxicity / carcinogenicity: Several investigations have shown that estragole-induced
hepatic tumours in CD-1 and B6C3F
1
mice aIter chronic dietary exposure and aIter i.p. or s.c.
injections prior to or aIter weaning. Weanling male mice were Iound to be more susceptible than
Iemales (Drinkwater et al., 1976; Miller et al., 1983; Wiseman et al., 1987). When Iemale CD-1
mice (groups oI about 50 animals) were Ied 0.23 oI 0.46 oI estragole or 0.25 oI 1`-
hydroxyestragole in the diet (resulting in doses oI approximately 350 and 700 mg estragole/kg
bw/day and 375 mg 1`-hydroxyestragole/kg bw/day) Ior 12 months, the incidences oI
hepatoma-bearing mice at 20 months were 56 and 71 Ior estragole treated animals and 56
Ior 1`-hydroxyestragole treated animals. No hepatomas were Iound in the controls. Four animals
in the high dose group showed angiosarcomas (Miller et al., 1983). In another study,
preweanling CD-1 mice (groups oI about 50 males and 50 Iemales) were administered 370 mg
estragole/kg bw ten times, twice weekly by gavage. By termination oI the study aIter 14 months,
the incidence oI hepatic tumours (mostly hepatomas) was 73 in the treated males vs. 24 in
the control males oI and 9 in the treated Iemales vs. 2 in the control Iemales. A small
increase oI lung adenomas was observed in treated males (15) (Miller et al., 1983). The 1`-
hydroxy metabolite oI estragole was shown to be a stronger hepatocarcinogen in mice than the
parent compound (Miller et al., 1983; Wiseman et al., 1987). For the carcinogenic potency oI
estragole in Iemale mice a TD
50
oI 50-100 mg/kg bw resulted Irom the above studies.
Mutagenicity / genotoxicity: In the majority oI studies, estragole was not mutagenic in the
Ames test with Salmonella tvphimurium tester strains TA1535, TA100, TA1537, TA1538
and/or TA98 at concentrations up to 300 micrograms/plate with or without exogenous metabolic
activation (Zeiger et al., 1987; Sekizawa and Shibamoto, 1982), in the Bacillus subtilis DNA
repair test (Rec-assay) in the presence or absence oI metabolic activation, and in the Escherichia
coli WP2 uvrA reversion test with or without metabolic activation (Sekizawa and Shibamoto,
1982). However, estragole was reported to be very weakly mutagenic in the Ames test with
Salmonella tvphimurium strain TA1535 only at concentrations up to 50 micrograms/plate in the
absence and in the presence oI a metabolic activation system prepared Irom rat liver
microsomes (S-9), with a marked increase in the mutagenicity upon addition oI 3`-
phosphoadenosine-5`-phosphosulIate in the presence oI S-9. Conversion oI estragole to DNA-
binding sulIuric acid esters under these conditions had been suggested (To et al., 1982). A very
weak mutagenic activity oI estragole at concentrations oI up to 20 micromoles/plate had been
reported in Salmonella tvphimurium strain TA 100 without metabolic activation, while no
mutagenic activity was observed Ior strain TA 98 at concentrations up to 30 micromoles/plate
(Swanson et al., 1979). PuriIied 1`-hydroxyestragole was reported to be mutagenic in S.
tvphimurium strain TA 100 without metabolic activation (Swanson et al., 1979), while negative
results were reported with strain TA100 and TA98 by others (Drinkwater et al., 1976).
Supplementation with NADPH-IortiIied rat liver microsomes and cytosol increased the
77
mutagenic activity Ior strain TA100 oI 1`-hydroxyestragole, as well as oI estragole (Swanson et
al., 1979). The 2`,3`-oxides oI estragole and 1`-hydroxyestragole showed dose-dependent
mutagenic activities in strain TA1535 in the absence oI IortiIied liver microsomes (Swanson et
al., 1979). Aqueous or methanolic extracts Irom Iennel (Foeniculum vulgare Mill.) did not show
any mutagenic activity in S. tvphimurium TA 98 and TA 100, with or without metabolic
activation, or in Bacillus subtilis rec-assay and in the in vivo micronucleus and chromosomal
aberration assay in mice (Morimoto et al., 1982; Yamamoto et al., 1982; Yin et al., 1991).
Mutagenic activity was reported Ior Iennel oil (2.5 mg/plate) in S. tvphimurium strain TA 100
with a liver-activating system (Sekizawa and Shibamoto, 1982), while negative results were
obtained in an in vitro chromosomal aberration test using a Chinese hamster Iibroblast cell line
(Ishidate et al., 1984). Estragole and its 1`-hydroxy metabolite induced in vitro unscheduled
DNS synthesis (UDS) in rat hepatocytes derived Irom male Fischer 344 rats in a dose-related
manner. 1`-Hydroxyestragole was a more potent genotoxin than its parent compound (Howes et
al., 1990; Chan and Caldwell, 1992). In an ex vivo liver UDS assay rats were treated with
estragole at doses oI 500, 1000 and 2000 mg/kg bw and UDS was induced only at the highest
dose (Mller et al., 1994).


Other studies: The Iormation oI DNA adducts in vivo and in vitro by metabolites oI estragole
has been demonstrated and the major hepatic DNA adducts have been characterized (Wiseman
et al., 1985; Phillips et al., 1981a, Fennel et al., 1985; Luo and Guenthner, 1996). A covalent
binding index (CBI) oI 30 has been determined (Randerath et al., 1984). Studies on the
structures oI the hepatic DNA adducts oI mice given 1`-hydroxyestragole indicate that sulIate
esters are the major electrophilic metabolites reacting with the hepatic DNA in vivo (Phillips et
al., 1981a and 1981b). These Iindings have been conIirmed by others. AIter loss oI the O-sulIate
moiety the resulting electrophilic carbonium ion reacts with purine bases in the DNA. The
major adduct Iormed Irom estragole is with N2 oI guanine (Wiseman et al., 1985).

TOXICOLOGICAL EVALUATION: Several studies with oral, i.p. or s.c. administration to
CD-1 and B6C3F
1
mice have demonstrated a weak genotoxic hepatocarcinogenic potential oI
estragole. The 1`-hydroxy metabolite is a stronger hepatocarcinogen than the parent compound.
DNA binding and carcinogenicity studies indicate that the carcinogenic potency oI estragole is
similar to that oI saIrole. Controversial results have been reported Ior the mutagenicity oI
estragole. However, the Iormation oI hepatic DNA adducts in vivo and in vitro with estragole
metabolites has clearly been demonstrated. It has been shown that metabolism oI estragole
depends very much on the dose level. ThereIore, the Iormation oI potential genotoxic
metabolites (e.g. 1`-hydroxyestragole and epoxide metabolites) in vivo might result in
considerably diIIerent concentrations than applied in in vitro genotoxicity studies. According to
recent metabolic data, the Iormation oI 2`,3`-epoxide metabolites (metabolic pathway III) seems
not to contribute to estragole genotoxicity in vivo.

(T)MDI: Not established (suspected genotoxic carcinogen).

MAIN OCCURRENCE: The highest concentrations oI estragole are Iound in tarragon
(Artemisia dracunculus L., CE No. 64) and sweet basil (Ocimum basilicum L., CE No. 308)
with approximately 0.7 and 0.4 oI estragole in the plant, respectively (Table I). High levels
oI estragole are Iound in the Iruits (seeds) oI various plants oI the UmbelliIerae Iamily such as
chervil (Anthriscus cerefolium (L.) HoIIm. ssp. cerefolium, CE No. 50; up to 0.8), Iennel
(Foeniculum vulgare Mill. var. dulce (Mill.) Batt. et Trab., CE No. 200; approx. 0.3), star
78
anise (Illicium verum Hook. I., CE No. 238; up to 0.25), and anise (Pimpinellum anisum L.,
CE No. 336). Considerably high levels oI estragole have also been reported in essential oil oI
anise hyssop (Agastache foeniculum (Pursh) Ktze., no CE No.), sweet chervil (Mvrrhis odorata
(L.) Scop., CE No. 299) and lemon balm (Melissa officinalis L., CE No. 280).

INTAKE ESTIMATION: Tarragon (and tarragon oil) is considered as the main source oI
estragole in the human diet, with basil also being a contributor (Council oI Europe, 1997b).
Maximum use levels oI tarragon oil and basil oil in Ioods and beverages have been provided by
industry (Council oI Europe, 1996b). Combined with data on estragole levels in the essential
oils (20-60 in tarragon oil and up to 85 in basil oil) concentrations oI estragole in Ioods and
beverages have been calculated (Table II) (Council oI Europe, 1999a). The highest levels oI
estragole are Iound in sauces and condiments (up to 500 mg/kg). According to a Swedish
survey, typical Iood categories such as ready made Iood, salads, gravies and dressings Ilavoured
with tarragon (use oI approx. 1.5 spice in Iood) contain estragole at levels oI up to 120 mg/kg
(Council oI Europe, 2000). Considerably high amounts oI estragole are Iound in alcoholic
beverages (50-150 mg/kg).
For alcoholic beverages traditionally Ilavoured with anise, star anise, sweet Iennel and bitter
Iennel, concentrations oI 20-50 mg estragole/kg have been reported, with Tsipouro (55
ethanol, 37 mg estragole/kg) and Ouzo (46 ethanol, 30 mg estragole/kg) being particular
examples (Council oI Europe, 1996). In aniseed Ilavoured conIectionery, estragole
concentrations oI 40-150 mg/kg in aniseed Ilavoured sweets in liquorice allsorts`, up to 40
mg/kg in herbal mixtures and winter mixtures oI sugar conIectionery, approx. 1 mg/kg in jelly
and gum products have been reported, whereas Ior other traditional products such as aniseed
balls and aniseed drops estragole levels are not known (inIormation Irom UK trade associations,
see Council oI Europe, 1999b).
Estragole concentrations in various Iood categories have also been reported by the French Iood
and Ilavouring industry ANIA-SNIAA (Table III). Based on these theoretically maximal levels
identiIied Ior various Iood categories, a mean maximal daily estragole intake oI
9 micrograms/kg bw/day or 0.45 mg/person (median 0.39 mg/person and 97.5 percentile
1.2 mg/person) has been calculated (Council oI Europe, 1998b). An intake estimation based on
general levels oI estragole in Ioods and beverages at the proposed limit oI determination (e.g.
0.05 mg/kg) results in a theoretical maximal mean exposure oI 0.19 micrograms/kg bw/day by
Iood in general (Ioods and beverages without exceptions) and 5.1 micrograms/kg bw/day by all
IoodstuIIs (sum oI Iood in general and exceptions) (Table III).
The intake oI estragole Irom spices and essential oils containing estragole has also been
estimated based on the assumption that meat products, condiments/relish and baked goods
contribute the most to the overall intake oI estragole (Tables VI a,b) (Council oI Europe,
1997a). These calculations have been carried out using Iood intake data Irom the dietary and
nutritional survey oI British adults (British adult study) in 1990 (Gregory et al., 1990). Since the
assumption has been made that all the Ioods in the three categories consumed in the given
amounts contain the spices tarragon, basil, star anise, Iennel, chervil and anise as well as
essential oil Irom basil and tarragon, the estimated estragole intake is largely overestimated.
Accordingly, estragole intake via spices and essential oils adds up to a mean daily intake oI 6
mg per person (extreme intake oI 18.3 mg).
Calculations carried out by industry resulted in an estimated daily estragole intake via Iood oI
1 mg Irom anise and 3 mg Irom star anise (IOFI estimates oI alkenyl benzene intakes Irom Iood,
see Council oI Europe, 1997a).

In conclusion, an approximate estimate oI the total intake oI estragole Irom all sources appears
to be in the order oI one milligram per person and day.
79
CONCLUSIONS: The available data indicate that estragole acts as a weak genotoxic
carcinogen in experimental animals aIter Iew repeated doses and chronic exposure. 1`-
Hydroxyestragole, the supposed proximate carcinogen, has been Iound in the urine oI men
dosed with estragole at levels achieved through normal diet. Estragole was classiIied by the
Committee oI experts at its 36
th
meeting as a weak genotoxic carcinogen. ThereIore, the
Committee oI experts concluded at its 48
th
meeting to classiIy estragole as a type I active
principle, Ior which no (T)MDI can be set. For a better assessment oI the risk associated with
human exposure to estragole via Ioods and beverages, long-term carcinogenicity studies in rats
and mice oI both sexes using a wide range oI dose levels are needed.
The estimated daily intake oI estragole by Ioods including essential oils is by a Iactor oI about
3000 below the TD
50
derived Irom the available carcinogenicity studies in mice. Since
metabolism via the activating pathway is disproportionally dependent on the estragole dose,
exposure to the putative carcinogenic metabolite 1`-hydroxyestragole is assumably lower.
EIIorts should be made to reduce the amount oI estragole in Iood as Iar as possible. It has to be
taken into account that the greatest proportion oI estragole intake derives Irom tarragon, Iennel
and chervil. ThereIore exception limits have been deIined Ior particular Ioods characteristically
Ilavoured with tarragon, Iennel or chervil.

DATA NEEDED: -

LIMITS: (mg/kg)

Foods and beverages ND
a

(not traditionally Ilavoured with
estragole containing herbs and spices)

Exceptions:
Anise Ilavoured conIectionery 100
Anise Ilavoured alcoholic beverages 35
Alcoholic beverages traditionally Ilavoured Ilavoured
with tarragon, Iennel or chervil 70
Flavoured baked goods 10
Cheese with herb 10
Flavoured preserves (with vegetable, Iish) 35
Flavoured ready meals (with Iish, meat) 35
Tarragon/basil Ilavoured oil 175
Condiments 200
Sauces 100

REFERENCES:
Anthony, A., Caldwell J., Hutt A.J. and Smith R.L. (1987) Metabolism oI estragole in rat and
mouse and inIluence oI dose size on excretion oI the proximate carcinogen 1'-
Hydroxyestragole. Fd. Chem. Toxic., 25, 799-806.
Caldwell, J., Howes A.J. and Hotchkiss S.A. (1990) The toxicological signiIicance oI
xenobiotic metabolism. Food Add. Contam., 7 (Suppl. 1), S116-S126.

a
80
Council oI Europe (1995) Committee oI Experts on Flavouring Substances. Intake data Ior
alkenylbenzenes - RD 4/2-36. Document submitted by IOFI, April 1995.
Council oI Europe (1996a) Committee oI Experts on Flavouring Substances. IOFI note on
content oI essentail oils Ior saIrole, estragole, methyleugenol and use in Europe - RD 4.1/3-
38.
Council oI Europe (1996b) Committee oI Experts on Flavouring Substances. InIormation
received Irom IOFI on natural source materials, Oct. 1996 - RD 6/4-39.
Council oI Europe (1997a) Committee oI Experts on Flavouring Substances. Overview on
toxicity and occurrence oI alkenylbenzenes: allylbenzenes and propenylbenzenes - RD
4.1/1-40.
Council oI Europe (1997b) Committee oI Experts on Flavouring Substances. RD 5/9-41.
Document submitted by IOFI, Oct. 1997.
Council oI Europe (1998a) Committee oI Experts on Flavouring Substances. Published
quantitative data oI active principles in essential oils - RD 4.2/13-43. Document submitted
by IOFI, Oct. 1998.
Council oI Europe (1998b) Committee oI Experts on Flavouring Substances. RD 4.2/17-43.
Document submitted by the Observatoire des Consommations Alimentaires.
Council oI Europe (1999a) Committee oI Experts on Flavouring Substances. Occurrence oI
saIrole, estragole and coumarin in Ioods and beverages - RD 4.1/2-44.
Council oI Europe (1999b) Committee oI Experts on Flavouring Substances. Note on
occurrence oI coumarin, saIrole and estragole in Ioods and beverages - RD 4.5/2-45.
Council oI Europe (2000) Committee oI Experts on Flavouring Substances. InIormation on the
occurrence oI saIrole, estragole and coumarin in Iood and beverages - RD 4.5/1-46.
Chan, V.S.W. and Caldwell J. (1992) Comparative induction oI unsceduled DNA synthesis in
cultured rat hepatocytes by allylbenzenes and their 1'-hydroxy metabolites. Fd. Chem.
Toxic., 30, 831-836.
Drinkwater, N.R., Miller E.C., Miller J.A. and Pitot H.C. (1976) Hepatocarcinogenicity oI
estragole (1-allyl-4-methoxybenzene) and 1'-hydroxyestragole in the mouse and
mutagenicity oI 1'-acetoxyestragole in bacteria. J. Natl. Cancer I., 57, 1323-1331.
Fenaroli (1995) Fenaroli`s Handbook oI Flavor Ingredients. Volume I, 3
rd
ed., Ed. G.A.
Fennel, T.R., Wiseman R.W., Miller J.A. and Miller E.C. (1985) Major role oI hepatic
sulIotransIerase activity in the metabolic activation, DNA adduct Iormation, and
carcinogenicity oI 1'-hydroxy-2',3'-dehydroestragole in inIant male C57BL/6J x C3H/HeJ
F1 mice. Cancer Res., 45, 5310-5320.
Guenthner, T.M. and Luo G. (2001) Investigation oI the role oI the 2',3'-epoxidation pathway in
the bioactivation and genotoxicity oI dietary allylbenzene analogs. Toxicology, 160 (1-3),
47-58.
Hall, R.L., Oser B.L. (1965) Recent progress in the consideration oI Ilavouring ingredients
under the Food Additives Amendment. 3. GRAS substances. Food Technol. 19 (2, Part 2),
151-197.
Howes, J.A., Chan V.S.W. and Caldwell J. (1990) Structure-speciIicity oI the genotoxicity oI
some naturally occurring alkenylbenzenes determined by the unsceduled DNA synthesis
assay in rat hepatocytes. Food Chem. Toxicol., 28, 537-542.
Ishidate, M. Jr., SoIuni T, Yoshikawa K, Hayashi M, Nohmi T, Sawada M. and Matsuoka A.
(1984) Primary mutagenicity screening oI Iood additives currently used in Japan. Food
Chem. Toxicol., 22, 623-636.
81
Leung, A.Y. (1996) Encyclopedia oI Common Natural Ingredients Used in Food, Drugs, and
Cosmetics. John Wiley & Sons, New York.
Luo, G. and Guenthner T.M. (1996) Covalent binding to DNA in vitro oI 2',3'-oxides derived
Irom allylbenzene analogs. Drug Metab. Dispos., 24 (9), 1020-1027.
Lutz, W.K. (1991) Dose-response relationships in chemical carcinogenesis: Irom DNA adducts
to tumor incidence. Adv. Exp. Med. Biol., 283, 151-156.
Miller, E.C., Swanson A.B., Phillips D.H., Fletcher T.L., Liem A. and Miller J.A. (1983)
Structure-activity studies oI the carcinogenicities in the mouse and rat oI some naturally
Res., 43, 1124-1134.
Morimoto, I., Watanabe F., Osawa T., Okitsu T. and Kada T. (1982) Mutagenicity screening oI
crude drugs with Bacillus subtilis rec-assay and Salmonella/microsome reversion assay.
Mutat. Res., 97, 81-102.
Mller, L., Kasper P., Mller-TegethoII K. and Petr T. (1994) The genotoxic potential in vitro
and in vivo oI the allyl benzene etheric oils estragole, basil oil and trans-anethole. Mutat.
Res., 325 (4), 129-136.
Phillips, D.H., Miller J.A., Miller E.C. and Adams B. (1981a) Structures ot the DNA adducts
Iormed in mouse liver aIter administration oI the proximate hepatocarcinogen 1`-
hydroxyestragole. Cancer Res., 41, 176-186.
Phillips, D.H., Miller J.A., Miller E.C. and Adams B. (1981b) N
2
atom oI guanine and N
6
atom
oI adenine residues as sites Ior covalent binding oI metabolically activated 1`-
hydroxysaIrole to mouse liver DNA in vivo. Cancer Res., 41, 2664-2671.
32
P-post-labelling analysis
oI DNA adducts Iormed in the livrs oI animals treated with saIrole, estragole and other
naturally-occurring alkenylbenzenes. I. Adult Iemale CD-1 mice. II Newborn male
B6C3F
1
mice. Carcinogenesis, 5, 1613-1622 and 1623-1628.
Sangster, S.A., Caldwell J., Hutt A.J., Anthony A. and Smith R.L. (1987) The metabolic
disposition oI |methoxy-14C|-labelled trans-anethole, estragole and propylanisole in
human volunteers. Xenobiotica, 17(10),1223-32.
SCF (2001) Opinion oI the ScientiIic Committee on Food on estragole.
Sekizawa J. and Shibamoto T. (1982) Genotoxicity oI saIrole-related chemicals in microbial test
systems. Mutat. Res., 101, 127-140.
Swanson, A.B., Chambliss D.D., Blomquist J.C., Miller E.C. and Miller J.A. (1979) The
mutagenicities oI saIrole, estragole, eugenol, trans-anethole, and some oI their known or
possible metabolites Ior Salmonella tvphimurium mutants. Mutat. Res., 60, 143-153.
Taylor, J.M., Jenner P.M. and Jones W.I. (1964) A comparison oI the toxicity oI some allyl,
propenyl and propyl compounds in the rat. Toxicol. Appl. Pharmacol., 2, 378-387.
To, L.P., Hunt J.P. and Andersen M.E. (1982) Mutagenicity oI trans-anethole, estragole,
eugenol and saIrole in the Ames Salmonella tvphimurium assay. B. Environ. Contam. Tox.,
28, 647-654.
administration to preweanling male C57BL/6J x C3H/HeJ F1 mice. Cancer Res., 47, 2275-
2283.
Wiseman, R.W., Fennel T.R, Miller J.A. and Miller E.C. (1985) Further characterisation oI the
DNA adducts Iormed by electrophilic esters oI the hepatocarcinogens 1`-hydroxysaIrole
and 1`-hydroxyestragole in vitro an in mouse liver in vivo, including new adducts at C-8
and N-7 oI guanine residues. Cancer Res., 45, 3096-3105.
Yamamoto, H., Mizutani T. and Nomura H. (1982) Studies on the mutagenicity oI crude drug
extracts. I. Yakugaku Zasshi; 102, 596-601.
82
Yin, X.J., Liu D.X., Wang H.C. and Zhou Y. (1991) A study on the mutagenicity oI 102 raw
pharmaceuticals used in Chinese traditional medicine. Mutat. Res., 260, 73-82.
Zangouras, A., Caldwell J., Hutt A.J. and Smith R.L. (1981) Dose dependent conversion oI
estragole in the rat and mouse to the carcinogenic metabolite, 1'-hydroxyestragole.
Biochem. Pharmacol., 30, 1383-1386.
Zeiger, E., Andersen B., Haworth S., Lawlor T., Mortelmans K. and Speck W. (1987)
Salmonella mutagenicity tests: III. Results Irom the testing oI 255 chemicals. Environ.
Mutagen., 9 (Suppl. 9), 1-109.

DATA BASES USED: Chemical Abstracts (1967-2001), Biosis (1973-2001), FSTA (1969-
2001), Toxline (1969-2001), Medline (1966-2001). Keywords: estragole, Ilavourings, toxicity
data.
83
APPENDIX:

Table I. Main occurrence oI estragole in natural sources oI Ilavourings
Botanical name Common name Essential oil
in plant
()
b

Estragole in
essential oil
()
a

Estragole
in part of
plant used
()
b

Artemisia dracunculus L.
(Compositae)
Tarragon 0.25-1
in herb
60-75 0.7
Ocimum basilicum L.
(Labiatae)
Sweet basil 0.8
in herb
20-89 approx. 0.4
Foeniculum vulgare Mill. subsp.
vulgare var. dulce (Mill.) Batt.
(syn. Foeniculum vulgare Mill. var.
dulce (Mill.) Batt. et Trab.)
(UmbelliIerae)
Sweet Iennel,
Roman Iennel
1.5-5.0
Foeniculum vulgare Mill. subsp.
vulgare var. vulgare (syn.
Foeniculum vulgare var. vulgare)
(UmbelliIerae)
Bitter Iennel,
Common Iennel
2-6
in Iruit
3.5-12.0 0.3
Pimpinella anisum L.
(UmbelliIerae)
Anise,
Sweet cumin
1-4
in Iruit
1-5 max. 0.04
Illicium verum Hook. I.
(Magnoliaceae)
Star-anise 5
in Iruit
5-6 max. 0.25
Agastache foeniculum (Pursh.) Ktze.
(syn. Lophantus anisatus. A.
anethiodora, A. anisata)
(Labiatae)
Anise hyssop,
Giant hyssop,
Liquorice mint
74
Anthriscus cerefolium (L.) HoIIm.
ssp. cerefolium
(UmbelliIerae)
(Garden) chervil 0.9
in Iruit
up to 85 max. 0.8
Melissa officinalis L.
(Labiatae)
Lemon balm 6.3
Mvrrhis odorata (L.) Scop.
(UmbelliIerae)
Sweet chervil up to 75
a
According to Council oI Europe (1998a) and secondary literature
b
According to Leung (1980), Council oI Europe (1997a) and Council oI Europe (1995)

84
Table II. Estragole concentrations in Ioods and beverages Ilavoured with tarragon oil or basil oil
Estragole concentration (mg/kg food)
a
Foodstuffs
flavoured with tarragon oil flavoured with basil oil
Alcoholic beverages 50-150 -
Soups 16-48 9
Sauces (e.g. Sauce bearnaise) 40-120 850
Tomato sauce - 13
Pesto sauce - 130
Snacks 20-60 170
Vegetable preserves 4-12 9
Ready meals 20-60 85
Meat products - 3
Cheese 3-9 -
Baked goods - 120
Candy - 2
Condiments 160-480 380
a
Calculation based on maximum use levels oI tarragon oil and basil oil in Ioods and beverages provided by
industry, estragole levels oI 20-60 in tarragon oil and up to 85 in basil oil (Council oI Europe, 1996b).
85
Table III. Reported maximum estragole levels in Iood and beverages according to the French
Iood industry and calculated maximal estragole intake levels
Foods / Beverages Maximum levels of
estragole (mg/kg)
Mean estragole intake
level (micrograms/kg
bw/day)
e

Foods 1
a
1.79
I
Food in general
Non-alcoholic beverages 1
a
2.09
I

Anise Ilavoured alcoholic
beverages (e.g. Pastis, Ouzo)
20 0.20
Other alcoholic beverages 100 0.23
Savoury biscuits 10 0.01
Cheese with herbs 10 0.13
Flavoured Iish preserves
b
50 0.75
Flavoured vegetable preserves
(seasoned coatings)
c
45 1.53
Ready meals with Iish 45 0.19
Ready meals with meat 45 0.73
Exceptions
Tarragon- or basil Ilavoured oil
d
250 1.12
Total All Exceptions 4.899
All IoodstuIIs 8.78
a
Provisional limit (as proposed in earlier Council oI Europe documents)
b
Flavoured part is usually the brine which is not consumed
c
Probably overestimated as all vegetable preserves covered incl. non-Ilavoured
d
Production given up in France according to inIormation by French industry, April 2002
e
Maximal estragole intake calculated using correction Iactors Ior the market share oI industrially prepared
products containing high levels oI estragole (4 oI the alcoholic beverages contain 100 mg/kg, 30 oI the
canned Iish contain 50 mg/kg, 1 oI Iats and oils contain 250 mg/kg) (Council oI Europe, 1998b).
I
Intake estimation based on general estragole levels in Ioods and beverages oI 0.05 mg/kg (e.g. proposed limit
oI determination) results in maximal estragole exposure oI 0.19 micrograms/kg bw/day by Iood in general
(IoodstuIIs 0.09 micrograms/kg and non-alcoholic beverages 0.10 micrograms/kg; without exceptions) and
5.09 micrograms/kg bw/day by all IoodstuIIs (sum oI Iood in general and all exceptions)

86
Table IV a. Intake oI estragole Irom spices (summarized Irom Council oI Europe, 1997a)
Mean (extreme) estragole intake (mg/day)
a
Foods Mean
(extreme)
intake of
food (g)
Tarragon Basil,
sweet
Star
anise
Fennel,
bitter
Chervil Anise
Total
mean
(extreme)
daily
intake
(mg)
Meat
products
24 (78) 0.1 (0.4) 0.2 (0.5) 0.1 (0.3) 0.1 (0.3) 0.2 (0.7) 0.2 (0.7) 0.9 (2.9)
Condiment,
relish
12 (32) 0.2 (0.6) 0.2 (0.4) - - - 0.1 (0.1) 0.5 (1.1)
Baked
goods
51 (154) (0.1) (0.1) (0.1) 0.1 (0.2) 1.5 (4.6) 1.6 (5.1)
a
Level oI use oI plant in mg/kg according to Fenaroli (1995), calculated estragole levels in Iood oI 3.6-9.2
mg/kg in meat products, 4.3-19.1 mg/kg in condiment, 0.4-0.7 mg/kg in baked goods (except anise 30.3
mg/kg).

Table IV b. Intake oI estragole Irom essential oils
Mean (extreme) estragole intake (mg/day)
a
Total mean
(extreme)
daily intake
(mg)
Foods Mean (extreme)
intake of food (g)
From tarragon
essential oil
b

From Sweet basil
essential oil
c

Meat products 24 (78) 1.8 (5.7) 0.3 (1.1) 2.1 (6.8)
Condiment
d
,
relish
12 (32) 0.8 (2.1) 0.1 (0.3) 0.9 (2.4)
a
Level oI use oI essentail oil in mg/kg according to Fenaroli (1995), calculated levels oI estragole in Ioods oI
14 and 73 mg/kg in meat products and 9 and 67 mg/kg in condiment/relish Ilavoured with basil and tarragon
oil, respectively.
b
Assuming 70 estragole in essential oil
c
Assuming 50 estragole in essential oil
d
Intake data Ior condiments reIer to intake oI white sauce, intake data Ior meat products reIer to intake oI
sausages, meat spreads, pates, bolognese sauce and curry sauce, baked goods are biscuits, Iruit pies, buns, cakes
and pastries.
87
Eucalyptol


SYNONYMS: 1,8-cineole; p-cineole; 1,8-epoxy-p-menthane, cajeputol, limonene oxide, 1,3,3-
trimethyl-2-oxabicyclo|2.2.2|octane

CAS No: 470-82-6

STRUCTURE:

O

REGULATORY / INTERNATIONAL STATUS: In the USA, eucalyptol was given GRAS
status by FEMA (1965) and is approved by FDA Ior Iood (CFR 172.510). SCF considered the
available toxicological studies as inadequate to derive an ADI and concluded that the available
animal data do not indicate a cause Ior concern about the daily eucalyptol intake Irom IoodstuIIs
including hard candies (SCF, 2002). FDA has concluded on the saIe use oI eucalyptol in a
variety oI products, such as the intake by lozenges at 0.2-15 mg every 0.5 to 1 hour or 1-30 mg
every 2 hours (FDA 1976-1990).


Metabolism:
Animal studies: In rats given eucalyptol at a dose oI 800 mg/kg bw intragastrically, 1,8-
dihydroxy-10-carboxy-p-menthane, 2-hydroxycineole and 3-hydroxycineole were Iound to be
the main metabolites in the urine as a consequence oI the opening oI the ether bridge
(Madyastha et al., 1988) . DiIIerent metabolites such as p-cresol, 9-hydroxycineole, cineol-9-oic
acid and benzoic acid were Iound in another experiment in the urine oI rats and brush tail
possums aIter oral administration (Southwell et al., 1980; Frigerio et al., 1985). In rabbits given
200 mg eucalyptol/kg bw by gavage, the hydroxylated metabolites 2- and 3-hydroxycineole
(endo- and exo-stereoisomers) were observed. Peak plasma concentrations oI the Iree 2- and 3-
hydroxycineole were observed aIter 1 hour (2400 micrograms/dl Ior 2-exo-hydroxy-1,8-
cineole), the conjugated Iorms reached a maximum aIter 1.5-2.0 hours (1250 micrograms/dl Ior
conjugated 2-exo-hydroxycineole) (Miyazawa, 1989).
Human studies: Eucalyptus oil is well absorbed orally and its gastrointestinal absorption is
rapid. Due to its lipid solubility, absorption is likely to be enhanced with Ioods such as milk.
Bioavailability is unknown (IPCS INCHEM, 1998). Eucalyptol is excreted via the lungs, urine,
skin and Ieces (Mc Pherson, 1925), yet, this has not been quantitatively determined.

88
Toxicology:
Subacute / subchronic toxicity: Fischer 344 rats (groups oI 6 animals per sex) were treated
with eucalyptol Ior 28 days either by gavage at doses oI 0, 150, 300, 600, 1200, 2400 mg/kg
bw/day or in encapsulated Iorm in the diet at concentrations oI 0, 3750, 7500, 15000 or 30000
mg/kg, corresponding to 380-3340 mg/kg bw/day in males and 350-3500 mg/kg bw/day in
Iemales. Administration by gavage resulted in high mortality (50) in both Iemale and male
rats in the highest dose group (2400 mg/kg bw/day) aIter the Iirst dose. A dose-related reduction
in the body weight gain and histopathologic damage oI the liver were observed in males at
treatment levels equal to or higher than 600 mg/kg bw. At lower dose levels (150 and 300
mg/kg bw/day) no treatment-related lesions were observed in the liver, and the reduction in
body weight gain compared to untreated controls was equal to that oI the vehicle (Trioctanoin
,
a glyceryl trioctanoate oil, 95 pure, dioctanoins indicated as possible impurities). Dietary
exposure to encapsulated eucalyptol was related to histopathological alterations in the liver,
kidneys and parotid gland at all dose levels in male rats (National Toxicology Program, 1987).
In another study, groups oI 10 male Wistar rats were given 0, 500 or 1000 mg eucalyptol/kg
bw/day by gavage Ior 28 days. Statistically signiIicant decreases in terminal body weight were
Iound in rats dosed with 500 and 1000 mg/kg bw/day. The relative liver and kidney weights
were signiIicantly increased in all dosed groups, whereas the relative brain weight was increased
only in the highest dose group (1000 mg/kg bw/day). In the kidneys oI all groups, eosinophilic
protein droplet accumulation was seen in the cytoplasma oI proximal tubule cells (Kristiansen et
al., 1973).
Hybrid B6C3F1 mice (groups oI 6 animals per sex) were treated with eucalyptol Ior 28 days
either by gavage at doses oI 0, 150, 300, 600 or 1200 mg/kg bw/day or in encapsulated Iorm in
the diet at concentrations oI 0, 3750, 7500, 15000 or 30000 mg/kg, corresponding to 60-5600
mg/kg bw/day in males and 700-6800 mg/kg bw/day in Iemales. In the gavage study, no
treatment-related lesions were detected in either sex. Administration oI eucalyptol in
encapsulated Iorm resulted in an increased relative liver weight in males at all but the lowest
dose level and in an increased relative brain weight in Iemales at the highest dose. Microscopic
examination revealed dose-related lesions in the liver with minimal hypertrophy oI centrilobular
hepatocytes in animals oI both sexes (National Toxicology Program, 1987).
Chronic toxicity / carcinogenicity: No data available.
Reproductive toxicity / teratogenicity: The eIIect oI eucalyptol on the liver microsomal
enzyme activity in Ioetal and newborn rats was studied in Sprague-Dawley rats. Dams were
treated subcutaneously with doses oI 500 mg/kg bw/day Ior 4 days at i) between day 10 and 14
oI pregnancy, ii) during the last 4 days oI pregnancy, or iii) between the 2
nd
and 6
th
day aIter
delivery. Liver microsomal enzyme activity oI the mothers was greatly enhanced, enzyme
activity oI the Ioetus livers was induced, but the microsomal activity oI the newborn rats was
not inIluenced by the treatment (Jori et al., 1973).
Mutagenicity / genotoxicity: Eucalyptol was not mutagenic in the Salmonella/microsome assay
(Ames test) in various tester strains with and without metabolic activation (Haworth et al., 1983;
Gomes-Carneiro et al., 1998) and did not cause DNA damage in bacteria (Oda et al., 1978; Yoo,
1986). Eucalyptol was not active in the rec-assay using B. subtilis up to a concentration oI
20 microlitres per plate (Yoo, 1986). Mitomycin C-induced sister chromatid exchanges (SCE) in
CHO K-1 cells were not increased by posttreatment with eucalyptol at 0 - 100 micromoles/l
(Sasaki et al., 1989). Induction oI sister chromatid exchange was observed in CHO cells only in
the absence oI metabolic activation at doses inducing cell cycle delay (Galloway et al., 1987).
Results oI an in vitro micronucleus assay in binuclei lymphocytes indicated the absence oI a
genotoxic eIIect oI eucalyptol. In this study, peripheral blood was incubated together with cell
medium at 37 degrees Ior 24 hours in order to let the cells divide. On day two, test substance
89
(eucalyptol) dissolved in 50 microliters oI ethanol was added at two concentrations. Colchicine
in PBS was used as positive control, and ethanol (solvent Ior eucalyptol) served as negative
control. 50 microliters oI PBS was also tested to ensure that this vehicle did not interIere with
positive control. Flasks were incubated Ior another 24 hours. Cytochalasine B was added to
prevent complete division oI cytoplasm, which makes the dividing cells easy to detect due to
their binucleate appearance. Micronuclei were counted aIter Iixation oI the cells. Eucalyptol did
not induce chromosomal damage in this study (Swedish National Food Administration, 2004).

Human data: Therapeutic dose recommendations Ior eucalyptus oil Ior adults are 0.05-0.2 ml
Ior single oral doses (equivalent to approx. 35 -150 mg eucalyptol) (British Pharmaceutical
Codex, 1979; Martindale, 1982) and 0.3-0.6 ml per day (Bundesgesundheitsamt, 1990).
Recommended therapeutic doses oI eucalyptol are 100 to 200 mg as single oral dose and up to
1.0 g per day (Pharmacopoeia Helvetica). Other reported uses are intranasally at 1 in drops,
one teaspoon in a hot steam vaporizer Ior inhalation and topically at 0.5 to 3 in ointments as
a rubeIacient (British Pharmaceutical Codex, 1979; Martindale, 1982). Intoxications have been
reported Iollowing accidental exposure to high doses oI eucalyptus oil (Table I).
The lowest lethal dose reported Ior adults was 4-5 ml eucalyptus oil, in general a dose oI 30 ml
was lethal (Mc Pherson, 1925). Thus, based on a body weight oI 60 kg, doses oI eucalyptus oil
in the range oI 0.05-0.5 ml/kg bw are considererd to be potentially lethal Ior adults. Fatal doses
cause a strong depression oI the central nervous system resulting in paralysis oI respiration and
heart. Eucalyptol intoxication aIIects the nervous system (loss oI consciousness,
hypoventilation, depression oI reIlexes and convulsions), the gastrointestinal system (abdominal
pain, vomiting and diarrhoea) and the respiratory system (respiratory depression, dyspnoea,
toxic pneumonia and bronchospasm). Further symptoms recorded are tachycardia and weak
irregular pulse, muscle weakness and ataxia, miosis and mydriasis (miosis being more
common), as well as nephritis (rarely recorded). Gastrointestinal eIIects are Irequently the initial
eIIects, although drowsiness may occur within a Iew and coma within 10 minutes aIter oral
exposure. Some symptoms such as central nervous system (CNS) depression and vomiting may
occur aIter a delay oI up to Iour hours, however, most patients recover Irom a eucalyptol
intoxication within 24 hours.

Other studies: Dietary addition oI eucalyptol had no signiIicant chemopreventive eIIect in rat
mammary carcinogenesis (Russin et al., 1989). Eucalyptol given as an aerosol to rats induced
the cytochrome P-450 system oI the liver but not oI the lung (Chada et al., 1984).

TOXICOLOGICAL EVALUATION: Toxicological data on eucalyptol in laboratory animals
and humans are limited and not oI optimal quality. Subacute oral toxicity studies with
eucalyptol in mice and rats revealed dose-related eIIects mainly in liver and kidneys. In these
experiments mice were less susceptible than rats to the toxic eIIects oI eucalyptol. Limited
studies on the metabolism oI eucalyptol indicate species-speciIic diIIerences in the metabolic
pathways. The Iormation oI hydroxylated metabolites oI eucalyptol have been reported in rats,
rabbits and brush tail possums. However, major diIIerences have been observed in the metabolic
patterns with various metabolites Iormed by ring opening. Limitations oI the toxicological
studies, including the presence oI histopathological alterations in male rats at all doses tested in
the 28-day diet study, do not allow the derivation oI a no-adverse-eIIect-level (NOAEL).
Adverse eIIects Iollowing accidental exposure oI humans to eucalyptus oil have been reported
in several studies. Death was reported in two cases aIter the ingestion oI only 3.5-5 ml oI
eucalyptus oil. However, a number oI recoveries have also been described aIter much higher
exposure. Eucalyptol was not active in several in vitro mutagenicity tests in bacteria. The results
on in vitro clastogenicity were inconclusive.
90
TMDI: Not established due to insuIIicient data.

MAIN OCCURRENCE: Eucalyptol is a primary constituent in the essential oils oI Tasmanian
blue gum (Eucalvptus globulus Labill., CE No. 185; 70-80), rosemary (Rosmarinus officinalis
L., CE No. 406; 12-47), yellow ginger (Hedvchium flavum Roxb., CE No. 224; 42),
cardamom (Elettaria cardamomum (L.) Maton, CE No. 180; 13-51) and Salvia lavandulifolia
Vahl, CE No. 413; 12-41) (Table II). Considerable contents oI eucalyptol have been reported
in the essential oils oI Roman mugwort (Artemisia pontica L., CE No. 70), common mugwort
(Artemisia vulgaris L., CE No. 72), and sage (Salvia officinalis L., CE No. 414) , sweet basil
(Ocimum basilicum L., CE No. 308)., peppermint (Mentha x piperita L., CE No. 282),
spearmint (Mentha spicata L., CE No. 286) and other Artemisia species.

INTAKE ESTIMATION: The estimated intake oI eucalyptol Irom Ioods and beverages, based
on maximum use levels provided by the Ilavouring industry (Council oI Europe, 2001, Table
III) is approximately 4.5 mg/person/day, corresponding to 75 micrograms/kg bw/day (Table
IV). Consumption Iigures, provided by the French Iood saIety agency, are based on a selection
oI 18 Iood categories identiIied to which eucalyptol might potentially be added. Correction
Iactors were applied Ior the percentage oI industrially prepared product Irom a particular Iood
category which is most likely Ilavoured with eucalyptol or eucalyptol containing herbs and
spices or essential oils (examples: 30 oI the market share oI canned Iish will contain
eucalyptol, 10 oI white/Iresh cheese contain eucalyptol mainly Irom basil or sage).

CONCLUSIONS: Due to the insuIIicient toxicological data a TMDI could not be allocated and
eucalyptol was classiIied as a class II active principle. The estimated daily intake oI eucalyptol
Irom Ioods and beverages oI approximately 4.5 mg per person is a Iactor oI 1000 below the
lowest exposure oI eucalyptol by ingestion oI eucalyptus oil reported to result in severe
intoxications or death in humans (e.g. 3.5-5 ml eucalyptus oil/person, corresponding to 2.45-
6.25 g eucalyptol per person) and is also much lower than the eucalyptol exposure resulting
Irom recommended therapeutic doses oI eucalyptus oil and eucalyptol Ior adults (in the range oI
35 to 200 mg eucalyptol per dose and up to 1000 mg eucalyptol/person/day).

DATA NEEDED: In order to reconsider the classiIication oI eucalyptol and to set an MDI, a
28-day oral study in the most sensitive species (i.e. in rats) as well as Iurther studies on the
metabolism to elucidate the species-speciIicity oI oxidation and elimination are required.

LIMITS: (mg/kg)

Foods and beverages 10

Exceptions:
Mint-Ilavoured syrups 30
Candies 1000
Micro-candies 10500
a

Chewing-gums 5000
Baked goods (salted biscuits) 50

a
Industry was asked to provide actual use levels oI micro-candies in time beIore the Iinal decision. In the absence
oI quantitative data Irom industry, restrictive limits have been set by the Committee on the basis oI reported
maximal use levels.
91
Pork meats 100
Sauces and condiments 250
White cheese, Iresh cheese, cream-cheese and Iresh spreads 100
Oils 60
Fish preserves 30

REFERENCES:
British Pharmaceutical Codex (1979) British Pharmaceutical Codex (BPC), 11
th
ed. The
Pharma-ceutical Press, London, p. 346.
Bundesgesundheitsamt (1990) Monographie: Eucalypti aetheroleum (Eucalyptusl). Bundes-
anzeiger, 50, 13.03.1990.
Chadha, A. and Madyastha K.M. (1984) Metabolism oI geraniol and linalool in the rat and
eIIects on liver and lung microsomal enzymes. Xenobiotica, 14 (5), 365-374.
Council oI Europe (2001) Committee oI Experts on Flavouring Substances. Evaluation des
quantites dèucalyptol ingerees a partir des denrees alimentaires aromatisees - RD 4.6 / 1-48
(including maximum use levels reported by the European Flavour and Fragrance
Association, EFFA, September 2000 and the French Food and Flavouring Industries,
ANIA/SNIA, March 2001).
Craig, J.O. (1953) Poisoning by the volatile oils in childhood. Arch. Dis. Child., 28 (142) 475-
483.
European Pharmacopoeia (1998) 3rd ed. Supplement. Council oI Europe, Strasbourg.
FDA (US Food and Drug Administration) (1976 1990) Fed. Reg. 41, 38312; 47, 22712; 47,
54646; 52, 30042; 55, 46914 (cited in TNO BIBRA, 1991).
rd
ed., Ed. G.A.
Frigerio, A., Paladino R., Testoni G., Cobelli L. Pastorello L. and Tolentino D. (1985) 1,8-
cineole (eucalyptol) metabolites in rat. Spectroscopy, 4, 43-56.
Galloway, S.M., Armstrong M.J., Reuben C., Colman C., Brown B., Cannon C., Bloom A.D.,
Nakamura F., Ahmed M., Duk S., Rimpo J., Margolin B.H., Resnick M.A., Anderson B.
and Zeiger E. (1987) Chromosome aberrations and sister chromatid exchanges in Chinese
hamster ovary cells: evaluations oI 108 chemicals. Environ. Mol. Mutagen., 10 (S10), 1-
175.
Gleason, M.N., Gosselin R. E. and Hodge H. C. (1963) Clinical Toxicology oI Commercial
Products. 2
nd
ed., William& Wilkins, Baltimore, p. 59.
Gomes-Carneiro, M.R., Felzenszwalb I. and Paumgartten F.J. (1998) Mutagenicity testing (/-)-
camphor, 1,8-cineole, citral, citronellal, (-)-menthol and terpineol with the Salmonella /
microsome assay. Mutat. Res., 416 (1-2), 129-136.
Gurr, F.W. and Scroggie J.G. (1965) Eucalyptus oil poisoning treated by dialysis and mannitol
inIusion. Aust. Ann. Med., 14 (3), 238-249.
Hall, R.L. and Oser B.L. (1965) Recent progress in the consideration oI Ilavouring ingredients
under the Food Additives Amendment. 3. GRAS substances. Food Technol. 19 (2, Part 2),
151-197.
Haworth, S., Lawlor T., Mortelmans K., Speck W. and Zeiger E. (1983) Salmonella
mutagenicity test results Ior 250 chemicals. Environ. Mutagen., Suppl. 1, 3-142.
IPCS INCHEM (1998) Eucalyptus oil. Poisons InIormation Monograph, Pharmaceuticals, PIM
031, WHO, Geneva.
Jori, A. and Briatico G. (1973) EIIect oI eucalyptol on microsomal enzyme activity oI Ioetal and
newborn rats. Biochem. Pharmacol. 22 (4), 543-544.
92
Kristiansen, E. and Madsen C. (1995) Induction oI protein droplet (alpha2-microglobulin)
nephropathy in male rats aIter short-term dosage with 1,8-cineole and l-limonene. Toxico.
Lett., 80 (1-3), 147-152.
Mack, M.D. (1988) Fair dinkum koala kruisine eucalyptus oil poisoning. N. C. Med. J., 49
(11), 599-600.
MacPherson, J. (1925) The toxicology oI eucalyptus oil. Med. J. Aust., 2, 108-110.
Madyastha, K.M. and Chadha A. (1986) Metabolism oI 1,8-cineole in rat: Its eIIects on liver
and lung microsomal cytochrome P-450 systems. B. Environ. Contam. Toxicol., 37 (5),
759-766.
Martindale (1982) The Extra Pharmacopoeia, Reynolds J.E.F. (ed.), 28
th
ed. The Pharmaceutical
Press, London.
Miyazawa, M., Kameoka H., Morinaga K., Negoro K. and Mura N. (1989) Hydroxycineole:
Iour new metabolites oI 1,8-cineole in rabbits. J. Agric. Food Chem., 37, 222-226.
National Toxicology Program (1987) Twenty-eight day gavage and encapsulated Ieed study on
1,8-cineole in Fischer 344 rats.
Oda, Y. et al. (1978) Osaka Furitsu Koshu Eisei Kenkyu Hokoku Skokuhin Eisei Hen. (Proc.
Osaka PreI. Inst. Publ. Lth, Ed. Fd. Sanit.) 9, 177 (cited in TNO BIBRA, 1991).
Patel, S. and Wiggins J. (1980) Eucalyptus oil poisoning. Arch. Dis. Child., 55, 405-406.
Pharmacopoea Helvetica (1971-1985) 6
th
ed. EDMZ, Bern.
Russin, W.A., Hoesly J.D., Elson C.E., Tanner M.A. and Gould M.N. (1989) Inhibition oI rat
mammary carcinogenesis by monoterpenoids. Carcinogenesis, 10 (11), 2161-2164.
Sasaki, Y.F., Imanishi H., Ohta T. and Shirasu Y. (1989) ModiIying eIIects oI components oI
plant essence on the induction oI sisterchromatid exchanges in cultured Chinese hamster
ovary cells. Mutat. Res., 226, 103-110.
SCF (2002) Opinion oI the ScientiIic Committee on Food on eucalyptol.
SCF/CS/FLAV/Flavour/20 ADD2 Final, 17 April 2002.
Southwell, I.A. and Flynn T.M. (1980) Metabolism oI alpha and beta-pinene, p-cymene and
1,8-cineole in the brushtail possum, Trichosurus vulpecula. Xenobiotica, 10 (1), 17-23.
Spoerke, D.G., Vandenberg S.A., Smolinske S.C., Kulig K. and Rumack B.H. (1989)
Eucalyptus oil: 14 cases oI exposure. Vet. Hum. Toxicol., 31 (2), 166-168.
Swedish National Food Administration (2004) Personal communication on an in vitro
micronucelus assay oI eucalyptol in binuclei lymphocytes.
TNO BIBRA (1991) Toxicity proIile. Eucalyptol.
Webb, N.J. and Pitt W.R. (1993) Eucalyptus oil poisoning in childhood: 41 cases in south-east
Queensland. J. Paediatr. Child H., 29 (5), 368-371.
Yoo, Y.S. (1986) Mutagenic and antimutagenic activities oI Ilavoring agents used in Iood-
stuIIs. J. Osaka City Med. Cent., 34, 267-288.

DATA BASES USED:
Chemical Abstracts (1967-2001), Biosis (1973-2001), FSTA (1969-2001), Toxline (1969-
2001), Medline (1966-2001). Keywords: eucalyptol, Ilavourings, toxicity data.
93
APPENDIX:

Table I. Toxicity oI eucalyptus oil in humans
Effects Amount ingested Number of cases (age) References
Death 4-5 ml 1 (adult) Mc Pherson, 1925
Survival aIter dialysis
Death
21-30 ml
3.5 ml
a

1 (8 years)
1 (age unknown)
Mack et al., 1988
Mack et al., 1988
Transient coma 1 ml 1 Gleason et al., 1953
25 min. aIter ingestion the subject
was pale and collapsed with rapid,
shallow respiration and a rapid and
Ieeble pulse. He was discharged on
the sixth hospital day.
a teaspoonIul
(about 1 ml)
1 (boy, 7 months) Craig, 1953
Gastrointestinal symptons Iollowed
by CNS depression
Death
5-25 ml
b

30 ml
9 (7 months to adult)

1 (8 months)
Spoerke et al., 1989
Severe poisoning with CNS
depression within 30 min.
10 ml 1 (boy, 3 years) Patel et al., 1980
33 oI the cases (80) remained
entirely asymptomatic.
8 cases (20) were manged with
gastrointestinal decontamination and
did not require admission to the
intensive care unit.
30 ml 41 (children 14 years) Webb et al., 1993
Survival aIter dialysis and mannitol
inIusion, coma Ior 3.5 days.
120-220 ml 1 (male, 18 years) Gurr et al., 1965
a
3.5 ml also cited as letal dose in Martindale (1982) and European Pharmacopoiea (1998)
b
5 ml ingested by 7 and 20 months old inIants, 25 ml ingested by 8 year old child

94
Table II. Main occurrence oI eucalyptol in natural sources oI Ilavourings
Botanical name Common name Part of plant
used
Eucalyptol content
in essential oil ()
Artemisia pontica L. Roman mugwort Herb 12-23
Artemisia absinthium L. Wormwood Herb 3.7
Artemisia herba-alba Asso White mugwort Herb 0.5-15
Artemisia vulgaris L. Common
mugwort
Herb 0.25-26.8
Eucalvptus globulus Labill. Tasman blue
gum, Eucalyptus
Leaves 70-80
Ocimum basilicum L. Sweet basil Leaves, Herb 8
Rosmarinus officinalis L. Rosemary Leaves 12-47
Salvia officinalis L. Sage Leaves 8-23
Salvia lavandulifolia Vahl Spanish sage Leaves 11.8-41.2
Elettaria cardamomum (L.)
Maton
Cardamon Seeds 13.1-51.3
Hedvchium flavum Roxb. Yellow ginger,
Yellow butterIly
lily
Rhizomes 42.2
Mentha x piperita L. Peppermint Herb 5-18
Mentha spicata L.
(syn. Mentha viridis L.)
Spearmint Herb 6

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97
Hydrocyanic acid


SYNONYMS: Hydrogen cyanide, prussic acid, HCN

CAS No: 74-90-8

STRUCTURE: H-CN

REGULATORY / INTERNATIONAL STATUS: Maximum use levels Ior HCN in Ioods and
beverages have been set in Annex II oI Directive 88/388/EEC (EEC, 1988) mainly in
accordance with the Council oI Europe, Blue Book (Council oI Europe, 1981), namely 1 mg/kg
in IoodstuIIs and beverages with the exceptions oI 5 mg/kg in canned stone Iruit (Blue Book: 5
mg/kg in stone Iruit juices), 50 mg/kg in nougat, marzipan or its substitutes or similar products
(Blue Book: 50 mg/kg in marzipan); 1 mg/ volume oI alcohol in alcoholic beverages. In the
Blue Book an additional exception limit oI 25 mg/kg has been recommended Ior conIectionery.
The EC regulation oI HCN in Ioods and beverages is currently being under revision.

International and European bodies considered the available experimental animal data and
epidemiologic studies in cassava-eating populations exposed to high levels oI cyanogenic
glycosides as inadequate to establish an ADI or a TDI in humans Ior chronic exposure to HCN
(JECFA, 1993; SCF, 2003; WHO 2004; EFSA, 2004). For acute eIIects, JECFA concluded that
a level oI up to 10 mg HCN equivalent/kg cassava Ilour was not associated with acute toxicity
(JECFA, 1993). EFSA stated that current exposure to cyanide Irom Ilavouring ingredients was
unlikely to give rise to acute toxicity. However, in view oI the lack oI adequate data on chronic
toxicity, EFSA supports the continued application oI limits to HCN levels in Ioods and
beverages (EFSA, 2004). Nevertheless, WHO concluded that acute cyanide intoxication may
arise Irom eating apricot kernels, choking cherries, and other stone Iruit kernels with high
concentrations oI cyanogenic glycosides. Inadequately prepared cassava, when constituting the
major part oI the diet, may be hazardous as well (WHO, 2004).


Metabolism:
Animal studies: Hydrocyanic acid is readily absorbed Iollowing inhalation, oral, and dermal
exposure. It is distributed in the body as hydrocyanic acid and is not present as the Iree cyanide
(CN
-
) under physiological pH (pK
a
oI 9.22). The distribution oI hydrocyanic acid to the various
tissues is rapid and Iairly uniIorm. Somewhat higher levels are generally Iound in the liver,
lungs, blood and brain. In mammals, hydrocyanic acid is mainly (about 80) detoxiIied in the
liver by the mitochondrial enzyme rhodanese, which catalyses the transIer oI the sulIane sulIur
oI thiosulIate to the cyanide ion to Iorm thiocyanate. While rhodanese is present in the
mitochondria oI all tissues, the species and tissue distributions oI rhodanese are highly variable.
The rate limiting step is the amount oI thiosulIate (EPA, 1990). Cyanide has not been Iound to
accumulate in the blood and tissues Iollowing oral exposure to inorganic cyanide (ATSDR,

98
1997). However, a cumulative eIIect oI exposure to thiocyanate Irom the breakdown oI
cyanogenic glycosides in Iood plants, resulting in thyroid toxicity, including goitre and
cretinism, has been reported (Nahrstedt, 1993). A halI-liIe time Ior hydrogen cyanide
elimination oI about 1 hour has been reported (Ansell and Lewis, 1970; IPCS, 1992). The
excretion oI an acute dose oI
14
C-labelled cyanide (two s.c. injections oI 8.3 micromoles
14
C-
KCN/animals at a 30 min interval) in urine, Ieces, and expired air was studied in male albino rats
exposed to daily doses oI 77 micromoles unlabelled KCN/rat in the diet Ior 6 weeks. Urinary
excretion was the main route oI elimination, accounting Ior 83 oI the total excreted radioactivity
aIter 12 hrs, and 89 aIter 24 hrs, respectively. The major metabolite in the urine was thiocyanate,
accounting Ior 70-80 oI the total excreted radioactivity aIter 12 and 24 hrs (Okoh, 1983). In rats
exposure to 160 mg KCN/kg bw/day via the drinking water Ior 13 weeks resulted in blood cyanide
concentrations in the range oI 16-25 nmol/ml blood, and thiocyanate concentrations in the range oI
340-880 nmol/ml plasma (Leuschner et al., 1991).
Human studies: The rate oI spontaneous detoxiIication oI cyanide in humans is about
1 microgram/kg bw per minute, which is considerably slower than in small rodents or dogs
(WHO, 2004). The normal average amount ot thiocyanate in urine varies Irom 0.85-14 mg per
24 hrs (ATSDR, 1997). Reported halI-liIe time values Ior the elimination oI the principal
metabolite thiocyanate in humans ranged Irom 4 hrs (Blaschle and Melmon, 1980) to 2 days
(Bdigheimer et al., 1979) and 2.7 days (Schultz et al., 1979).

Toxicology:
Acute toxicity: Oral LD
50
values have been reported in the range oI 3-4 mg cyanide/kg bw Ior rats,
6 mg cyanide/kg bw Ior mice and about 2.6 mg cyanide/kg bw Ior rabbits (WHO, 2004).
Symptoms oI acute intoxication by oral doses oI cyanide are cardiovascular, respiratory, and
neuroelectric alterations. Several studies have shown that the brain is the most sensitive organ.
Death Irom cyanide poisoning is believed to result Irom central nervous system depression,
subsequent to inhibition oI brain cytochrome oxidase activity (Way, 1984).
Subacute / subchronic toxicity: Groups oI 9 male Wistar rats received 0, 0.3, 0.9, 3, or 9 mg
KCN/kg bw/day (corresponding to 0, 0.12, 0.36, 1.2 or 3.6 mg cyanide/kg bw/day) in the drinking
water Ior 15 days. Body weight gain was reduced by 70 in the highest dose group. No apparent
eIIect on the body weight gain was observed in the other groups. An increase in thiocyanate levels
in the plasma was veriIied in all experimental groups (p0.05). Aspartate aminotransIerase (AST)
activity was signiIicantly increased (p0.05) in the dose-range oI 0.33.0 mg KCN/kg bw/day and
signiIicantly decreased at 9.0 mg KCN/kg bw/day. Alanine aminotransIerase (ALT) activity was
not apparently aIIected by KCN-treatment. No signiIicant changes were observed in serum
triiodothyronine (T
3
), thyroxine (T
4
), creatinine and urea levels. Qualitative histological analysis,
however without statistical treatment and morphometric analysis, revealed hydropic degeneration
oI renal tubular epithelial cells in rats oI the 3 and 9 mg KCN/kg bw/day group and hydropic
degeneration oI hepatocytes in rats oI the 9 mg KCN/kg bw/day group, however, no necrosis was
observed in liver tissue. A dose-dependent increase in the number oI reabsorption vacuoles oI
Iollicular colloid in the thyroid gland was noted in all treated animals, but no hyperplasia and
hypertrophy oI the thyroidal Iollicles was seen. Due to the described deIiciencies oI the study and
the mainly unspeciIic changes observed, the validity oI the results and their biological relevance is
questioned (Sousa et al., 2002).
Groups oI about 30 male Sprague-Dawley rats were administered 0, 40, 80 or 160/140 KCN mg/kg
bw/day (equivalent to 16, 32 and 64/56 mg cyanide/kg bw/day) in the drinking water Ior 13 weeks
(the dose oI 160 mg KCN/kg bw/day was reduced to 140 mg KCN/kg bw/day at the beginning oI
week 12 due to high mortality). Reduced body weight gain, increased protein in urine and dose-
dependently increased relative weights oI several organs were seen in all treatment groups,
however in the absence oI histopathological changes (Leuschner et al., 1989).

99
In another study (NTP, 1993), groups oI 10 F344/N rats received 0, 3, 10, 30, 100 or 300 mg
NaCN/litre in the drinking water (equivalent to 0, 0.2, 0.5, 1.4, 4.5 and 12.5 mg cyanide/kg bw/day
in males and 0, 0.2, 0.5, 1.7, 4.9 and 12.5 mg cyanide/kg bw/day in Iemales) Ior 13 weeks. At the
end oI the treatment complete necropsies were perIormed on all animals. Complete
histopathological examinations were perIormed on all animals in the control and highest dose
groups. Vaginal cytology and sperm motility evaluations were perIormed on rats and mice Irom the
control and three highest dose groups. Males oI these groups were also evaluated Ior reproductive
tissue weights, epididymal spermatozoal data and spermatogenesis, and Iemales Ior estrous cycle
length, and the percentage oI cycle spent in the various stages. No deaths, clinically signiIicant
eIIects on body or organ weights, gross or histopathological lesions or clinical pathology changes
attributable to cyanide exposure were noted. In particular, no lesions were observed in the brain or
thyroid gland. Cyanide treatment caused a slight but signiIicant reduction in the weight oI the cauda
epididymidis (7-13; p_0.05 at 30 and 100 mg/l, p_0.01 at 300 mg/l) and a marginal decrease in
sperm motility (p_0.05) in male rats. The reported reduction oI sperm motility was not dose-related
and thereIore oI doubtIul relevance. In males oI the highest dose group, a signiIicant decrease in the
weight oI the leIt epididymis, leIt cauda epididymidis, leIt testis (all p_0.01), and the number oI
spermatid heads oI per testis (p_0.05) was seen. The authors noted that the changes observed in
male rats are consistent with a small but measurable adverse eIIect on reproduction. However, the
changes were insuIIicient to decrease the Iertility. The two highest dose levels caused a signiIicant
increase in the relative length oI time spent by Iemale rats in proestrus and diestrus relative to estrus
and metestrus (p0.03). The Iindings in Iemale rats were not considered adverse (ATSDR, 1997).
The same study protocol was used in a Ieeding study with B6C3F
1
mice receiving 0, 3, 10, 30, 100
or 300 mg NaCN/kg in the drinking water (equivalent to 0, 0.3, 1, 3, 9 and 26 mg cyanide/kg
bw/day) Ior 13 weeks. In male mice a signiIicant decrease in the leIt cauda epididymidis weights
was noted at the highest dose level, but no changes in sperm motility or spermatid head density
were observed. No changes in the estrous cycle length in Iemale mice were observed (NTP, 1993).

Chronic toxicity / carcinogenicity: Groups oI 10 male and 10 Iemale albino rats received a diet
containing 0, 73 and 183 mg hydrogen cyanide/kg (equivalent to 0, 4.3 and 10.8 mg cyanide/kg
bw/day) Ior two years. No treatment-related eIIects on survival or growth rate, signs oI toxicity, or
haematological or histopathological changes in the organs examined (heart, lung, liver, spleen,
gastrointestinal tract, kidneys, adrenals, thyroid, testes, uterus, ovaries, cerebrum, cerebellum and
brain) were observed (Howard and Hanzal, 1955).
Reproductive toxicity / teratogenicity: Fetuses oI pregnant albino rats receiving cassava
powder as 50 and 80 oI their diet during the 15 days oI gestation showed limb deIects, open
eyes, microcephaly and growth retardation. Body weight gain oI the dams was lowered. No
Iurther inIormation on maternal toxicity and no indication oI the cyanide content oI the diet was
stated (Singh, 1981).
Pregnant Wistar rats were Ied a cassava diet liberating 21 mg hydrogen cyanide/kg diet, IortiIied
with 500 mg KCN/kg diet throughout gestation and lactation (49 day study in adults and 28 day
study in pubs). This is equivalent to 16 mg cyanide/kg bw/day. The high dietary cyanide level did
not have any marked eIIect on gestation and lactation perIormance oI Iemale rats. No eIIects were
observed on the number, mortality at birth, or body weight oI oIIspring or weight gain oI pups
during lactation. However, a signiIicant reduction oI the Ieed consumption and the daily growth
rate was observed in the oIIspring that were Ied high cyanide-containing diet during the post-
weaning period (Tewe and Maner, 1981).
Pregnant hamsters were Ied cassava meals with low (0.6-0.7 mmol/kg, equivalent to 16-18 mg
cyanide/kg diet) or high (5-11 mmol/kg, equivalent to 130-290 mg/kg diet) cyanide
concentrations during days 3-14 oI gestation. Reduced body weight gain in cassava Ied dams
compared to control animals (Ied diet oI similar nutritional value but without cyanogenic

100
glycosides) and Ioetotoxic eIIects (with reduced Ioetal body weight and retarded ossiIication)
were observed with both doses. High cyanide-containing cassava diet produced a signiIicant
increase in the number oI runts compared with litters Irom dams Ied either low-protein (i.e. 4,
comparable to cassava diet) or laboratory-stock diets (25 protein content) (Frakes et al., 1986).
Mutagenicity / genotoxicity: In vitro: Potassium cyanide was Iound to be negative in Ames tests
with Salmonella tvphimurium strains TA1535, TA1537, TA1538, TA98 and TA100 with and
without metabolic activation (NTP, 1993; Leuschner et al., 1983a). Another study reported a
marginal mutagenic activity oI HCN to Salmonella tvphimurium strain TA100 in the absence oI
metabolic activation, whereas in the same study no mutagenic activity oI HCN to strain TA98 with
or without metabolic activation was observed (Kushi et al., 1983). Cyanide was negative in the
Bacillus subtilis rec-assay (EPA, 1990).
In vivo. HCN did not induce chromosomal aberrations in Chinese hamsters aIter oral
administration oI 0.4 mg HCN/kg bw (Leuschner, 1983b).

Human data: The lowest published lethal doses oI HCN in humans aIter intravenous and oral
administration are 0.055 mg/kg bw and 0.570 mg/kg bw, respectively (RTECS, 2004). Based on
analyses oI cyanide contents in tissues and in gastrointestinal tract contents among Iatal (oral)
poisoning cases (and comparative kinetics with dogs) it was estimated that death occurred aIter
absorption oI an average oI 1.4 mg hydrogen cyanide/kg bw in humans, and that the lowest Iatal
absorbed dose was 0.54 mg hydrogen cyanide/kg bw (Gettler and Baine, 1938). The eIIects oI
acute cyanide exposure are dominated by central nervous system and cardiovascular disturbances.
Typical signs oI acute cyanide poisoning include tachypnoea, headache, vertigo, lack oI motor
coordination, weak pulse, convulsions, and coma (Ballantyne, 1983; Way, 1984; Johnson and
Mellors, 1988; ATSDR, 1991).
Consumption oI Iood containing cyanogenic glycosides (i.e. consumption oI cassava in AIrica) has
been attributed to diseases aIIecting mainly the nervous system, such as tropical ataxic neuropathy,
spastic paraparesis (also called Mantakassa or Konzo disease), retrobulbar neuritis and optic
atrophy associated with pernicious anaemia. Cyanides have also been implicated in thyroid eIIects
such as goitre and even cretinism (ATSDR, 1991; Tylleskr, 1992; Ernesto, 2002). Outbreaks oI
Konzo disease in Congo occurred during droughts and dry seasons when populations had almost
only relied on cassava roots as the staple Iood. A daily intake oI 0.19-0.37 mg cyanide/kg bw/day
has been estimated and sulphate deIiciency has been suggested to be an important contributory
Iactor in the development oI Konzo disease (Tylleskr et al., 1992). An epidemic oI Mantakassa
disease with 1102 acute cases occurred in Mozambique 1981-1982 in a cassava-eating population.
The estimated intake oI cyanide was 0.2-0.5 mg/kg bw/day. Also, because oI the drought, there was
generally a lack oI Iood, especially oI protein-rich Iood. There was no correlation between the
disease severity and serum thiocyanate level (Ministry oI Mozambique, 1984a,b). Several surveys
in areas oI endemic iodine deIiciency and goitre, hypothyroidism, and cretinism have demonstrated
that there is a strong correlation between cassava consumption and the thyroid eIIects (JECFA,
1993). It has been demonstrated that a cassava meal diminished the uptake oI
131
I in the thyroid
(Delange et al., 1971).

TOXICOLOGICAL EVALUATION: Hydrocyanic acid was acutely toxic in laboratory
animals with oral LD
50
s oI 2.6-6 mg cyanide/kg bw in rabbits, rats and mice. From the two 13-
week studies in rats and mice, 12.5 mg cyanide/kg bw/day was considered as the lowest dose
leading to biologically signiIicant adverse eIIects on reproductive organs in male rats and 4.5 mg
cyanide/kg bw/day as the NOAEL in rats. A NOAEL oI 10.8 mg cyanide/kg bw/day was derived
Irom the chronic Ieeding study with rats. The biological relevance oI eIIects observed in other
repeated-dose toxicity studies is considered questionable. There is a slight indication oI
teratogenicity in hamsters and rats at maternally toxic doses. Mutagenicity tests were negative in

101
bacteria and Chinese Hamsters in vitro and in vivo. From studies in cassava-eating human
populations there is an indication oI an association between neurological complications and the
intake oI cyanogenic glycosides at doses corresponding to 0.2-0.5 mg cyanide/kg bw/day, although
dietary deIiciencies may be involved.
Based on the NOAEL oI 4.5 mg cyanide/kg bw/day Iound in the 13-week study in rats and a saIety
Iactor oI 200 a TMDI oI 0.023 mg cyanide/kg bw/day was established. The saIety Iactor oI 200
takes into account the small but measurable eIIects on reproduction in male rats, which were
insuIIicient to decrease Iertility, and also the recognised higher sensitivity oI humans, as compared
to rats, to such changes. This TMDI is a Iactor oI 10 below the lowest estimated intake level oI 0.19
mg cyanide/kg bw/day leading to neurological disorders in cassava-eating populations as observed
in the Konzo study. This saIety margin is considered adequate since Konzo disease is associated
with sulphate deIiciency making it less likely to occur in adequately nourished populations.

TMDI: 0.023 mg cyanide/kg bw/day

MAIN OCCURENCE: Cyanide occurs naturally as cyanogenic glycosides in at least 2000
plants. Hydrogen cyanide can be produced by hydrolytic reaction catalysed by one or more
enzymes Irom the plant containing cyanogenic glycosides. This reaction can occur when kernels
are crushed and moistened or chewed. When the enzymes are activated the hydrolysis reaction
is rapidly completed, particularly in alkaline environment. The most important sources oI HCN
in the human diet are the cyanogenic glycosides linamarin or prunasin (see Table I). However,
also amygdalin has been Iound in about 1000 plants. The highest levels oI HCN (up to 3000-
4000 mg HCN/kg) are Iound in seeds oI lima beans (Phaseolus lunatus L., no CE No.) as
linamarin and in kernels oI bitter almonds (Prunus dulcis (Mill.) D.A. Webb var. amara (DC.)
Buchheim, CE No. 366) and apricots (Prunus armeniaca L., CE No. 368) as prunasin. High levels
oI HCN bound in linamarin are Iound in cassava roots (Manihot utilissima Pohl, no CE No.; up
to 200 mg HCN/kg) and in Ilax seeds (Linum usitatissimum L., CE No. 263; over 500 mg
HCN/kg), whereas the content in common beans (Phaseolus vulgaris L., no CE No.) is much
lower (20 mg HCN/kg). High levels oI HCN, bound in prunasin, have been reported in kernels
oI peaches (Prunus persica (L.) Batsch., CE No. 374; 470 mg HCN/kg).

INTAKE ESTIMATION: HCN contents in various categories oI Ioods and beverages are
presented in Table II. The highest levels are Iound in marzipan and similar products made Irom
apricot kernels and in nougat (up to 50 mg/kg). Lower levels have been reported in stone Iruit
products such as purees and preserves (up to 1 mg/kg) and alcoholic beverages (maximum oI 10
mg/l).
The overall intakes oI HCN Irom Ioods and beverages by mean and high level (97.5th
percentile) consumers have been estimated to be 46 and 214 micrograms/person/day,
respectively (equivalent to 0.7 and 3.3 micrograms/kg bw/day), using the limits proposed below
and consumption data Irom the dietary and nutritional survey oI British adults (British adult
study) (Gregory et al., 1990; Council oI Europe, 1999a).

CONCLUSIONS: Both the estimated mean and the 95th percentile intakes oI hydrocyanic acid
(i.e. 0.7 and 3.3 micrograms/kg bw/day) are well below the TMDI oI 0.023 mg/kg bw/day. No
adverse eIIects are expected Irom regular consumption oI Ioods and beverages containing
cyanide within the proposed limits. However, acute intoxication may arise Irom eating apricot
kernels, choking cherries, and other stone Iruit kernels with high concentrations oI cyanogenic
glycosides. Inadequately prepared cassava, when constituting the major part oI the diet, may be
hazardous as well.

102
DATA NEEDED: In order to reconsider the classiIication oI HCN a reproductive toxicity study
in rodents should be carried out in conIormity with OECD guidelines to elicit the relevance oI
the observed eIIects on male reproductive organs in rats.

LIMITS: (mg/kg, expressed as HCN)

Foods 0.5
Beverages 0.05

Exceptions:
Stone Iruit juices 0.5
Canned stone Iruit and stone Iruit preserves and purees 2
Marzipan, nougat and similar products 50
Almond and/or marzipan (or other similar products)-containing
conIectionery and baked goods 10
Special` almond- and/or marzipan, (or other similar products)- containing
conIectionery and baked goods e.g. amaretti`, Dresdner Christstollen`,
Schwarzbrtchen, chocolate enrobed marzipan, marzipan novelties 40
For every 1 alcohol by volume in alcoholic beverages 0.5

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Public Health Service, Agency Ior Toxic Substances and Disease Registry, Atlanta, GA.
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Nitroprussid-Natrium-Metaboliten Thiocyanat. Dtsch. Med. Wochenschr., 104, 939-943.
Conn, E. (1973) Cyanogenic glycosides. In: Toxicants occurring naturally in Ioods, National
Academy oI Science, 299-308.
Corradi, C. and Micheli G. (1982) Sul contenuto di acido cianidrico totale degli amaretti. Industrie
alimentari, 21, 459-465.
Book, 3
rd
ed., Strasbourg.
Council oI Europe (1998) Committee oI Experts on Flavouring Substances. InIormation received
Irom the UK marzipan manuIacturers, personal communication.
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datasheet on hydrocyanic acid RD 4.10/1-45.
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content oI HCN in IoodstuIIs RD 4.2/28-44.

103
Council oI Europe (1999c) Committee oI Experts on Flavouring Substances. Note on HCN (use
levels) RD 4.2/31-44. InIormation received Irom the British SoIt Drinks Association
(BSDA).
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levels) RD 4.2/32-44. InIormation received Irom the UK preserves manuIacturers
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Council oI Europe (1999e) Committee oI Experts on Flavouring Substances. InIormation
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thyrotropin level, the prevalence oI goiter and the pattern oI iodine metabolism in Idjwi
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and Materials in Contact with Food (AFC) on hydrocyanic acid in Ilavourings and other
Iood ingredients with Ilavouring properties adopted on 7 October 2004
EPA (1990) Summary Review oI Health EIIects Associated with Hydrogen Cyanide, Health Issue
Assessment Environmental Criteria and Assessment OIIice, OIIice oI Health and
Environmental Assessment OIIice oI Research and Development, US Environmental
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Ernesto, M., Cardoso A.P., Nicala D., Mirione E., Massaza F., CliII J., Haque M.R. and Bradbury
H.R. (2002) Persistent konzo and cyanogen toxicity Irom cassava in northern Mozambique.
Acta Trop., 82, 357-362.
Frakes, R.A., Sharma R.P., Willhite C.C. and Gomez G. (1986) EIIect oI cyanogenic glycosides
and protein content in cassava diets on hamster prenatal development. Fundam. Appl.
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Gessner, O. and Orzechowski G. (1974) GiIt- und ArzneipIlanzen von Mitteleuropa, Carl Winter
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Lebensmitteln. Z. Lebensm. Unters. Forsch., 134 (2), 69-80.
Holzbecher, M.D., Moss M.A. and Ellenberger H.A. (1984) The cyanide content oI laetrile
preparations, apricot, peach and apple seeds. J. Toxicol. Clin. Toxicol., 22, 341-347.
Howard, J.W. and Hanzal R.F. (1955) Chronic toxicity Ior rats oI Iood treated with hydrogen
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Johnson, R.P. and Mellors J.W. (1988) Arteriolization oI venous blood gases: A clue to the
diagnosis oI cyanide poisoning. J. Emerg. Med., 6, 401-404.
Kushi, A., Matsumoto T. and Yoshida D. (1983) Mutagen Irom gaseous phase oI protein
pyrolysate. Agr. Biol. Chem., 47, 1979-1982.

104
Leuschner, F., Neumann B.W. and Liebsch M. (1983a) Mutagenicity study oI hydrocyanic acid in
the Ames Salmonella / microsome plate test (in vitro). Unpublished study, Laboratory oI
Pharmacology and Toxicology, Hamburg, August 1983 (cited in JECFA, 1993).
Leuschner, F., Neumann B.W. and Liebsch M. (1983b) Mutagenicity study oI hydrocyanic acid in
Chinese hamster cells (chromosome aberration) by oral administration. Unpublished study,
Laboratory oI Pharmacology and Toxicology, Hamburg, August 1983 (cited in JECFA, 1993).
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cyanide administered to Spraque-Dawley rats in the drinking water. Unpublished study,
Laboratory oI Pharmacology and Toxicology, Hamburg, July (cited in JECFA, 1993).
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associated with chronic cyanide intoxication in a cassava staple area oI Mozambique. 1.
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477-484.
Ministry oI Health, Mozambique (1984b) Mantakassa: An epidemic oI spastic paraparesis
associated with chronic cyanide intoxication in a cassava staple area oI Mozambique. 2.
Nutritional Iactors and hydrocyanic acid content oI cassava products. Bulletin oI the WHO,
62, 485-492.
Nahrstedt, A.F. (1993) Cyanogenesis and Iood plants. In: van Beek, T.A., Breteler, H. (eds).
Proceedings oI the International Symposium on Phytochemistry and Agriculture, 22-24
April 1992, Wageningen, OxIord University Press, 107-129.
NTP Technical Report (1993) No. 37, NIH Publ. No. 94-3386, National Toxicology Program.
Ogunsua, A.O. (1989) Total cyanide levels in bread made Irom wheat/cassava composite Ilours.
Intern. J. Food Sci. Technol., 24, 361-365.
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potassium cyanide. Toxicol. Appl. Pharmacol., 70 (2), 335-339.
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http://www.cdc.gov/niosh, mw682428.html, reIerences Ior i.v.: National Technical
InIormation Service, SpringIield, VA 22161, Iormerly U.S. Clearinghouse Ior ScientiIic &
Technical InIormation; oral: Pesticide Chemicals OIIicial Compendium, Association oI the
American Pesticide Control OIIicials, Inc., 1966, Topeka, KS.
Schultz, V., Bonn R. and Kindler J. (1979) |Kinetics oI elimination oI thiocyanate in 7 healthy
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SCF (2003) Minutes oI the 137th plenary meeting oI the scientiIic committee on Iood held on
2/3/4 april 2003 in Brussels (SCF/CS/FLAV/FLAVOUR/48 Final).
Singh, J.D. (1981) The teratogenic eIIects oI dietary Cassava on the pregnant albino rat: a
preliminary report. Teratology, 24 (3), 289-291.
Sousa, A.B., Soto-Blanco B., Guerra J.L., Kimura E.T. and Gorniak S.L. (2002). Does
prolonged oral exposure to cyanide promote hepatotoxicity and nephrotoxicity?
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Sturm, W. (1969a) Bittere Mandeln 1. Mitteilung. Ernhrungsumschau, 16 (11), 450-453.
Sturm, W. (1969b) Bittere Mandeln - Schluss. Ernhrungsumschau, 16 (12), 483-486.
Tewe, O.O. and Maner, J.H. (1981) Long-term and carry-over eIIect oI dietary inorganic cyanide
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105
Tylleskr, T., Banea M., Bikangi N., Cooke R.D., Poulter N.H. and Rosling H. (1992) Cassava
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DATA BASES USED: Medline (1966-1999), Toxline (1969-1999), Embase (-1999), Biosis
(1973-1999), Chemical Abstracts (-1999). Keywords: hydrocyanic acid, hydrogen cyanide.

106
APPENDIX:

Table I. Hydrocyanic acid content in some edible plants

Botanical name Common name Plant
part used
Cyanogenic
glycoside
HCN in plant
part (mg/kg)
Manihot utilissima Pohl
(syn. Manihot esculenta Crantz)
Cassava, manjok root Linamarin 10-200
a, b
Phaseolus lunatus L. Lima bean seed Linamarin 100-3000
b, c, d, e
Phaseolus vulgaris L. Common bean,
Garden bean
seed Linamarin 20
b
Linum usitatissimum L.. Flax seed Linamarin ~500
h

Prunus dulcis (Mill.) D.A.
Webb var. amara (DC.)
Buchheim
(syn. Prunus amvgdalus var.
amara (DC.) Focke)
Bitter almond kernel Prunasin 300-3400
b, I, g

Prunus armeniaca L. Apricot kernel Prunasin 120-4000
d, e
Prunus persica (L.) Batsch. Peach kernel Prunasin 470
d, e

According to
a
Ogunsua, 1989;
b
Lindner, 1986;
c
Conn, 1973;
d
Gupta, 1987;
e
Holzbecher et al., 1984;
I
Sturm,
1969a und b;
g
Hanssen and Sturm, 1967;
h
Gessner and Orzechowski, 1974.

107
Table II. HCN concentration in Ioods and beverages

Foods / Beverages

HCN concentration in foods (mg/kg)
and beverages (mg/l)
Ground almonds
1
1.4
Marzipan
2
(in the UK diIIerent grades oI marzipan
contain diIIerent concentrations oI HCN). Other
similar products made Irom apricot kernels

Marzipan novelties
3

Almond paste
3

up to 50
15-20 (retail)
30-35 (higher grade, manuIacturing)
~50 (baker`s raw paste)
0.8
3.0

Peach/apricot/plum/cherry juices/purees
4

Peach puree
4

0.1

0.043

Stone Iruit preserves
5
1

Canned stone Iruit 0.4-1.5
3

0.01-0.02
6

Alcoholic beverages manuIactured with stone Iruits:
Kirsch (distilled Irom cherries with up to 61
alcohol)
7
Calvados and williams (distilled Irom pears with up to
40 alcohol)
7
Stone Iruit brandies
3

10
0.5

3 (80 oI the samples had undetectable
levels oI HCN)
'Special almond and/or marzipan (or other similar
products)-containing conIectionery and baked goods
8

Chocolate enrobed marzipan
3

up to 40
median 44 (in amaretti)
1.3
Nougat up to 50

According to
1
Tokyo Metropolitan Research Laboratory oI Public Health, 1975;
2-7
Council oI Europe, (
2
1998,
3
1999b,
4
1999c;
5
1999d,
6
1999e,
7
1999I);
8
Corradi and Micheli, 1982.

108

109
Hypericin


SYNONYMS: Hypericum red; cyclo-werrol; cyclosan; 4,5,7,4`,5`,7`-hexahydro-2,2`-dimethyl-
naphthodianthrone; phenanthro|1,10,9,8-opqra|perylene-7,14-dione; 1,3,4,6,8,13-hexahydroxy-
10,11-dimethyl-(6CI, 7CI, 8CI, 9CI)

CAS No: 548-04-9

STRUCTURE:

O
O
OH
OH
OH
OH
HO
HO

REGULATORY / INTERNATIONAL STATUS: In the USA, the hypericin-Iree alcoholic
distillate Irom St. John`s Wort is listed as GRAS by the FDA Ior use as a Ilavouring in alcoholic
beverages only (CFR 172.510). Annex II oI EC Directive 88/388/EEC on Ilavourings speciIies
limits Ior hypericin in Ioods to which Ilavourings or Iood ingredients with Ilavouring properties
have been added as Iollows: 10 mg/kg in alcoholic beverages, 1 mg/kg in conIectionery and
0.1 mg/kg in IoodstuIIs and non-alcoholic beverages. Isolated hypericin may not be added to
IoodstuIIs (EEC, 1988). The SCF recently considered the saIety oI hypericin in Ilavourings and
Iood ingredients and concluded that the database was too limited to allow an adequate saIety
assessment, and no ADI was established (SCF, 2002). Regulation oI the use oI St. John`s Wort
(Hvpericum perforatum L.) in Ioods and beverages in the EU is currently being under revision.


Metabolism:
In vitro studies: No data Iound.
Animal studies: A pharmacokinetics study in mice given a single i.v. dose oI 17.5 mg/kg bw
hypericin determined the terminal biological halI-liIe to be 38.5 hours (Liebes et al., 1991).
Human studies: Twelve healthy male volunteers were given single oral doses oI 4.2, 12.5 or
25 micrograms hypericin/kg bw (as LI 160/PK tablets containing a standardized extract oI
Hvpericum perforatum L., subsequently named Hvpericum). The median lag time Ior absorption
was about 2 hours and median elimination halI-lives were 24.5 (range 14.7-57.8), 43.1 (28.2-

110
57.8) and 48.2 (22.9-57.8) hours when volunteers were dosed with 4.2, 12.5 or 25 micrograms
hypericin/kg bw, respectively. The pharmacokinetics oI hypericin Iollowing i.v. injection was
investigated in two oI the subjects. Comparison oI areas under the plasma-time curve (AUC)
Iollowing a single oral dose (12.5 micrograms/kg bw) and a single i.v. dose (2 micrograms/kg
bw) oI hypericin indicated that the systemic availability Iollowing oral exposure was
approximately 14. When 13 volunteers were given 12.5 micrograms hypericin/kg bw/day (as
LI 160/PK tablets containing Hvpericum extract) orally Ior 14 days, plasma hypericin reached a
steady state concentration oI 7.9 micrograms/l aIter 7 days (Kerb et al., 1996). In another study
in which 13 human volunteers were given single oral doses oI 0, 18, 36 or 73 micrograms
hypericin/kg bw/day, the halI-liIe oI hypericin was estimated to be 28 hours. Intact hypericin
was not detected in the urine, nor were any possible glucuronidation or sulphation metabolites.
The authors also conducted a multiple dosing study, in which 50 volunteers were dosed with
36 micrograms hypericin/kg bw/day Ior 15 days. The estimated halI-liIe was 42 hours
(Brockmller et al., 1997).
Hepatitis C patients received 50 micrograms hypericin/kg bw/day (12 volunteers) or
100 micrograms hypericin/kg bw/day (7 volunteers) orally Ior 8 weeks. Mean plasma halI-lives
were 36.1 and 33.8 hours, respectively (Jacobson et al., 2001).

Toxicology:
Acute toxicity: Single doses oI 926, 1852 or 2778 mg St. John`s Wort extract/kg bw
(equivalent to approximately 1, 2 or 3 mg hypericin/kg bw) given to male rats (8-12 animals per
group) by gavage were associated with an increased locomotor activity in the open Iield and an
anxiolytic activity in the light-dark test. No eIIects were observed when rats were given 3 mg
pure hypericin/kg bw by gavage (Vandenbogaerde et al., 2000).
Two rats were Ied total amounts oI 30 or 60 mg hypericin in three divided doses over a period
oI 6 hours. The Iollowing day they were exposed to sunlight. Within 5 minutes oI sunlight
exposure they developed erythema oI the ears, began scratching vigorously and sought out
shade (Pace, 1942).
Calves were given single oral doses oI 1000, 3000 or 5000 mg/kg bw oI dried St. John`s Wort
(equivalent to approximately 0.124, 0.372 and 0.620 mg hypericin/kg bw, respectively) and
exposed to sunlight. At the two higher doses adverse eIIects were noted including increased
temperature and respiration rate, restlessness and skin reddening around eyes and nostrils and in
white areas oI the body. The lowest dose produced no adverse eIIects (Araya and Ford, 1981).
Groups oI 11 ewes were dosed by gavage with ground, dried St. John`s Wort equivalent to
approximately 2.65, 3.7 or 5.3 mg hypericin/kg bw, then exposed to bright sunlight Ior up to 5
hours/day on 5 successive days or shorter iI moderately severe clinical signs developed. All
sheep showed increased body temperature and signs oI skin irritation as well as restlessness,
pawing oI the ground, head shaking, head rubbing and oedema around the Iorehead and eyes.
EIIects persisted Ior up to 4 days (Bourke, 2000).
Subacute / subchronic toxicity: Groups oI 8 male rats were Ied a diet containing 0 or 10
dried and Iinely ground St. John`s Wort (hypericin content not indicated) Ior 12 days, and then
the amount was reduced to 5 due to unpalatability. AIter 17 weeks, 4 animals per group were
sacriIiced and autopsied. The remainder were sacriIiced aIter 25 weeks. Animals treated with St.
John`s Wort showed a signiIicantly decreased body weight gain compared to controls. Survival
time oI rats Ied St. John`s Wort was not decreased. No signiIicant tissue lesions were Iound.
Liver copper levels were not directly aIIected, and no major eIIects on liver zinc or iron levels
were observed (Garrett et al., 1982).
Groups oI 3 sheep were given Iresh St. John`s Wort at doses oI 0, 4, 8, 12 or 16 g plant/kg
bw/day Ior up to 14 days and exposed to day light. The amount oI hypericin in the Ieed was not
determined. EIIects observed in all animals aIter 7 and 14 days oI treatment included

111
restlessness, photophobia, tachycardia, polypnoea, congested mucous membranes, diarrhoea,
hyperthermia, skin redness oI exposed parts oI tail and legs, oedema oI the eyelids and swelling
and loss oI serum Irom the ears. Symptoms progressed aIter one week, with eIIects including
crusts and ulcers oI the skin, salivation, alopecia oI the Iace and around ears and eyes, severe
congestion oI mucous membranes, keratoconjunctivitis, loss oI eyelashes, corneal opacity and
blindness. A decrease oI haemoglobin, red blood cell count and packed cell volume was
observed in all treated animals. Total protein, glucose, cholesterol, triglycerides and serum
alkaline phosphatase activity were all decreased. Blood urea nitrogen, sodium, potassium,
bilirubin, and activities oI aspartate aminotransIerase, alanine aminotransIerase, lactate
dehydrogenase and gamma glutamyltransIerase were all increased (Kako et al., 1993).
Reproductive toxicity / teratogenicity: No adequate studies Iound. In a preliminary study
which was only published as an abstract, reduced litter size and reduced body size at birth were
observed when 25 CD-1 mice were dosed with approximately 136 mg dried St. John`s Wort/kg
bw/day via their diet (equivalent to approximately 0.4 mg hypericin/kg bw/day) Irom 2 weeks
beIore mating throughout gestation (Gonzalez et al., 1998).
Forty Iemale CD-1 mice were randomised to receive either 0 or approximately 180 mg St.
John`s Wort/kg bw/day (equivalent to approx. 0.54 mg hypericin/kg bw/day) in their diet Irom 2
weeks beIore mating through gestation. The impact oI Hvpericum on certain cognitive tasks was
tested in the oIIspring. No signiIicant diIIerences in Iinal perIormances in various
neurodevelopment trials were observed, although exposed Iemale oIIspring took longer to learn
the Morris maze task than non-exposed oIIspring (Rayburn et al., 2001).
Mutagenicity / genotoxicity: In vitro: Hypericin was negative in an Ames test with Salmonella
tvphimurium strains TA98 and TA100 with and without metabolic activation (Turek et al.,
1997). St. John`s Wort extract gave a negative result in an HPGRT test in Chinese hamster V79
cells with and without metabolic activation, in an unscheduled DNA assay in rat hepatocytes
and in a Syrian hamster embryo cell assay with an without metabolic activation (Okpanyi et al.,
1990). However, phototoxicity and a slight increase (doubling) oI the number oI micronucleated
cells was observed in Chinese hamster V79 cells exposed to 100 and 158 ng hypericin/ml and
irradiated with 300/10 mJ UVA/UVB per cm
2
. Lower concentrations oI 10-30 ng/ml exerted no
eIIect whereas higher concentrations oI 320-3200 ng/ml were cytotoxic (Kersten et al., 1999).
In vivo: St. John`s Wort extract was negative in a mouse Iur spot test and a bone marrow
chromosome assay in mice (Okpanyi et al., 1990). An in vivo mouse micronucleus test with St.
John`s Wort extract was positive but showed no dose-relationship. Since no Iurther details were
provided the relevance oI this observation cannot be assessed (Turek et al., 1997).

Human data: In a review article the incidence oI adverse eIIects amongst people taking
preparations oI St. John`s Wort was examined and adverse reactions oI the skin exposed to light
were described as the most common adverse eIIect (1 per 300`000 cases treated with St. John`s
Wort preparations). Less common, potentiation oI coumarin-type anticoagulants, breakthrough
bleeding in women taking contraceptive pills, gastrointestinal eIIects and reduced cyclosporine
levels in organ transplant patients occurred. From the reviewed results oI investigations in
volunteers it was concluded that the threshold dose Ior an increased risk oI photosensitisation is
about 2-4 g/day oI a usual commercial Hvpericum extract (equivalent to approximately 5-10 mg
hypericin/day and 80-170 micrograms hypericin/kg bw/day) (Schulz, 2001).
In a study in which volunteers with hepatitis C were orally administered 50 or 100 micrograms
hypericin/kg bw/day Ior 8 weeks (hypericin as de novo synthesized substance), signs oI
phototoxicity were reported at both dose levels (5/12 subjects receiving the lower dose, and
6/7 subjects receiving the higher dose). EIIects included dermatitis, burning and/or tingling
sensations in the skin. Three volunteers in the higher dose group showed darkened coloration oI

112
exposed skin and one patient had pruritic nodules. All symptoms resolved Iollowing
discontinuation oI hypericin treatment (Jacobson et al., 2001).
No adverse skin reactions were reported in a single-dose pharmacokinetics study in which
12 volunteers were given oral doses oI St. John`s Wort extract (standardized dried extract LI
160), equivalent to hypericin intakes oI 0.25, 0.75 or 1.5 mg (corresponding to 4.2, 13 or 25
micrograms hypericin/kg bw iI a bodyweight oI 60 kg is assumed) (Kerb et al., 1996).
Three HIV-inIected adults were given oral doses oI 50 micrograms hypericin/kg bw/day
(hypericin as de novo synthesized substance) in a phase I clinical trial and all withdrew Irom the
trial within the Iirst 8 weeks due to phototoxicity. The reaction resolved in all patients Iollowing
cessation oI treatment (Gulick et al., 1999).
In a placebo-controlled randomised double-blind trial to test the potential oI St. John`s Wort
extract to produce photosensitivity, each oI 13 volunteers received a single dose oI 0, 900, 1800
or 3600 mg St. John`s Wort extract LI 160 (equivalent to hypericin intakes oI approximately 0,
18, 36 and 73 micrograms/kg bw iI a bodyweight oI 60 kg is assumed). BeIore and 4 hours aIter
dosing, small areas oI the backs oI volunteers were exposed to solar simulated irradiation
(containing UVA and UVB light), and, in another area, to UVA light only. A slight reduction in
the median minimal tanning dose oI UVA was observed at the highest dose oI hypericin. No
eIIect oI hypericin on the minimal dose oI solar simulated light or UVA only to produce
erythema was observed (Brockmller et al., 1997).
In a repeated-dose study, 50 volunteers were given oral doses oI 600 mg/day oI St. Johns Wort
extract (equivalent to approximately 36 micrograms hypericin/kg bw/day based on a body
weight oI 60 kg) Ior 15 days. At the end oI the trial, a slight reduction in the median minimal
dose oI solar simulated irradiation required to produce erythema and a 21 reduction in the
mean minimal tanning dose oI UVA were observed, compared to beIore dosing (Brockmller et
al., 1997).
St. John`s Wort extract (standardized dried extract LI 160) was either given orally to
24 volunteers at an initial dose corresponding to 90 micrograms hypericin/kg bw Iollowed by
45 micrograms hypericin/kg bw/day Ior 7 days or once orally to 48 volunteers at doses oI 90 or
180 micrograms hypericin/kg bw. Prior to dosing and 6 hours Iollowing dosing the volunteers
were tested on their Iorearms Ior skin sensitivity to UVB, UVA, visible light or solar simulated
irradiation. Erythema index and melanin-index was assessed using a mexameter. In the
repeated-dose study, a marginal eIIect on UVB-induced pigmentation (p0.0471) and a possible
marginal eIIect on visible-light induced erythema (p0.0568) was observed. These eIIects were
not dose-related. In the single dose study, there were no apparent eIIects on erythema or
pigmentation (Schempp et al., 2003).
Other adverse eIIects have been in limited reports oI clinical trials oI St. John`s Wort Ior
treating depression, including dry mouth, dizziness, gastrointestinal symptoms, skin redness
with pruritis, tiredness with Iatigue and other unspeciIied symptoms. Estimated hypericin
intakes ranged Irom approximately 6.7 to 45 micrograms/kg bw/day Ior treatment periods oI 2
to 12 weeks (Linde et al., 1996; SCF, 2002; Schrader et al., 1998).
Furthermore, 5 cases oI adverse eIIects were reported in elderly persons in the USA who
combined the use oI St. John`s Wort extract with prescription antidepressants. Four oI the cases
were using a serotonin re-uptake inhibitor when they started taking 600-900 mg St. John`s Wort
extract/day. Within 2 to 4 days, they developed symptoms including nausea, vomiting,
conIusion and restlessness. The symptoms were diagnosed as being the result oI a central
serotonin excess or serotonin syndrome`, characterised by Lantz et al. (1999).

Other studies: Mechanism of toxicitv. Hypericin produced singlet oxygen in vitro when
irradiated with light. This is thought to be at least partly responsible Ior the photosensitive and

113
phototoxic eIIects oI hypericin (Ehrenberg et al., 1998; Fernandez et al., 1997; Wills et al.,
2001).
Induction of en:vme activitv. Hypericin inhibited CYP2C9, CYP2D6, CYP3A4 and dopamine-
beta-hydroxylase in vitro (Obach, 2000; Denke et al., 2000). It also inhibited CYP1A1-catalysed
diolepoxide-2-Iormation Irom benzo|a|pyrene-7,8-dihydrodiol (Schwarz et al., 2003). However,
in vivo, St. John`s Wort induced CYP3A4 activity (twoIold increase) but had no signiIicant
eIIect on CYP2D6 in healthy human volunteers (6 men, 6 women) receiving 900 mg St. John`s
Wort extract per day (containing 0.3 hypericin, equivalent to 45 micrograms hypericin/kg
bw/day) Ior 16 days (Roby et al., 2000; Markowitz et al., 2003).
Psvchotropic effects and MAO-inhibition. Monoaminooxidase (MAO) inhibition has been
proposed as a possible mechanism by which St. John`s Wort exhibits an antidepressant activity.
MAO was inhibited in vitro by Hvpericum Iractions either oI high or low hypericin content, and
it is suggested that other components than hypericin with known MAO-inhibiting properties
(e.g. xanthone derivatives) could be responsible Ior this eIIect (Bladt and Wagner, 1994; Thiede
and Walper, 1994; Suzuki et al., 1981; Demish et al., 1989). Hypericin on its own did not
signiIicantly aIIect MAO activity in either in vitro or ex vivo studies (Bladt and Wagner, 1994).
However, in another study with commercially available hypericin (80 purity only) a 50
irreversible inhibition oI rat brain mitochondrial type A and type B monoamine oxidase (MAO)
was shown in vitro at concentrations oI 68 and 420 micromoles/l (e.g. 34 and 212
micrograms/ml, respectively) (Suzuki et al., 1984). Up to now there is no conclusive evidence
on whether hypericin has MAO-inhibiting potency and whether it is responsible Ior the
psychotropic activity oI Hvpericum.
In vitro studies of reproductive toxicitv: The potential Ior hypericin to cause teratogenicity was
investigated in vitro using a whole rat embryo culture model. Rat embryos were explanted at
gestation day 9.5, cultivated in vitro Ior 48 hours in medium containing 0, 14.2, 28.4, 71 or 142
ng hypericin/ml and then examined. Embryos exposed to 71 or 142 ng hypericin/ml had a
signiIicantly lower total morphological score and number oI somites than controls (p0.05).
Trend analysis showed a negative linear trend Ior total morphological score (p0.001), number
oI somites (p0.001) and crown-rump length (p0.01), but not Ior yolk sac diameter (Chan et
al., 2001).
Potential to cause cataracts. Hypericin at a concentration oI 50 micromoles/l (e.g.
25 micrograms/ml) caused polymerization oI calI lens alpha-crystalline only when exposed to
light. Mass spectrometry indicated oxidation oI methionine, tryptophan and histidine residues,
which increased with irradiation time (Schey et al., 2000).

TOXICOLOGICAL EVALUATION: Data on biotransIormation, elimination and toxicity oI
hypericin is limited. From various studies there is evidence that Hvpericum induces enhanced
photosensitivity, both in animals and in humans. Several clinical studies with human volunteers
have shown that the lowest doses leading to adverse skin reactions are in the range oI 25 to
50 micrograms hypericin/kg bw/day. The available data indicate that single dose administration
is tolerated at higher hypericin levels than repeated-dose administration. Overall, a LOAEL oI
25 micrograms hypericin/kg bw/day was set Ior the observed adverse skin reactions in humans.
Other side eIIects were not reported at the above mentioned dose levels.
Limited genotoxicity studies with hypericin or Hvpericum extract provided only negative
results. Data on chronic toxicity and carcinogenicity are not available. Reproductive toxicity
studies are limited to a preliminary study on neurobehavioral developmental toxicity in mice
indicating that Hvpericum may elicit adverse eIIects on Ioetal physical development at very
high doses oI 136 mg/kg bw/day.
It has been demonstrated that St. John`s Wort extract may have psychotropic and in particular
an anti-depressing activity. The current knowledge does not yet explain the mechanism oI the

114
observed psychotropic activity oI Hvpericum and Hvpericum extract and it has been impossible
to attribute this activity to any oI its particular constituents and certainly not to hypericin.
Hvpericum extract (900 mg Hvpericum extract/day Ior 16 days, equivalent to 45 micrograms
hypericin/kg bw/day) induced CYP3A4 activity (twoIold increase) in healthy volunteers, but
had no signiIicant eIIect on CYP2D6.
The available data indicate that photosensitivity and enzyme induction are the critical endpoints
in animals and most possibly also in humans. Based on a LOAEL oI
36 micrograms hypericin/kg bw/day Ior enhanced photosensitivity and phototoxicity in humans
derived Irom the most reliable repeated-dose study in healthy volunteers (Brockmller et al.,
1997) a TMDI oI 0.002 mg hypericin/kg bw/day was established using a saIety Iactor oI 20. The
saIety Iactor oI 20 comprises a Iactor oI 10 Ior interindividual diIIerences and a Iactor oI 2 Ior
the use oI a LOAEL instead oI a NOAEL, taking into account that the observed eIIects were
minor skin reactions.

TMDI: 0.002 mg/kg bw/day.

MAIN OCCURRENCE: Hypericin is a napthodianthrone (anthraquinone derivative) which
occurs in Hvpericum perforatum L. (St. John`s Wort) at concentrations oI 0.0095 to 0.466 in
the whole plant (Duke, 1989) and 0.02-0.18 in dried Ilowers (Hlzl and Ostrowski, 1987).
Drying St. John`s Wort can reduce the hypericin content by up to 80 (Araya and Ford, 1981).
Other major constituents in Hvpericum are tannins (up to 16), hyperoside, hyperIorin,
adhyperIorin and Ilavonoids (up to 4), pseudohypericin and pinene (up to 0.6). Only traces
oI xanthones have been reported in Hvpericum (USDA, 2004).
Most clinical trials have been conducted with LI 160/PK tablets (commercial product Jarsin),
containing 300 mg oI a Hvpericum extract standardized to 0.3 hypericin.

INTAKE ESTIMATION: Very limited data on the use oI St. John`s Wort as a Ilavouring
substance are available. The plant and its preparations are believed to be no major Iood sources,
but used as Ilavourings in some liqueurs. II it is assumed that liqueurs are the only source oI
hypericin, that all liqueurs contain hypericin at the current maximum limit oI 10 mg/kg
permitted according to EU Directive 88/388/EEC (EEC, 1988), and that 42.4 g oI liqueurs are
consumed per day (high intake level Irom a UK survey), the intake oI hypericin would be 0.424
mg/day, equivalent to 7.1 micrograms/kg bw/day. II the new limit oI 2 mg/kg set by the Council
oI Europe Ior hypericin in alcoholic beverages is applied, the intake oI high level liqueur
consumers (42.4 g/day) would be 0.085 mg/day, equivalent to 1.4 micrograms/kg bw/day, and
the intake oI mean consumers (10 g liqueurs/day) would be 0.020 mg/day, equivalent to 0.33
micrograms/kg bw/day. Hypericin-containing Hvpericum extracts or dried plant products are
used in herbal teas and in Over-The-Counter (OTC) anti-depression medication. A herbal tea
marketed in the Netherlands has been reported to contain 2 g St. John`s Wort (dried leaves) per
tea bag, providing an intake oI about 250 microgram hypericin per cup (SCF, 2002). Consumers
are advised to take 1 to 2 cups, 3 times a day, and may thus be exposed to a maximum level oI
1500 micrograms hypericin/day, equivalent to 25 micrograms hypericin/kg bw/day.
Consumption oI one cup per day results in a intake oI 4.2 micrograms hypericin/kg bw/day Ior a
person oI 60 kg body weight.

CONCLUSIONS: Consumption oI liqueurs containing the maximum level oI 10 mg
hypericin/kg permitted according to EU Directive 88/388/EEC, may result in hypericin intakes
in high level consumers which are by a Iactor oI 4 above the TMDI. At the maximum level oI
2 mg hypericin/kg in alcoholic beverages recommended by Council oI Europe, the TMDI will
not be exceeded neither in high level liqueur consumers nor in mean consumers. However, the

115
TMDI may be largely exceeded in individuals consuming one or more cups per day oI a herbal
tea prepared Irom dried leaves oI Hvpericum perforatum L..

DATA NEEDED: Further studies on metabolism and subchronic and possibly chronic toxicity,
a validated study assessing the photogenotoxicity, as well as Iurther studies on the
photosensitivity in humans, preIerably using the pure substance are needed. Data on any other
Iood uses oI St. John`s Wort are also required.

LIMITS: (mg/kg)

a

a

Exceptions:

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Schwarz, D., Kisselev P. and Roots I. (2003) St. John`s Wort extracts and some oI their
constituents potently inhibit ultimate carcinogen Iormation Irom benzo|a|pyrene-7,8-
dihydrodiol by human CYP1A1. Cancer Res, 63, 8062-8068.
Suzuki, O., Katsumata Y. and Oya M. (1981) Inhibition oI type A and type B monoamine
oxidases by naturally occurring xanthones. Planta Medica, 42, 17-21.
Suzuki, O., Katsumatra Y., Oya M. (1984) Inhibition oI monoamine oxidase by hypericin.
Planta Medica, 46, 272-274.
Thiede, H.M. and Walper A. (1994) Inhibition oI MAO and COMT by Hypericum extracts and
hypericin. J. Geriatr. Psychiatry Neurol., 7 (S 1), 54-56.
Turek, B., Barta I., Smerak P., Kovacova E., Sedmikova M. and Sestakova H. (1997)
|Mutagenic activity oI substances oI plant origin|. Potrav. Vedy, 15, 271-288.
USDA, ARS, National Genetic Resources Program. (2004) Phytochemical and Ethnobotanical
Databases. |Online Database| National Germplasm Resources Laboratory, Beltsville,
Maryland. 09 September 2004.
Vandenbogaerde, A., Zanoli P., Puia G., Truzzi C., Kamuhabwa A., De Witte P., Merlevede W.
and Baraldi M. (2000) Evidence that total extract oI Hypericum perIoratum aIIects
exploratory behavior and exerts anxiolytic eIIects in rats. Pharmacol. Biochem. Behav., 65,
627-633.
Wills, N.J., Park J., Wen J., Kesavan S., Kraus G.A., Petrich J.W. and Carpenter S. (2001)
Tumor cell toxicity oI hypericin and related analogs. Photochem. Photobiol., 72, 216-220.

DATABASES USED: Medline (1966-2003), Toxline (1969-2003). Keywords: hypericin, St.
John`s Wort, Hypericum, toxicity.

118

119
Isosafrole


SYNONYMS: 5-(1`-Propenyl)-1,3-benzodioxole; 1,2-(methylenedioxy)-4-(1`-propenyl)
benzene

CAS No: 120-58-1

STRUCTURE:

O O

O O
(Z)

(E) - IsosaIrole (Z) IsosaIrole

IsosaIrole exists as a trans-(E-)isomer (beta-isosaIrole, CAS No. 4043-71-4) and as a
cis-(Z-)isomer (alIa-isosaIrole, CAS No.17627-76-8).

REGULATORY / INTERNATIONAL STATUS: IsosaIrole is carcinogenic in mice and rats,
producing liver tumours Iollowing its oral administration (IARC, 1976). IARC concluded in
1987 Ior isosaIrole that there were no adequate human carcinogenicity data but limited evidence
oI animal carcinogenicity, and isosaIrole was classiIied in group 3, not classiIiable as to its
carcinogenicity Ior humans` (IARC, 1987). In 1981 JECFA also concluded that isosaIrole is
carcinogenic in rats and mice and no ADI was allocated (WHO, 1981).
In 1999, the Council oI Europe Committee oI Experts on Flavouring Substances was inIormed
by the International Organisation oI Flavour Industry (IOFI) that isosaIrole does not occur in
any natural source material Ior Ilavourings used by the Ilavour industry and thereIore the
Committee deleted isosaIrole Irom the list oI active principles (Council oI Europe, 1999). In
2002, the Council oI Europe Committee oI Experts on Flavouring Substances was inIormed that
isosaIrole does occur in relevant source materials Ior Ilavourings and decided to reconsider
isosaIrole at its next meeting (Council oI Europe, 2002).

120
IsosaIrole is listed in the Directive 88/388/EEC on Ilavourings in Annex II with maximum
limits Ior isosaIrole (and saIrole) oI 1 mg/kg in IoodstuIIs and beverages with exceptions oI
2 mg/kg and 5 mg/kg in alcoholic beverages with not more than and with more than 25
volume oI alcohol, respectively, and 15 mg/kg in IoodstuIIs containing mace and nutmeg (EEC,
1988). SCF considered isosaIrole as weakly hepatocarcinogenic in rodents, probably mediated
by a non-genotoxic mechanism. A NOEL could not be derived Ior the hepatic eIIects in the
long-term studies and thus no TDI was established (SCF, 2003).


Metabolism:
studies: In epithelial cells Irom adult rat liver, the major metabolite was 1`,2`-dihydro-
1`,2`-dihydroxyisosaIrole with lesser amounts oI 1`,2`-epoxyisosaIrole and 1`-hydroxysaIrole.
The metabolites were identiIied by gas chromatography-mass spectrometry, but no quantitative
data are given (Janiaud et al., 1976).
Animal studies: At 1 mmol (162 mg) isosaIrole (cis-trans mixture`)/kg bw given to three male
albino rats (Wistar strain derived) by stomach tube, metabolite excretion accounted Ior 89 oI
the dose in urine in 72 hours. Demethylenation leading mainly to 1,2-dihydroxy-4-(1`-
propenyl)benzene was the most prominent reaction (92 oI the urinary metabolites were
demethylenated) but also allylic hydroxylation and epoxide-diol pathway took place. Allylic
hydroxylation took place at the 3`-position, but this was a minor pathway in the present rat
study. Only 1.3 oI the dose was recovered as 3`-hydroxyisosaIrole and, contrary to the above
in vitro study, no 1`-hydroxysaIrole was detected (Klungsoyr and Scheline, 1982). Although
Klungsoyr and Scheline (1982), contrary to Janiaud et al. (1976) in the above in vitro study,
could not detect any 1`-hydroxysaIrole, they did not exclude that some Iormation oI this
metabolite may occur when very large doses oI isosaIrole are administered. This is supported by
the study by Peele and Oswald (1978) who did in Iact demonstrate excretion oI traces oI 1`-
hydroxysaIrole in the urine oI rats to which they had administered 3`-hydroxyisosaIrole. It has
also been shown that 1`-hydroxysaIrole may undergo chemical rearrangement to 3`-
hydroxyisosaIrole (Borchert et al., 1973b).

Toxicology:
50
oI 2.47 g/kg bw was reported in mice and oI 1.34 g/kg bw in rats
(Jenner et al., 1964; Hagan et al., 1965).
Subacute / subchronic toxicity: Rats (Osborne-Mendel strain, 10 males and 10 Iemales) given
10000 mg isosaIrole/kg in the diet showed growth retardation in both sexes. No rats survived
beyond 11 weeks oI treatment. The livers were enlarged and microscopically slight hepatic cell
hypertrophy, which was usually Iocal and resulted in the Iormation oI nodules, was shown
(Hagan et al., 1965; Hagan et al., 1967).
Rats (3 males and 3 Iemales) given daily doses oI 460 mg isosaIrole/kg bw by stomach tube Ior
Iour days produced severe liver lesions consisting oI discolouration, enlargement, and loss oI
normal texture. No histopathology was perIormed. Two oI the rats died during the test period.
Apparently, no control group was included in the study (Taylor et al., 1964). Oral intubation to
young male and Iemale Osborne-Mendel rats oI 500 mg isosaIrole/kg bw/day Ior 41 days and oI
250 mg isosaIrole/kg bw/day Ior 34 days resulted in mortality ratios oI 8/10 rats and oI 2/10
rats, respectively. All ten rats oI the control group survived. The Iollowing eIIects were
observed: liver hypertrophy and slight Iocal necrosis and Iibrosis, slight degree oI Iocal Iatty
metamorphosis and bile duct proliIeration (Hagan et al., 1965).

121
Chronic toxicity / carcinogenicity: Two strains oI mice (C57BL/6 x C3H/AnI)F
1
and
(C57BL/6 x AKR) F
1
(18 males and 18 Iemales per group) isosaIrole (vehicle: water) were
given 215 mg/kg bw by stomach tube Irom day 7 to day 28 oI age, then subsequently Ied ad
libitum at 517 mg/kg diet Ior up to 82 weeks. The study included control groups oI up to 18
males and up to 18 Iemales per strain. Liver cell tumours occurred in 5/18 males and 1/16
Iemales and in 2/17 males and 0/16 Iemales, pulmonary tumours in 3/18 males and 1/16 Iemales
and in 0/17 males and 0/16 Iemales, and lymphomas in 1/18 males and 0/16 Iemales, and in
1/17 males and 0/16 Iemales, oI the two strains, respectively. The diIIerence Irom controls was
only statistically signiIicant Ior the liver tumours (p0.05) in (C57BL/6 x C3H/AnI)F
1
mice
(males and Iemales combined) (Innes et al., 1969). No hepatocarcinogenic activity was Iound in
male B6C3F1 mice given a single i.p. injection (solvent: trioctanoin) oI isosaIrole (52 cis-
/48 trans-isomer) to a group oI 29 animals or trans-isosaIrole (90 trans-/10 cis-isomer) to
a group oI 32 animals, at 12 days oI age (dose: 0.75 mmol/kg bw equal to 122 mg/kg bw) and
killed at 10 months. Thirty-two animals, only given the solvent, served as a control group
(Wiseman et al., 1987).
IsosaIrole was given in the diet Ior two years to Osborne-Mendel rats at 0, 1000, 2500 or 5000
mg isosaIrole/kg (control group: 35 males and 35 Iemales). At the two lowest dose levels (10
males and 10 Iemales per group) there was slight growth retardation reported in Iemales.
Microscopically, slight hepatic cell hypertrophy but no primary hepatic tumours were reported.
At 2500 mg/kg in the diet there was also slight hyperplasia in the thyroid. At the highest dose oI
5000 mg/kg in the diet (25 males and 25 Iemales) growth retardation was reported in both sexes.
The liver was enlarged and microscopically slight hepatic cell hypertrophy which was usually
Iocal and resulted in the Iormation oI nodules was shown. Five rats had primary hepatic tumours
(two adenomas and three carcinomas). Slight hyperplasia in the thyroid and an increase in the
incidence oI chronic nephritis were demonstrated. An increased number oI interstitial cell
tumours was Iound in testes. The study is poorly reported, and it does not allow a clear NOEL to
be established (Hagan et al., 1965; Hagan et al., 1967). No local tumours were observed in 18
male rats (Charles River CD, random bred) given a total oI 20 (twice weekly) s.c. injections,
each oI 3 mg isosaIrole (trioctanoin solution) per rat. The rats were examined aIter surviving 18
months (Borchert et al., 1973a).
Mutagenicity / genotoxicity: In vitro. IsosaIrole (19.7 cis / 78.2 trans-isomer) did not
induce gene mutations in Salmonella tvphimurium strains TA 98, TA 100, TA 1535, TA 1537
and TA 1538 or in Escherichia coli WP 2 uvr A with or without S9. It was negative in a
Bacillus subtilis DNA repair test (Rec assay) without S9 (Sekizawa and Shibamoto, 1982).
IsosaIrole did not induce UDS in cultured rat hepatocytes, in contrast to saIrole, estragole and
methyleugenol (Howes et al., 1990). DNA adduct Iormation using
32
P post-labelling analysis
was studied in livers Irom adult Iemale CD1 mice, isolated 24 hours aIter i.p. administration oI
diIIerent alkenylbenzenes, including isosaIrole (2 or 10 mg/mouse). AIter administration oI
isosaIrole, only low binding to the mouse liver DNA was demonstrated, with the two major
DNA adducts Iormed in the N2-position oI guanine. The low DNA binding could also be
expressed by the covalent binding index (CBI) value oI about 1 Ior isosaIrole. For comparison,
the CBI values Ior saIrole, estragole and methyleugenol were all about 30 (Randerath et al.,
1984).


Other studies: IsosaIrole is a known inducer oI some oI the liver enzymes oI the cytochrome P-
450 group in rodents, especially CYP1A2 (cI. Ryan et al., 1980; Waxman et al., 1985; Murray
and Reidy, 1989; Ishida et al., 1998; Allis et al., 2002).

122
TOXICOLOGICAL EVALUATION: IsosaIrole is an eIIicient inducer oI some oI the liver
cytochrome P450s, and is a weak liver carcinogen in rats and mice. Liver DNA adduct
Iormation (
32
P post-labelling) is low. IsosaIrole gave negative results in the reported
genotoxicity tests. These data provide support Ior a non-genotoxic mechanism oI
hepatocarcinogenicity associated with hepatic enzyme induction, but the available data Irom
carcinogenicity studies in mouse and rat do not allow the derivation oI a clear NOEL. Finally, it
cannot be completely excluded that high exposure to isosaIrole may give rise to some
isomerisation oI 3`-hydroxyisosaIrole to 1`-hydroxysaIrole, the anticipated proximate
carcinogen oI saIrole. As no NOAEL could be established no MDI or TMDI could be set.


MAIN OCCURRENCE: According to the inIormation received Irom industry, natural source
materials Ior Ilavourings used by the Ilavour industry do not contain isosaIrole (Council oI
Europe, 1999a). Former inIormation on the possible occurrence oI isosaIrole in ylang-ylang oil
(Cananga odorata (Lam.) Hook I. et Thoms., CE No. 103), Dong Quai roots (Angelica sinensis
(oliv.) Diels.; 'danggui', no CE No.) and bark essential oil oI sassaIras (Sassafras officinale Th.
Nees et Eberm., syn. Sassafras albidum (Nutt.) Nees, S. albidum (Nutt.) Nees var. molle (RaI.)
Fern., CE No. 424) (Council oI Europe, 1999b) could not be conIirmed (MAFF, 1996; Carlson
and Thompson, 1997). On the other hand, small amounts oI isosaIrole were recently Iound in
some samples oI nutmeg oils (range 0.1-3.4) and oleoresins (range 0.1-2.7) (MAFF,
1996). In contrast to this report Lawrence (1990) did not Iind isosaIrole in nutmeg oil. In a study
by Adam and Postel (1992) isosaIrole was Iound in trace amounts (0.01 and 0.03 mg/kg) in two
out oI 24 alcoholic beverages. According to available data isosaIrole only occurs sporadically
and then together with saIrole but at much lower concentrations than saIrole, roughly an order
oI magnitude lower (MAFF, 1996). It has thereIore been speculated, whether isosaIrole occurs
as an arteIact, Iormed due to heating during the analytical process and/or during the preparation
oI source material extracts, essential oils, processed Ioods and others, containing saIrole. This
supposition is backed up by the Iact that isosaIrole can be produced by isomerisation oI saIrole
under hot alkaline conditions (Bert, 1941; Naves and Ardizio, 1957) and hence the possibility
that traces oI isosaIrole may be Iormed even under neutral conditions (Council oI Europe,
1999a).

INTAKE ESTIMATION: A rough intake estimate Ior isosaIrole could be based on the
estimated average saIrole intake (Ior consumers only) (SCF, 2001), assuming that the intake oI
isosaIrole is one tenth oI the saIrole intake. Using this assumption, the estimated average per
capita intake oI isosaIrole amounts to 0.03 mg/day and the 97.5 percentile to 0.05 mg/day.

CONCLUSIONS: According to the available data isosaIrole occurs together with saIrole, but
at much lower concentrations. ThereIore measures to restrict saIrole would also restrict
exposure to isosaIrole.

DATA NEEDED: -

LIMITS: It is not considered necessary to set limits Ior isosaIrole as its main intake would be
restricted via the limits set Ior saIrole.

123
REFERENCES:
Adam, L. and Postel, W. (1992) Bestimmung von alpha- und beta-Thujon, SaIrol, IsosaIrol,
beta-Asaron und Cumarin in weinhaltigen Getrnken und Spirituosen. Die
BranntweinwirtschaIt, 132, 202-206.
Allis, J.W., Anderson B.P., Zhao G., Ross T.M. and Pegram R.A. (2002) Evidence Ior the
involvement oI CYP1A2 in metabolism oI bromodichloromethane in rat liver. Toxicology,
176, 25-37.
Bert, M.L. (1941) Sur une nouvelle methode generale de synthese des essences allylique et
propionylique. Compt. Rend., 213, 873-874.
Borchert, P., Miller J.A., Miller E.C. Shires and T.K. (1973a) 1`-HydroxysaIrole, a proximate
carcinogenic metabolite oI saIrole in the rat and mouse. Cancer Res., 35, 590-600.
Borchert, P., Wislocki P.G., Miller J.A. and Miller E.C. (1973b) The metabolism oI the
naturally occurring hepatocarcinogen saIrole to 1`-hydroxysaIrole and the electrophilic
reactivity oI 1`-acetoxysaIrole. Cancer Res., 33, 575-589.
Carlson, M. and Thompson R.D. (1997) Liquid chromatographic determination oI saIrole in
sassaIras-derived herbal products. J. AOAC, 80, 1023-1028.
Council oI Europe (1999a) Committee oI Experts on Flavouring Substances. IsosaIrole intake
Irom natural source materials RD 4.11/1-45. Document submitted by IOFI.
Council oI Europe (1999b) Committee oI Experts on Flavouring Substances. Occurrence oI
IsosaIrole in plants RD 4.2/25-44.
Council oI Europe (2002) Committee oI Experts on Flavouring Substances. 50th Session.
Record.
the member States relating to Ilavourings Ior use in IoodstuIIs and to source materials Ior
Hagan E.C., Jenner P.M., Jones W.I., Fitzhugh O.G., Long E.L., Brouwer J.G. and Webb W.K.
(1965) Toxic properties oI compounds related to saIrole. Toxicol. Appl. Pharmacol., 7, 18-
24.
Hagan, E.C., Hansen W.H., Fitzhugh O.G., Jenner P.M., Jones W.I., Taylor J.M., Long E.L.,
Nelson A.A. and Brouwer J.B. (1967) Food Ilavourings and compounds oI related
structure. II. Subacute and chronic toxicity. Food Cosmet. Toxicol., 5, 141-157.
Howes, A.J., Chan V.S.W. and Caldwell J. (1990) Structure-speciIicity oI the genotoxicity oI
some naturally occurring alkenylbenzenes determined by the unscheduled DNA synthesis
assay in rat hepatocytes. Food Chem. Toxicol., 28, 537-542.
IARC (1976) IARC Monographs on the Evaluation oI the Carcinogenic Risk oI Chemicals to
Man: Some Naturally Occurring Substances, 10, 231-244.
IARC (1987) IARC Monographs on the Evaluation oI the Carcinogenic Risk oI Chemicals to
Humans: Overall Evaluations oI Carcinogenicity: An Updating oI IARC Monographs
Volumes 1-42, Supplement 7, 65.
Innes, J.M., Ulland B.M., Valerio M.G., Petrucelli L., Fishbein L., Hart E.R., Pallotta A.J.,
Bates R.R., Falk H.L., Gart J.J., Klein M., Mitchell I. and Peters, J. (1969) Bioassay oI
pesticides and industrial chemicals Ior tumorigenicity in mice: A preliminary note. J. Nat.
Cancer Inst., 42, 1101-1114.
Ishida, T., Tasaki K., Fukuda A., Ishii Y. and Oguri K. (1998) Induction oI a cytosolic 54 kDa
protein in rat liver that is highly homologous to selenium-binding protein. Environ.
Toxicol. Phar., 6, 249-255.
Janiaud, P., DelaIorge M., Levi P., Maume B.F. and Padieu P. (1976) Etude comparative en
culture cellulaire de Ioie de Rat du metabolisme de diIIerents analogues et metabolites dùn
hepatocancerogene naturel: le saIrol. CR Soc. Biol., 170, 1035-1041.

124
Jenner, P.M., Hagan E.C., Taylor J.M., Cook E.L. and Fitzhugh O.G. (1964) Food Ilavourings
and compounds oI related structure. I. Acute oral toxicity. Food Cosmet. Toxicol., 2, 327-
343.
Klungsoyr, J. and Scheline R.R. (1982) Metabolism oI isosaIrole and dihydrosaIrole in the rat.
Biomed. Mass Spectrom., 9, 323-329.
Lawrence, B.M. (1990) Progress in essential oils. PerIumer and Flavorist, 15, 63-69.
MAFF (1996) Ministry oI Agriculture Fisheries and Food, Food Surveillance Paper No. 48,
Flavourings in Food, London, HMSO.
Murray, M. and Reidy G.F. (1989) In vitro Iormation oI an inhibitory complex between an
isosaIrole metabolite and rat hepatic cytochrome P-450 PB-B. Drug Metab. Dispos., 17,
449-454.
Naves, Y.-R. and Ardizio P. (1957) Etudes sur les matieres vegetales volatiles CXLVII(I) Sur
les cis et trans-isosaIroles. Bull. Soc. Chim. France, 1053-1057.
Peele, J.D. and Oswald E.O. (1978) Metabolism oI the proximate carcinogen 1`-hydroxysaIrole
and the isomer 3`-hydroxyisosaIrole. Bull. Environ. Contam. Toxicol., 19, 396-402.
32
P-Post-labelling analysis
oI DNA adducts Iormed in the livers oI animals treated with saIrole, estragole and other
naturally-occurring alkenylbenzenes. I. Adult Iemale CD-1 mice. Carcinogenesis, 5, 1613-
1622.
Ryan, D.E., Thomas P.E. and Levin W. (1980) Hepatic microsomal cytochrome P-450 Irom rats
treated with isosaIrole. J. Biol. Chem., 255, 7941-7955.
SCF (1979) Report oI the ScientiIic Committee Ior Food on Flavourings (opinion expressed on
21 September 1979) Reports oI the ScientiIic Committee Ior Food (9
th
Series), OIIice Ior
OIIicial Publications oI the European Communities, Luxembourg, October 1979.
SCF (2001) Opinion oI the ScientiIic Committee on Food on the saIety oI the presence oI
saIrole (1-allyl-3,4- methylene dioxy benzene) in Ilavourings and other Iood ingredients
with Ilavouring, expressed 12 December 2001. Available at
http://europa.eu.int/comm/Iood/Is/sc/ scI/out116en.pdI
SCF (2003) Opinion oI the ScientiIic Committee on Food on IsosaIrole expressed 4 April 2003.
Sekizawa, J. and Shibamoto J. (1982) Genotoxicity oI saIrole-related chemicals in microbial test
systems. Mutat. Res., 101, 127-140.
Taylor, J.M., Jenner P.M. and Jones W.I. (1964) A comparison oI the toxicity oI some allyl,
propenyl, and propyl compounds in the rat. Toxicol. Appl. Pharm., 6, 378-387.
Waxman, D.J., Dannan G.A. and Guengerich F.P. (1985) Regulation oI rat hepatic cytochrome
P-450: Age-dependent expression, hormonal imprinting, and xenobiotic inducibility oI sex-
speciIic isoenzymes. Biochemistry, 24, 4409-4417.
WHO (1981) Evaluation oI Certain Food Additives. Twenty-FiIth Report oI the Joint
FAO/WHO Expert Committee on Food Additives. Technical Report Series No. 669. World
Health Organization, Geneva.
administration to preweanling male C57BL/6J x C3H/HeJ F
1
mice. Cancer Res., 47, 2275-
2283.

DATABASES USED: FSTA (1969-2002), Toxline plus (1985-2000), Biosis (1999-2002),
Medline (1999-2002). Keywords: IsosaIrol, isosaIrole.

125
Myristicin

ACTIVE PRINCIPLE: I

SYNONYMS: MethoxysaIrole, 1-allyl-5-methoxy-3,4-methylene-dioxybenzene

CAS No: 607-91-0

STRUCTURE:

O
O
O

REGULATORY / INTERNATIONAL STATUS: -


Metabolism:
studies: The metabolism oI myristicin has been investigated in several types oI in vitro
systems (Braun et al., 1973, Casida et al., 1966, Kamienski et al., 1970). In one experiment,
using perIused rat liver or rat liver homogenates, myristicin was shown to be converted to an
amphetamine derivative: 3-methoxy-4,5-methylene-dioxyamphetamine by both liver
preparations (Braun et al., 1973). Oxygenation increased the yield oI the metabolite indicating
that an oxidation reaction precedes the transamination.
Animal studies: The metabolism oI myristicin in vivo has been studied in guinea pigs, rats and
mice. Single doses oI myristicin in the range oI 5 - 20 mg/kg bw were administered i.p. to rats
and guinea pigs (Oswald et al., 1971), which resulted in the Iollowing metabolites in the urine:
1) 3-piperidyl-1-(3`-metoxy-4`,5`-methylenedioxyphenyl)-1-propanone (the major metabolite in
rats) and 2) 3-pyrrolindinyl-1-(3`-methoxy-4`,5`-methylendioxyphenyl)-1-propanone (the major
metabolite in guinea pigs). In addition, the rats and guinea pigs excreted trace quantities oI the
pyrrolidinyl ketone, and the piperidyl ketone, respectively. No amphetamine-type compounds
were detected. The proposed pathway Ior the Iormation oI these tertiary aminopropiophenones
is allylic oxidation oI the allylbenzene compounds Iollowed by condensation with a secondary
amine to produce the tertiary aminoketone.
The excretion oI
14
C-labeled myristicin has been studied in rats and mice (Casida et al., 1966,
Kamienski et al., 1970; Peele, 1976). In both species the excretion was rapid, as it was nearly
completed within 48 hours. Excretion occurred largely via the urinary route in rats (Kamienski
et al., 1970), and via the respiratory air in mice (Braun et al., 1973; Casida et al., 1966).
In mice, 61-76 oI the administered radioactivity (the methylenedioxy group was labelled) was
recovered as CO
2

in the expired air. In contrast, only 25 oI the the label was expired as CO
2

in

126
rats. Even less (2) was expired via this route when 3-
14
C-myristicin (ringlabelled myristicin)
was administered to rats (Peele, 1976). In rats very little retention oI myristicin was observed in
the tissues aIter 48 hours (Peele, 1976). OI the radioactivity remaining in the tissues halI oI the
activity was localized in the liver (Peele, 1976).
The experimental results suggest the Iollowing metabolic pathways: 1) addition oI ammonia to
the allyl group resulting in an amphetamine derivative (3-methoxy-4,5-methylenedioxy-
amphetamine) (Braun et al., 1973), 2) extensive demethylenation resulting in Iormate and the
corresponding cathecol (5-hydroxyeugenol) (Casida et al., 1966; Kamienski et al., 1970;
Peele, 1976); 3) epoxidation leading to several epoxides and their resultant diols, Ior example:
myristin glycol and Iinally a methylendioxy derivative oI phenylacetic acid (in rats) (Peele,
1976); 4) allylic hydroxylation to 1-hydroxymyristicin, 3-hydroxymyristicin and Iinally
piperonylic acid (also described as the metylendioxyderivative oI benzoic acid) (in rats) (Peele,
1976); 5) allylic oxidation Iollowed by condensation with a secondary amine to produce a
tertiary aminoketone (3-piperidyl-1-(3`-methoxy-4`,5`-methylenedioxylphenyl)-1-propanone
and 3-pyrrolidinyl-1-(3`-methoxy-4`,5`-methylene-dioxy-phenyl)-1-propanone) (in rats and
guinea pigs) (Peele, 1976; Oswald et al., 1971); and 6) possibly a reduction oI the allylic double
bond to dihydromyristicin (in rats) (Peele, 1976).
In conclusion, the metabolism oI myristicin broadly seems to resemble that oI saIrole. No
inIormation is, however, available concerning the quantitative importance oI the individual
metabolic pathways.
Human studies: No inIormation is available regarding kinetics or metabolism oI myristicin in
humans.

Toxicology:
Acute toxicity: An LD
50
~1000 mg/kg i.p. resulted Irom an experiment with 25 white male rats
(strain not mentioned). Myristicin was given intraperitoneally at dose levels Irom 200 to 1000
mg/kg bw. Large doses elicited hyperexcitability Iollowed by central nervous system depression
(Truitt et al., 1960).
Subacute / subchronic toxicity: Twelve white

rats (strain not mentioned) were administered
10 mg/kg bw/day oI myristicin daily in the Iood Ior 26 days. There were no diIIerences in body
weights between the animals receiving myristicin and controls. Histological studies oI livers and
kidneys showed no abnormalities that could be attributed to myristicin (Truitt et al., 1960).
Chronic toxicity / carcinogenicity: No chronic and carcinogenicity studies with liIe-long
adminstration oI myristicin are available. In the reIerred short-term studies, special assays have
been used, which are not regarded as conclusive but could be used as indicators oI carcinogenic
activity. Male B6C3F
1
mice were given myristicin i.p. during the preweaning period in two
separate experiments. This experimental design has, according to the authors, proved to be a
sensitive assay Ior induction oI hepatic tumours.
Myristicin was injected i.p. on days 1, 8, 15 and 22 post partum to 33 male B6C3F
1
mice. A
total dose oI 3.75 micromoles was given during the experiment. The amounts injected per dose
were in the ratio oI 1:2:4:8. The average body weights oI mice oI both sexes were 1.4 g (day 1),
3.5 g (day 8), 7 g (day 15), and 13 g (day 22). Thus, the applied doses were 0.25, 0.5, 1.0 and
2.0 micromoles/animal, equivalent to 34.3, 27.4, 27.4 and 29.5 mg myristicin/kg bw on days 1,
8, 15 and 22 post partum, respectively. The mice were weaned at 4 weeks oI age. The animals
were examined 12 months aIter the start oI the experiment. In 21 oI the mice, hepatomas were
observed in comparison with 15 oI the control mice only receiving vehicle (trioctanoin). The
average number oI hepatomas/mouse was 0.2 in mice treated with myristicin, in comparison
with 0.1 in control mice. This result was not statistically signiIicant (Miller et al., 1983).
In a second experiment carried out in 45 male B6C3F
1
mice, myristicin was injected i.p. on days
1, 8, 15 and 22 post partum. A total dose oI 4.75 micromoles was given to each animal during

127
the experiment. The amounts injected per dose were in the ratio oI 1:2:4:12. The average body
weights oI mice oI both sexes were 1.4 g (day 1), 3.5 g (day 8), 7 g (day 15), and 13 g (day 22).
Thus, the applied doses were 0.25, 0.5, 1.0 and 3.0 micromoles/animal, equivalent to 34.3, 27.4,
27.4 and 44.3 mg myristicin/kg bw on days 1, 8, 15 and 22 post partum, respectively. The mice
were weaned at 4 weeks oI age. Hepatomas were observed at 13 months (examination oI the
livers by laparotomy) and at 13 to 18 months. The average number oI hepatomas/mouse at
13 months was 0.2 and on the later occasion 0.4. Neither oI these numbers was statistically
signiIicant in comparison with the control group (0.5 hepatomas/mouse). Thus, myristicin had
no detectable activity Ior the initiation oI hepatic tumours on administration to male mice prior
to weaning (Miller et al., 1983).
In another study the eIIect oI myristicin and dihydromyristicin on benzo(alpha)pyrene (B|a|P)-
induced carcinogenicity was investigated in mice. Female A/J mice (7 weeks oI age at the
beginning oI the study) were administered orally (gavage) 1 mg oI B|a|P (in 0.3 ml cottonseed
oil) twice a week Ior 4 weeks and/or 10 mg oI myristicin or dihydromyristicin (in 0.3 ml oI
cottonseed oil) three times a week Ior 4 weeks. The Iirst dose oI B|a|P was given aIter three
doses oI test compounds were administered. The control mice were given 0.3 ml oI cottonseed
oil. Mice were killed 18 weeks aIter the Iirst dose oI B|a|P was administered. The lungs and
Iorestomachs were observed and checked histopathologically. Myristicin treatment resulted in a
signiIicant reduction oI 65 in the mean number oI lung-tumours in the tumour-bearing
animals. Dihydromyristicin produced a small or insigniIicant reduction oI lung tumour
Iormation. In the Iorestomach, myristicin treatment resulted in a 31 inhibition oI tumour
Iormation, while dihydromyristicin exhibited a 27 inhibition. These results were in
accordance with the Iact that myristicin given every second day at 10 mg/dose Ior a total oI
three doses increased the activity oI glutathione S-transIerase (GST) to 4.3 and 3.2 times the
control values in the liver and the small intestinal mucosa in Iemale A/J mice. In this study,
there was no indication oI carcinogenicity oI myristicin itselI (Zheng et al., 1992).
Mutagenicity / genotoxicity: Oleoresins prepared Irom nutmeg seed were tested in vitro in two
streptomycin-dependent Salmonella strains (SM
d
): SD 1018 and SD 7823 (which were isolated
Irom S. tvphimurium TA 100 and TA 98, respectively). These strains were used Ior the spot test
and the plate incorporation test. No metabolic activation oI the samples was perIormed in these
studies. In the spot tests, the oleoresins were Iound to be mutagenic in both strains. In the plate
incorporation test, oleoresins prepared Irom the raw seeds oI the nutmeg kernel showed a dose-
response eIIect, while those Irom dried and stored seeds did not have any eIIects. In addition,
myristicin was regarded as positive in the spot test Ior both SM
d
strains (Damhoeri et al., 1985).
The genotoxicity oI the alkenylbenzenes alpha- and beta-asarone, myristicin and elemicin as
determined by the unscheduled DNA synthesis assay (UDS) was investigated in cultured rat
hepatocytes. Alpha- and beta-asarone and elimicin gave positive responses in the UDS assay
while myristicin was negative (Hasheminejad et al., 1994).

Human data: In a study with 10 subjects given a single dose oI 400 mg myristicin or a placebo
in a crossover design, deIinite reactions to myristicin were experienced by 4 subjects. Two
persons experienced generally pleasant reactions such as increased alertness, a Ieeling oI
irresponsibility, Ireedom and euphoria. The other two subjects reported symptoms oI an
unpleasant character such as nausea, diIIiculty in concentrating, tremor, tachycardia, anxiety
and Iear. The onset was 1-2 hours aIter ingestion. The duration oI the symptoms was 7 hours to
one day` (Truitt et al., 1960).
Several cases oI nutmeg intoxication have been reported. Accidental and voluntary (in order to
experience hallucinogenic eIIects) intoxications have generally been reported aIter the ingestion
oI 5-15 g oI nutmeg. As the myristicin content oI nutmeg is approximately 1-2.8, the ingested

128
amount oI myristicin might be considerably less (i.e. 50-420 mg) than in the study on human
volunteers above. It is thereIore believed that there are several other components oI nutmeg that
contribute to the adverse eIIects. The major eIIects oI nutmeg ingested orally at the dose levels
mentioned above are Ilushing oI the skin, tachycardia, salivary inhibition, central excitation,
burning epigastric pain with or without vomiting, restlessness, giddiness and hallucinations. The
clinical course oI nutmeg intoxication may be shock, coma and acidosis. The onset oI the
symptoms is commonly reported to occur 2-6 hours aIter ingestion. The duration oI the
symptoms may last Ior 9 hours to several days, depending on the dose and other parameters
(Painter et al., 1971).

Other studies: DNA-binding capacitv. The binding oI a series oI alkenylbenzenes to liver DNA
Irom adult Iemale CD-1 mice was investigated. The test compounds saIrole, estragole,
methyleugenol, allylbenzene, anethole, myristicin, parsley apiol, dill apiol, eugenol and
elemicin were administered i.p. at 2 or 10 mg/mouse. The known hepatocarcinogens saIrole,
estragole and methyleugenol exhibited the strongest binding, with 200-300 pmol adduct/mg
DNA aIter a 10 mg dose, while all the other investigated compounds except Ior eugenol showed
intermediate to low binding levels in this
32
P-postlabelling assay. The DNA-binding oI
myristicin was estimated to approximately 50 pmol adduct/mg DNA (Randerath et al., 1984).
The binding oI a series oI alkenylbenzenes to mouse liver DNA (Irom male C57B1 x
C3H/HeF
1
-mice) was investigated. The mice were administered the compounds i.p. on days 1,
8, 15 and 22 aIter birth. A total dose oI 4.75 micromoles was given to each animal. The highest
levels oI adducts were detected with methyleugenol, estragole and saIrole (72.7, 30 and 17.5
pmoles/mg DNA, respectively). The DNA-binding oI myristicin was Iound to be 7-8 pmol/mg
DNA. In comparison with the three Iirst-mentioned (carcinogenic) compounds, the adducts oI
myristicin were less persistent, which has turned out to be oI importance in the process oI
tumour initiation (Phillips et al., 1984).
A series oI experiments was carried out with mice given cola drinks instead oI water. The
development oI signiIicant levels oI covalent liver DNA adducts in mice consuming cola drinks
up to 8 weeks and in Ietal liver when pregnant mice were administered myristicin was observed.
Myristicin adducts were Iound to be the main type oI adduct, amounting to about 80 in these
experiments. The liver adduct levels increased in a time-dependent manner in mice chronically
exposed to cola beverages. These results strongly suggest that the cola drinks contained
myristicin. In this study, myristicin was also Iound to induce transplacental liver DNA damage.
It was also noted that pregnancy caused an increase in the binding oI myristicin to mouse liver
(Randerath et al., 1993). Induction oI transplacental DNA damage in mouse liver and increased
binding in the liver DNA oI pregnant mice have previously been reported Ior saIrole (Lu et
al.,1986).
Effects of mvristicin on sleeping time. In an experiment with 10 rats treated with myristicin
(100 mg/kg i.p.), the sleeping time was signiIicantly reduced in comparison with rats treated
with phenobarbital only (Truitt et al., 1960). These observations might indicate that myrisiticin
could act as an inducer oI the cytochrome P-450 system. In contrast, a single treatment oI mice
with myristicin at a dose oI 300 mg/kg i.p. (myristicin was isolated Irom the seed oI Mvristica
fragrans Houtt. by column chromatography oI the hexane Iraction over silica gel) caused a
signiIicant prolongation oI hexobarbital-induced sleeping time (Shin et al., 1988). In addition to
this eIIect, an inhibition oI aminopyrine N-demetylase and hexobarbital hydroxylase activities
was shown. Furthermore, myristicin was also shown to cause sleeping episodes even at a
subhypnotic dose oI hexobarbital. These results suggest that myristicin exerts CNS depressant
activity.
Effects of mvristicin on the expression of liver cvtochrome P450s and its mRNA levels. Male
Sprague-Dawley rats were treated i.p. with myristicin at 500 micromols/kg bw (Jeong et al.,

129
1995). The eIIect oI myristicin on the expression oI inducible liver P450 enzymes (1A1/2,
2B1/2 and 2E1) was investigated using speciIic enzymatic activity assays, immunoblot analysis
and Northern blot analysis. It was concluded that myristicin is an inducer oI all the examined rat
liver P450 enzymes and that the induction involves increases in mRNA levels except in the case
oI P450 2E1.
Effects of mvristicin on lipid peroxidation. Mice were given myristicin p.o. at doses oI 25, 50
and 100 mg/kg bw respectively Ior 3 successive days. As a positive control, DL-alpha-
tocopherol was given at a dose oI 50 mg/kg bw/day. Control groups administered vehicle only,
were also included in the experiment. Lipid peroxidation was induced by repeated injections oI
FeCl
2
, ascorbic acid and ADP. The Iormation oI thiobarbituric acid in the liver tissues was
measured as an indicator oI lipid peroxidation. It was shown that myristicin signiIicantly
inhibited the lipid peroxidation. Since myristicin did not appreciably alter the superoxide
dismutase level in the liver it was thought that the anti lipid peroxidative action may be
attributed to the Iree radical-scavenging property oI myristicin (Hattori et al., 1993).

TOXICOLOGICAL EVALUATION: Toxicological inIormation on myristicin is very
limited. Many oI the studies reported here are inadequate and studies concerning chronic
toxicity, reproductive toxicity and carcinogenicity are lacking.
The metabolism oI myristicin broadly seems to resemble that oI saIrole. No inIormation is,
however, available concerning the quantitative importance oI the individual metabolic
pathways.
The acute toxicity oI myristicin appears to be low. No toxic eIIects were observed aIter peroral
administration oI 10 mg/kg bw/day Ior 26 days in rats. Myristicin at doses oI 100 mg/kg bw i.p.
signiIicantly reduced the sleeping time in rats pretreated with phenobarbital. According to one
in vitro study on the mutagenicity oI nutmeg, it is possible that both nutmeg and myristicin may
be weakly mutagenic. Important inIormation concerning the perIormance oI this study is,
however, lacking. On the other hand, myristicin was not Iound to be genotoxic in an UDS assay.
In order to examine the mutagenic potential oI myristicin, more studies would be required.
There are no indications that myristicin may exert carcinogenic activity in shortterm assays
using mice. Myristicin showed no detectable activity Ior the initiation oI hepatic tumours on
administration to male mice prior to weaning. In Iemale mice pretreated or simultaneously
treated with myristicin beIore/during exposure to benzo(alpha)pyrene, the mean number oI
lung-tumours in tumour bearing animals was signiIicantly reduced. In
32
P-postlabelling assays
oI some alkylbenzenes the DNA-binding oI myristicin was considerably weaker than that oI
known hepatocarcinogens such as saIrole, estragole and methyleugenol. The adducts oI
myristicin were also less persistent. Oral doses oI 400 mg myristicin (corresponding to 6-7
mg/kg bw) produced mild cerebral stimulation` in 4 out oI 10 human subjects. Several
intoxications have, however, been reported aIter the ingestion oI approximately 5 g oI nutmeg
corresponding to approximately 1-2 mg myristicin/kg bw. The reason Ior this may be combined
eIIects oI myristicin and other components oI nutmeg.
In conclusion, no toxic eIIects were observed in rats administered myristicin perorally at a dose
level oI 10 mg/kg bw, while 6-7 mg/kg bw evidently may be enough to cause transitional
psychopharmacological eIIects in man.

(T)MDI: Not established due to insuIIicient data (suspected genotoxic carcinogen).

MAIN OCCURRENCE: The highest concentrations oI myristicin are Iound in nutmeg
(Mvristica fragrans Houtt., CE No. 296) which contains approximately 1.3 in the seed and
2.7 in mace (dried arillodes oI the ripe nutmeg see) (Table I). Myristicin is also Iound in a
number oI plants oI the carrot Iamily (Umbelliferae), Ior instance in dill (Anethum graveolens L.

130
var. hortorum AleI., CE No. 42), celery (Apium graveolens L. var. dulce (Mill.) Pers., CE No.
52), parsnip (Pastinaca sativa L.), parsley (Petroselium crispum (Mill) Nym. Ex A.W. Hill, CE
No. 326), and carrot (Daucus carota L., CE No. 173) (Hall, 1973). Myristicin has also been
reported as a minor constituent in black pepper (Piper nigrum L., CE No. 347) (Richards and
Jennings, 1971), but no quantitative data have been Iound. Myristicin has also been reported to
be present in some plants that are unlikely to be used Ior Ilavouring purposes, such as
Levisticum scoticum (syn. Licusticum scoticum, Scotch or Scottish lovage), Ridolfia segetum
(L.) Moris. (harvest Iennel, corn parsley, Ialse carraway), and Oenanthe stolonifera (no CE
numbers) (Shulgin, 1966).

INTAKE ESTIMATION: The intake oI myristicin Irom spices (Table II) and essential oils
(Table III) was estimated using Iood intake data Irom the dietary and nutritional survey oI
British adults (British adult study) (Gregory et al., 1990). Food items spiced with nutmeg/mace
or dill were considered to be the main sources oI myristicin. However, myristicin intake Irom
dill appears to be very limited (see Table II). The highest levels oI myristicin are Iound in Iood
spiced with mace, with non-alcoholic beverages being the most important single source oI
intake. Due to its lower content oI myristicin, Iood spiced with nutmeg generally contains lower
levels oI myristicin than Iood spiced with mace.
In addition to the Iood groups listed in Table II, which were considered to contribute most to the
intake oI myristicin, mace and nutmeg are also reported to be used in condiments, soups, snack
Ioods, gravies, breakIast cereals, Iats, oils, milk products, Iruit juices, sweet sauce, gelatins,
puddings and alcoholic beverages (Fenaroli, 1995).
The intake oI myristicin Irom essential oils seems to be lower than that Irom spices (Table III).
In accordance with the intake Irom spices, dill essential oil contributes to a very minor degree to
the total intake oI myristicin, and again mace appears to be the main source oI intake, with non-
alcoholic drinks contributing most to the total intake. Apart Irom the Iood groups included in
Table III, which were considered to result in the largest intakes oI myristicin, mace and nutmeg
essential oils are also reported to be used in condiments, gelatins, puddings, sweet sauce,
alcoholic beverages and chewing gum (Fenaroli, 1995).
In conclusion, an approximate estimate oI the total intake oI myristicin Irom all sources appears
to be in the order oI a Iew milligrams per person and day.

CONCLUSIONS: The estimated daily intake oI myristicin seems to be considerably smaller
than the doses inducing eIIects in experimental studies. As essential toxicological data are still
lacking, it is, however, not possible to assess whether the present intake oI myristicin may
represent a health risk.

DATA NEEDED: Further studies on quantitative metabolism in diIIerent species, including
humans and mutagenicity studies with appropriate activation systems are required. II the results
on myristicin metabolism and mutagenicity studies are inconclusive, a long-term
carcinogenicity study would possibly be needed.

LIMITS: It is not considered necessary to set limits Ior myristicin as its main intake Irom
nutmeg would be restricted via the limits set Ior saIrole.

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Lu, L.-J.W., Disher R.M. and Randerath K.(1986) DiIIerences in the covalent binding oI
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Phillips, D.H., Reddy M.V. and Randerath K. (1984) 32 p-post-labelling analysis oI DNA
adducts Iormed in the livers oI animals treated with saIrole, estragole and other naturally-
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DATA BASES USED: Medline (1966-1997), Toxline (1969-1997). Keywords: myristicin,
myristicin and toxicity, nutmeg and toxicity

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135

136

137
Teucrin A

ACTIVE PRINCIPLE: III

SYNONYMS: (5aS-(5àlpha,6`beta(R*),7`beta,8`beta,8àlpha))-5-(3-Iuranyl)-
3`,4,5,5`,5à,7`,8`,8à -octahydro-8`-hydroxy-7`-methylspiro(Iuran-3(2H),6`-(6H)naphtho(1,8-
bc)Iuran)-2,2`(4`H)-dione

CAS No.: 12798-51-5 (Teucrin A); 84929-80-6 (Teucrium chamaedrvs extract)

STRUCTURE:
O
H
O
O
H
CH
3
OH
O
O
H
1
2
3
8
7
6
5
19
18

REGULATORY / INTERNATIONAL STATUS: SCF evaluated the toxicological data
available on teucrin A and the hydroalcoholic extract oI Teucrium chamaedrvs L. (germander,
wall germander) and considered it insuIIicient to establish an ADI. However, SCF concluded
that, due to the hepatotoxic properties oI teucrin A, use oI germander preparations including
teucrin A should be restricted to alcoholic beverages (SCF, 2003). As yet, there is not speciIic
EU regulation Ior teucrin A in Iood and beverages. In the USA, teucrin A is approved Ior Iood
use with limitation to alcoholic beverages only (CFR 172.510).


Metabolism:
studies: The Iurano-neoclerodane diterpenoids present in germander, oI which teucrin
A is a major compound, are transIormed by cytochromes P450, particularly CYP3A4, into
unidentiIied hepatotoxic metabolites. The metabolites, probably epoxides, are partially
inactivated by glutathione and probably epoxide hydrolase (Loeper et al., 1994).

All data described in this paper reIer to the whole plant Teucrium chamaedrvs L. (germander) or to the plant extract.

138
Animal studies: No data Iound.

Toxicology:
Acute toxicity: No LD
50
values are available. Single intragastric administrations oI the
germander plant lyophilisate (1.25 g/kg bw), the Iurano-neoclerodane diterpenoid Iraction
(0.125 g/kg bw) or teucrin A (150 mg/kg bw) produced midzonal liver cell necrosis at 24 hrs in
mice (Loeper et al., 1992; Kouzi et al., 1994).
Subacute / subchronic toxicity: A hydroalcoholic extract oI T. chamaedrvs L. (containing
7140 mg teucrin A/kg) was given once a day by gavage Ior 4 weeks to Sprague Dawley rats
(groups oI 10 males and 10 Iemales) at oral doses oI 0.014, 0.14 and 1.4 g/kg bw/day
corresponding to about 0.1, 1, and 10 mg/kg bw/day oI teucrin A. The control group received
the vehicle only. At 0.014 g/kg bw/day no eIIects that could be related to the test substance
administration were Iound in either sex at the various clinical, laboratory and post-mortem
investigations. At 0.14 g/kg bw/day the only treatment-related modiIications noted were a slight
increase in gamma glutamyl transpeptidase serum activity in males and a slight increase in total
serum protein in Iemales. At 1.4 g/kg bw/day, T. chamaedrvs L. induced liver modiIications,
which were generally slight and mainly oI degenerative origin, and associated with aspects oI
hypertrophy. Moreover, necroses oI isolated hepatocytes were Iound (RBM SpA, 1997a).
A hydroalcoholic extract oI T. chamaedrvs L. (containing 7140 mg teucrin A/kg) was given to
Sprague Dawley Crl:CD (SD) BR rats (groups oI 20 males and 20 Iemales) once a day by
gavage Ior 13 weeks at oral doses oI 0.056, 0.28 and 1.4 g/kg bw/day. The highest dose level oI
1.4 g/kg bw/day corresponding to about 10 mg/kg bw/day oI teucrin A, was poorly tolerated by
both males and Iemales, causing severe toxic eIIects on liver (hypertrophy associated with
diIIuse steatosis and other degenerative changes in males and changes in the hepatobiliary
system in Iemales) and related clinical alterations (mainly in blood chemistry parameters). At
0.28 g/kg bw/day corresponding to about 2 mg/kg bw/day oI teucrin A, no changes oI
toxicological signiIicance were observed at hematology, blood chemistry and urinalysis. At post
mortem examinations, liver changes were observed which mainly consisted oI hepatocellular
hypertrophy and steatosis. At 0.056 g/kg bw/day corresponding to about 0.4 mg/kg bw/day oI
teucrin A, a slight hepatocellular hypertrophy in the Iemale group was observed. Hepatocellular
hypertrophy, in the absence oI other morphological modiIications, can be considered as an
adaptive metabolic rather than a toxic change. AIter 6 weeks oI withdrawal (5
animals/sex/group maintained at the end oI the treatment period), the changes observed during
treatment with doses oI 0.28 g/kg bw/day were no longer observable (RBM SpA, 1997b).
Mutagenicity / genotoxicity: In vitro. A number oI mutagenicity tests were carried out with a
hydroalcoholic extract containing teucrin A at a concentration oI 1200 mg/l. An Ames test using
S. tvphimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 with and without liver
homogenate (S9 mix) was negative with up to 240 micrograms test extract/plate (corresponding
to 1.7 micrograms teucrin A/plate) (RBM SpA, 1996). The test extract did not induce any
signiIicant increase in gene mutations in V79 Chinese hamster lung cells in the concentration
range oI 0.01 to 0.9 micrograms/ml in the absence and in the presence oI hepatic microsomal
enzymes (S9 mix). The higher concentrations applied (up to 24 micrograms/ml) were highly
cytotoxic to V79 cells (RBM SpA, 1997c). The test extract up to the concentration oI 24
micrograms/ml, both in the absence and in the presence oI metabolic activation (S9 mix), did
not induce a statistically signiIicant increase in the number oI cells with chromosome
aberrations or in the number oI polypoid or endoreduplicated cells in cultured human
lymphocytes. At levels oI 8 and 24 micrograms/ml (corresponding to 0.06 and 0.17 microgram

139
teucrin A/ml), a reduction oI the mitotic index oI about 35 was recorded with and without S9
mix (RBM SpA, 1997d).
In vivo. In a micronucleus test, the T. chamaedrvs L. hydroalcoholic extract administered to
Charles River rats by oral route at the doses oI 2.5, 5 and 10 mg/kg bw, corresponding to 17.5,
35 and 70 microgram teucrin A/kg bw/day, did not induce any statistically signiIicant increase
in the Irequency oI micronucleated cells in the bone marrow 24 and 48 hours aIter the
administration. The ratio oI polychromatic to normochromatic erythrocytes remained unaIIected
in males and Iemales, indicating that the test article is not toxic to or has not reached the bone
marrow cells under the experimental conditions (RBM SpA, 1997e).

Human data: According to various case reports, the use oI T. chamaedrvs L. in herbal teas or
in tablets to Iacilitate weight loss can occasionally cause cytolytic hepatitis. A considerable
number oI patients came down with hepatitis three to 18 weeks aIter ingesting the herb at the
recommended doses oI 600 to 1620 mg/day as powdered herbal plant or dried aqueous extract,
corresponding to about 2 to 6 mg teucrin A/day) (Castot and Larrey, 1992; Larrey et al., 1992;
Dao et al., 1993; SCF 2003). Readministration caused early recurrence oI hepatitis in about halI
oI the patients, suggesting an immunoallergic type oI hepatitis (Castot and Larrey, 1992). A
letal case oI a 68-year old woman with massive hepatic necrosis aIter ingestion oI 450 mg/day
oI dried wild germander in a tea preparation Ior two weeks and repeated aIter six months has
been reported (MosteIa-Kara et al., 1992).

Other studies: Midzonal liver cell necrosis observed 24 hrs aIter single intragastric
administration oI 150 mg teucrin A/kg bw in mice was prevented by pre-treatment with a single
dose oI troleandomycin (speciIic inhibitor oI CYP3A) and enhanced by pretreatment with
dexamethasone (inducer oI CYP3A) (Loeper et al., 1994). Sera oI patients who had developed
cytolytic hepatitis aIter longterm ingestion oI T. chamaedrvs L. preparations were Iound to
contain anti-microsomal epoxide hydrolase (EH) autoantibodies. Human microsomal EH
(hmEH) was shown to be present in plasma membranes oI human hepatocytes and to be
exposed on the cell surIace where aIIinity puriIied germander autoantibodies recognized it as
their autoantigen. This Iinding could suggest (auto)antibody participation in an immune cell
destruction. In a yeast strain expressing human CYP3A4 and hmEH teucrin A was metabolised
to at least one reactive metabolite that Iormed adducts with hmEH and induced a time-
dependent inactivation oI EH, suggesting that a reactive oxide Irom teucrin A could alkylate the
enzyme and trigger an immune response. Antibodies were Iound to recognize teucrin A-
alkylated EH (De Berardinis et al., 2000).

TOXICOLOGICAL EVALUATION: Ingestion oI Teucrium chamaedrvs L. as a herbal
medication has been associated with hepatitis and liver cell necrosis, including a Iatal case in
humans.
Teucrin A is considered to be responsible Ior the hepatotoxicity oI T. chamaedrvs L. observed
in mice and rats as well as in man. An in vivo study in mice indicated that teucrin A is
speciIically transIormed by CYP3A4 into hepatotoxic metabolites (probably epoxides) that
induce apoptosis. DetoxiIication oI these metabolites depends on thiol groups (glutathione) and
epoxide hydrolase. There is some evidence Irom mechanistic studies Ior the involvement oI
immunological reactions in the destructive hepatotoxicity oI teucrin A.
All genotoxicity tests carried out with a hydroalcoholic extract oI T. chamaedrvs L. were
negative. In a 13-weeks oral toxicity study in rats no toxicological signs were observed upon
treatment with a hydroalcoholic extract oI T. chamaedrvs L. at doses oI 0.056 g/kg bw/day
corresponding to 0.4 mg/kg bw/day oI teucrin A. Hepatocellular hypertrophy and steatosis as
well as degenerative changes were Iound at the higher doses. There are no data available on
chronic toxicity, carcinogenicity and reproductive and developmental toxicity. Based on a

140
NOAEL oI 0.4 mg/kg bw/day derived Irom the 13-weeks oral toxicity study in rats and by
applying a saIety Iactor oI 200, an MDI oI 0.002 mg/kg bw/day or 0.12 mg/person/day oI
teucrin A was established.

MDI: 0.002 mg/kg bw/day

MAIN OCCURRENCE: Teucrin A represents 60-70 oI the Iurano neo-clerodane
diterpenoids present in the aerial parts oI Teucrium chamaedrvs L. (Popa and Reimbold, 1972;
Reimbold and Popa, 1974; Rodriguez et al., 1984a). The content oI teucrin A in Teucrium
chamaedrvs L. has been reported to be 0.03 in Italian and 0.15 in Spanish plant material
(dried and Iinely ground) (Rodriguez et al., 1984b).

INTAKE ESTIMATION: T. chamaedrvs L. extracts (or the dried plant material) are used in
Ilavoured wines, bitters and liqueurs. No other Iood uses are known. Use levels oI the dried
plant material per litre oI beverage were reported by the Ilavour industry to be 0.1-1 g Ior
Ilavoured wines and 1-2 g Ior bitters and liqueurs (Council oI Europe, 1996). According to the
Italian liqueur industry levels oI teucrin A Iound in alcoholic beverages range Irom 1 to 5 mg/l
(Council oI Europe, 2000). Levels oI 6.10.8 mg teucrin A per litre (n10) have been reported
in bitter alcoholic beverages prepared with herbs containing T. chamaedrvs L. (SCF, 2003).
II Ior a rough high intake estimate oI teucrin A a daily consumption oI 50 to 100 ml alcoholic
beverages Ilavoured with T. chamaedrvs L. and a teucrin A concentration oI 0.05 mg/kg Ior
every 1 oI alcohol by volume were assumed, the daily per capita intake would be 0.075 to
0.1125 mg (assumed consumption oI 100 ml 45 Vol, 75 ml 30 Vol or 50 ml 15 Vol). II
Ior an estimate oI the teucrin A intake Ior mean and 97
th
percentile consumers consumption data
on alcoholic beverages Irom the Dietary and Nutritional Survey oI British Adults (Gregory et
al., 1990) were used, the assumed daily consumption oI vermouths and liqueurs would be 18.5
g/day and 80.4 g/day, respectively. Based on a teucrin A content oI maximal 0.05 mg/kg Ior
every oI alcohol in each oI these beverages and a mean alcohol content oI 20 the estimated
daily per capita intakes would be 0.0185 mg/person/day Ior mean consumers and 0.0804
mg/person/day Ior 97
th
percentile consumers.

CONCLUSIONS: The estimated daily intakes oI teucrin A Ior mean and 97
th
percentile
consumers based on the concentration limits set Ior alcoholic beverages are well within the MDI
established Ior teucrin A.

DATA NEEDED: -

LIMITS: (mg/kg)

a

a

Exceptions:
Alcoholic beverages 0.05 Ior every 1 oI alcohol by volume

141

REFERENCES:
Castot, A. and Larrey D. (1992) Hepatites observees au cours dùn traitement par un
medicament ou une tisane contenant de la Germandree petit-chne. Bilan des 26 cas
rapportees aux Centres Regionaux de Pharmacovigilance. Gastroenterol. Clin. Biol., 16,
916-922.
Council oI Europe (1996) Committee oI Experts on Flavouring Substances. Use levels oI
Teucrium chamaedrys extract RD 4.2/X-38. InIormation submitted by IOFI.
Council oI Europe (2000) Committee oI Experts on Flavouring Substances. Comments on
Teucrium chamaedris - RD 4.10/1-46. Traditional use oI Teucrium chamaedris - RD
4.10/2-46. Documents submitted by FEDERVINI.
Dao, T., Peytier A., Gorlateau F. and Valla A. (1993) Hepatite chronique cirrhogene a la
germandree petit-chne. Gastroenterol. Clin. Biol., 17, 609-610.
De Berardinis, V., Moulis C., Maurice M., Beaune P., Pessayre D., Pompon D. and Loeper J.
(2000) Human microsomal epoxide hydrolase is the target oI germander-induced
autoantibodies on the surIace oI human hepatocytes. Mol. Pharmacol. 58 (3), 542-551.
Gregory, J., Foster K., Tyler, H. and Wiseman, M. (1990). The dietary and nutritional survey oI
British adults. HMSO, London.
Kouzi, S.A., McMurtry R.J. and Nelson S.D. (1994) Hepatotoxicity oI germander (Teucrium
chamaedrys L.) and one oI its constituent neoclerodane diterpenes teucrin A in the mouse.
Chem. Res. Toxicol., 7 (6), 850-867.
Larrey, D., Vial T., Pauwels A., Castot A., Biour M., David M. and Michel H. (1992) Hepatitis
aIter germander (Teucrium chamaedrvs) administration: another instance oI herbal
medicine hepatotoxicity. Ann. Intern. Med., 117, 129-132.
Loeper, J., Descatoire V., Letteron P., Moulis C., Degott C., Dansette P., Fau D. and Pessayre
D. (1994) Hepatotoxicity oI germander in mice. Gastroenterology, 106 (2), 464-472.
MosteIa-Kara, N., Pauwels A., Pines E., Biour M. and Levy V.G. (1992) Fatal hepatitis aIter
herbal tea. Lancet, 340 (8820), 674.
Popa, D.P and Reimbold A.M. (1972) Bitter substances Irom Teucrium chamaedrvs. Khim. Prir.
Soedin. 8, 67-69.
RBM SpA (1996) Ames test. Institute oI Biomedical Research Antoine Marxer`, Doc. No
960550.
RBM SpA (1997a) Dose range-Iinding study in rats by oral route oI 4 weeks duration. Institute
oI Biomedical Research Antoine Marxer`, Doc. No 960554.
RBM SpA (1997b) 13-week oral toxicity study in rats Iollowed by 6 weeks oI recovery.
Institute oI Biomedical Research Antoine Marxer`, Doc. No 960555.
RBM SpA (1997c) Gene mutation in V79 cells. Institute oI Biomedical Research Antoine
Marxer`, Doc. No 960552.
RBM SpA (1997d) Chromosome aberrations in humans lymphocytes cultured in vitro`.
Institute oI Biomedical Research Antoine Marxer`, Doc. No 960551.
RBM SpA (1997e) Micronucleus test in rat bone marrow cells treated by oral route. Institute oI
Biomedical Research Antoine Marxer`, Doc. No 960553.
Reimbold, A.M. and Popa D.P. (1974) Teucrium chamaedrvs. Khim. Prir. Soedin. 5, 589-591.
Rodriguez, M.C., Barluenga J., Pascual C., Rodriguez B., Savona G. and Piozzi F. (1984a) Neo-
clerodane diterpenoids Irom Teucrium chamaedrvs: The identity oI teucrin B with
dihydroteugin. Phytochemistry, 23 (12), 2960-2961.
Rodriguez, M.C., Barluenga J., Savona G., Piozzi G., Servettaz O. and Rodriguez B. (1984b)
IsoteuIlidin, a neo-clerodane diterpenoid Irom Teucrium chamaedrvs, and revised
structures oI teucrins F and G. Phytochemistry 23, 1465-1469.

142
SCF (2003) Opinion oI the ScientiIic Committee on Food on Teucrin A, major component oI
hydroalcoholic extracts oI Teucrium chamaedrvs (wild germander) expressed on 5 March
2003.

DATABASES USED: Biosis (1973-1997), Chemical Abstracts (1967-1997), Toxline (1969-
1997), Pascal (1973-1997), FSTA (1969-1997), Medline (1966-1997), RTECS, IPA, CAB, LiIe
Science, Napralert. Keywords: teucrin A, germander, Teucrium chamaedrvs.

143
alpha- and beta-Thujone


SYNONYMS: (-)-3-thujone and ()-3-isothujone ( beta-stereoisomer); 1-isopropyl-4-
methylbicyclo(3.1.0)hexan-3-one

CAS No: 546-80-5

STRUCTURE:
O
CH
3
H
3
C CH
3
O
H
3
C CH
3
H
CH
3
H

alpha-thujone beta-thujone

REGULATORY / INTERNATIONAL STATUS: Thujone is listed in Annex II oI the
Council Directive 88/388/EEC on Ilavourings (EEC, 1988) with maximum limits oI 0.5 mg/kg
in IoodstuIIs and beverages and the exceptions oI 5 mg/kg in alcoholic beverages with 25
volume oI alcohol, 10 mg/kg in alcoholic beverages with ~25 volume oI alcohol, 25 mg/kg in
IoodstuIIs containing preparations based on sage, and 35 mg/kg in bitters, in accordance with
Council oI Europe, Blue Book (Council oI Europe, 1981). Thujone may not be added to Iood as
such. The same maximum levels in Iinal products Ior consumption have been recommended by
WHO/FAO Codex Alimentarius in 1979 (value oI 25 mg/kg in IoodstuIIs containing
preparations on sage not included). SCF considered the available data inadequate to derive a
TDI/ADI (SCF, 2003). No ADI has been allocated by JECFA which considered that the
amounts oI thujone isomers in Ioods and beverages resulting Irom the addition oI thujone-
containing Ilavouring agents (e.g., sage) should be reduced to the lowest practicable level
(JECFA, 1981). Thujone is not authorised Ior use as a Ilavouring substance in the USA.


Metabolism:
studies: Alpha-thujone is rapidly metabolised by mouse liver microsomes with
NADPH Iorming 7-hydroxy-alpha-thujone as the major product plus Iive minor ones (4-
hydroxy-alpha-thujone, 4-hydroxy-beta-thujone, two other hydroxythujones and 7,8-dehydro-
alpha-thujone). Hydroxythujones appear to be detoxiIication products oI lower toxicity than
alpha-thujone and are expected to be Iinally excreted in conjugated Iorm. Incubation oI alpha-
thujone with rabbit (but not mouse) liver cytosol resulted in the Iormation oI thujol and

144
neothujol, however, enzymatic reduction was NADPH-dependent and gave only a small yield
(Hld et al., 2000).
Animal studies: AIter intraperitoneal injection oI alpha-thujone in mice, dose and time
dependent concentrations oI alpha-thujone and 7-hydroxy-alpha-thujone were measured in the
brain. At severely toxic doses oI alpha -thujone (40-60 mg/kg) the levels in the brain aIter 30
minutes were 0.3-1.0 mg/kg Ior alpha-thujone and 1.5-8.4 mg/kg Ior 7-hydroxy-alpha-thujone;
much higher levels (11 and 29 mg/kg, respectively) were measured 2.5 minutes aIter the
injection when the poisoning signs were most intense (Hld et al., 2000). AIter oral
administration oI 10 g alpha- and beta-thujone (ratio 9:2) to rabbits, three metabolites were
Iound in the urine, namely neoisothujanol, thujanol and neoisothuylic alcohol acetate (dose was
lethal Ior some animals) (Ishida et al., 1989).
Human studies: Little is known about the pharmacokinetics oI these terpenes in humans. Based
on the assumption that there is an appreciable hepatic Iirst-pass extraction Ior these compounds
and/or iI their rates oI hepatic metabolism are not unusually rapid, concentrations achieved in
the livers oI oI 19th century absinthe drinkers in the range oI 20-200 micromol/l (equivalent to
3-30 mg/l) have been estimated (Bonkovsky et al., 1992).

Toxicology:
50
oI 192 mg/kg bw (alpha/beta-thujone) has been reported in the rat
(Margaria, 1963). Acute toxicity oI thujone was Iound to be much higher upon parenteral
administration with an intraperitoneal LD
50
oI 140 mg/kg bw (alpha/beta-thujone) observed in
the rat (Sampson and Fernandez, 1939). Similar sensitivity was observed in mice with oral
LD
50
s oI 230 and 250 mg/kg bw (alpha/beta-thujone and alpha-thujone, respectively) (Margaria,
1963; Le Bourhis and Soenen, 1975) and an intraperitoneal LD
50
oI about 45 mg/kg bw (Hld et
al., 2000) as well as in guinea-pigs (Margaria, 1963) and dogs (Ionescu et al., 1958). Symptoms
oI thujone-induced acute intoxication are convulsions oI epileptiIorm character, preceeded by
general vasodilatation, Iall in blood pressure, slowed heart rate and increased respiration (Pinto-
Scognamiglio, 1968). Doses required to produce convulsions in mice and rats aIter
intraperitoneal application were Iound to be slightly below the LD
50
s, e.g. 100 mg/kg bw Ior rats
(Sampson and Fernandez, 1939) and 30 mg/kg bw Ior mice (Hld, 2000).
Subacute / subchronic toxicity: Two oral gavage studies have been conducted in rats. In the
Iirst study, administration oI 0, 12.5, 25.0 or 50.0 mg/kg bw/day oI a commercial mixture oI
alpha- and beta-thujone on 5 days a week Ior 13 weeks (20 animals/dose/sex) resulted in the
death oI 12 Iemales (60) and 8 males (37) at the highest dose within 38 days and 68 days,
respectively. There was no treatment related death at the lower doses. Convulsions were
observed at each dose in Iemales in relation to the dose and in males at 25 and 50 mg/kg
bw/day. The convulsive ED
50
was estimated as 35.5 mg/kg bw/day Ior males and 26.3 mg/kg
bw/day Ior Iemales. No correlation was seen between the number oI convulsions and mortality.
The haematology and the histology oI the organs including CNS were normal. The NOAEL Ior
convulsions was 12.5 mg/kg bw/day Ior males. No NOAEL could be derived Ior Iemales
(Surber, 1962).
In the second study, oral administration oI 0, 5, 10 or 20 mg oI thujone/kg bw/day (no
speciIication on isomeric composition) on 6 days a week Ior 13 weeks (10 animals/dose/sex)
resulted in the death oI oI 3 Iemales and 1 male at the highest dose. Convulsions were observed
in 9 Iemales and 6 males at the highest dose, in the 10 mg/kg bw/day dose group one convulsion
occurred in only one Iemale. At termination no signiIicant diIIerence was observed between
groups in haematology or weights oI main organs. No treatment related changes in gross
pathological or histopathological lesions were observed. The NOAEL Ior convulsions was 10
mg/kg bw/day Ior males and 5 mg/kg bw/day Ior Iemales (Margaria, 1963).

145
Mutagenicity / genotoxicity: Negative results have been reported Irom in vitro mutagenicity
tests in Salmonella tvphimurium with alpha-thujone and alpha/beta-thujone (NTP, 2003). In vivo
mouse micronucleus test was negative in males and positive in Iemales with alpha/beta-thujone
(NTP, 2003).

Human data: Cases with severe intoxications in humans, even with coma, have been reported
aIter the consumption oI essential oil rich in thujone (Centini and Laurini, 1987; Millet et al.,
1981). Convulsions resembling epilepsy were also observed in humans with toxic doses oI
absinthe (Magnan, 1869). The same eIIects have been reported aIter the ingestion oI isolated
thujone (Cobb, 1922). Estimation oI hepatic concentrations oI thujone demonstrated that
porphyrogenic concentrations oI thujone could readily have been achieved in absinthe drinkers
(Bonkovsky et al., 1992).

Other studies: Biochemistrv. Thujone is a porphyrogenic terpenoid that was shown to increase
the porphyrin production in primary cultures oI chicken embryonic liver cells at concentrations oI
1 mM (152 mg/l) and to induce the synthesis oI 5-aminolevulinic acid synthase, the rate
controlling enzyme oI the pathway. The accumulated porphyrins were predominantly copro- and
protoporphyrins. Thujone also induced benzphetamine demethylase and heme oxygenase by a
heme-dependent mechanism. Thujone could thus pose a risk to individuals with underlying
deIects in hepatic heme synthesis (Bonkovsky et al., 1992).
Neurotoxicitv. Alpha-thujone was shown to be a competitive inhibitor oI
ethynylbicycloorthobenzoate binding to mouse brain membranes. It was Iurther demonstrated that
alpha-thujone acts as a modulator oI the gamma-aminobutyric acid (GABA) type A receptor.
GABA-induced peak currents in rat dorsal root ganglion neurons were suppressed by alpha-
thujone with complete reversal aIter washout (Hld et al., 2000). The convulsions induced by
thujone originate Irom the central nervous system as proven by electrocortical records in
unanesthetized rats (Millet et al., 1981). Administration oI thujone (no speciIication on isomeric
composition) in seven rabbits and seventeen cats (i.v. application oI 0.1 ml oI 20 dilution, i.e. 20
mg/animal; body weight oI 2.7 to 4.5 kg) induced convulsions at several times. When the brains
were examined histologically aIter recovery, changes observed ranged Irom simple disappearance
to widespread degeneration oI nerve cells, characteristic Ior human epilepsy. The severity oI the
observed lesions was inversely proportional to the time between the last convulsion and the
sacriIice and less clearly dependent on the number and intensity oI seizures (Opper, 1939).
Thujone administered orally during 25 days (10 mg/kg bw/day) did not qualitatively modiIy either
spontaneous activity or conditioned behaviour oI the rat, but produced an improvement in
coordination (Pinto-Scognamiglio, 1968).

TOXICOLOGICAL EVALUATION: Alpha-thujone is generally considered to be the
principal active ingredient oI wormwood oil and the toxic principle in absinthe. Beta-thujone
appears to be oI lower toxicity than the alpha-isomer (Arnold, 1988). However, the database is
insuIIicient and literature reports on the relative potency oI the two isomers are inconsistent.
Older publications considered beta-thujone as more toxic than alpha-thujone.
The toxicological data on thujone are limited and the quality oI the available studies, mainly
published in the 1960s, was considered insuIIicient to set an MDI. Thujone has a neurotoxic
potential and produces convulsions in animals and man by acting on the central nervous system.
No data are available on long-term toxicity, carcinogenicity and reproductive toxicity. From a 90-
day oral study in rats a NOAEL oI 5 mg/kg bw/day Ior convulsive eIIects could be derived Ior
Iemales and oI 10 mg/kg bw/day Ior males. The isomeric composition oI the material applied was
not speciIied. However, these Iindings are supported by the NOAEL Ior convulsions oI 12.5
mg/kg bw/day in male rats Iound in another study carried out with a commercial mixture oI alpha-
and beta-thujone. On the basis oI the oral NOAEL oI 5 mg/kg bw/day in Iemale rats and an

146
increased saIety Iactor oI 500 due to the poor quality oI the database, a TMDI oI 0.01 mg/kg
bw/day was derived.

TMDI: 0.01 mg/kg bw/day

MAIN OCCURRENCE: Thujone is a primary constituent oI the essential oils oI various
Artemisia species with contents in the essential oils ranging Irom 0 in A. vulgaris L. (common
mugwort, CE No. 72) and 1.3 in A. absinthium L. (wormwood, CE No. 61) to nearly 90 in A.
umbelliformis Lam. (alpine wormwood, CE No. 68), A. spicata WulI. (syn. A. genipi Weber,
black juniper, CE No. 66) and A. eriantha Ten. (syn. A. petrosa Fritsch., A. petrosa (Baumg.) Jan
Ex D.C. ssp. eriantha. (no CE No.) (Mucciarelli et al., 1995). Thujone is also present in high
concentrations in the essential oil oI Salvia officinalis L. (sage, CE No. 414) at approx. 50, oI
Chrvsanthemum vulgare (L.) Bernh. (syn. Tanacetum vulgare L., tansy, CE No. 446) at nearly
80, and oI Juniperus species (junipers), Cedrus species (cedars) and Thufa occidentalis L.
(white cedar, arborvitae, CE No. 453; yields cedar leaI oil) at 64 (Albert-Puleo, 1978; Pinto-
Scognamiglio, 1967). Thujone occurs in nature as a mixture oI the alpha- and beta-isomer. In
cedar leaI and sage essential oil alpha-thujone is present in considerably higher amounts than the
beta-isomer, whereas in essential oil oI tansy and wormwood the beta-isomer is dominating
(Pinto-Scognamiglio, 1967).

INTAKE ESTIMATION: The intake oI thujone Irom spices and essential oils was estimated
using consumption data on selected Ioods and alcoholic beverages Irom the dietary and nutritional
survey oI British adults (British adult study) (Gregory et al., 1990). Sage-containing IoodstuIIs
were considered to be the main sources oI thujone. The highest levels oI thujone are Iound in
sweets spiced with sage. This Iood category may be the most important single source oI intake iI
consumed. In general, the main contributors to the total thujone intake are sage Ilavoured sausages
and other meat products, sage stuIIing, salad dressing, vermouth, liqueurs and bitters (Table I and
II). In addition to the Iood groups listed in Table I and II, sage is also used in some baked goods,
casseroles, soups, condiments and sauces. The thujone intake Irom wormwood (Artemisia
absinthium L.) and in particular Irom wormwood containing liqueurs such as absinthe appears to
be very limited.
Calculations oI daily intakes oI consumers were based on the limits set Ior selected Iood
categories and alcoholic beverages. The thujone intake Ior mean consumers was estimated to be
3.6 to 3.9 micrograms/kg bw/day (excluding and including sweets, respectively). Estimated
intakes Ior high-level consumers (97.5th percentile) were 13.5 to 14.2 micrograms/kg bw/day
(excluding and including sweets, respectively). In conclusion, the total intake oI thujone Irom all
sources appears to be in the order oI 0.25 mg/person/day Ior mean consumers and up to 1
mg/person/day Ior high-level consumers.

CONCLUSIONS: Estimated mean intakes Ior consumers are well within the TMDI Ior
thujone. Those Ior high-level consumers (97.5
th
percentile), which are believed to be
overestimated, slightly exceed it.

DATA NEEDED: In order to reconsider the classiIication oI thujone, a 28 day oral study in
rodents and two in vitro mutagenicity study in conIormity with OECD guidelines Ior testing oI
chemicals should be carried out. (The NTP report on genotoxicity studies is not available yet,
and NTP short-term and longterm exposure studies Ior alpha-thujone and alpha/beta-thujone in
mice and rats are ongoing). Further data are needed on the content oI thujone in prepared dishes
and Ioods containing sage and on the consumption oI such products in Europe.

147
LIMITS: (mg/kg)
General limits in Ioods and beverages 0.1

Exeptions:
Alcoholic beverages 25 (vermouths) 2
Alcoholic beverages ~ 25 (liqueurs, bitters) 10
Herb vinegar 5
Sage Iood and stuIIing 10
Sweets (lozenges) 50

REFERENCES:
Albert-Puleo, M. (1978) Mythobotany, pharmacology, and chemistry oI thujone-containing
plants and derivatives. Economic Botany, 32, 65-74.
Arnold, W.N. (1988) Vincent van Gogh and the thujone connection. JAMA, 260 (20), 3042-44.
Bonkovsky, H.L., Cable E.E., Cable J.W., Donohue S.E., White E.C, Greene Y.J., Lambrecht
R.W., Scrivastava K.K. and Arnold W.N. (1992) Porphyrogenic properties oI the terpenes
campor, pinene and thujone (with a note on historic implications Ior absinthe and the
illness oI Vincent van Gogh). Biochem. Pharmacol., 43 (11), 2359-2368.
Centini, F. and Laurini G.P. (1987) Una intossicazione da olio di salvia. Zacchia, 60, 263-274.
Cobb, S. (1922) A case oI epilepsy with a general discussion oI the pathology. Med. Clin. N.
Am., 5, 1403-1420.
Council oI Europe (1981) Flavouring Substances and Natural Sources oI Flavourings, Blue
Book, 3
rd
ed., Strasbourg.
Council oI Europe (2000) Committee oI Experts on Flavouring Substances. Note on estimated
intakes oI thujone Irom Ioods and alcoholic beverages - RD 4.11/1-46 rev.
the member States relating to Ilavourings Ior use in IoodstuIIs and to source materials Ior
British adults. HMSO, London.
Hld, K.M., Sirisoma N.S., Ikeda T., Narahashi T. and Casida J.E. (2000) Alpha-thujone (the
active component oI absinthe): gamma-aminobutyric acid type A receptor modulation and
metabolic detoxiIication. PNAS, 97 (8), 3826-3831.
Ionescu, C.N., Anitescu, Lungu and Stoican (1958) Extragerea principiilor activi din Tanacetum
vulgare. Communie Acad. Rep. Pop. Rom., 8, 279.
Ishida, T., Toyota M. and Asakawa V. (1989) Terpenoid transIormation in mammals. V
Metabolism oI ()-citronellal, ()-7-hydroxycitronellal, citral, (-)-perillaldehyde, (-)-
myrtenal, cuminaldehyde, thujone and ()-carvone. Xenobiotica, 19 (8), 846-855.
JECFA (1981) Thujone. WHO Food Additive Series 16 (FAS 16-JECFA 25/227-233, TRS 669-
JECFA 25/26).
Le Bourhis, B. and Soenen A.-M. (1973) Recherches sur làction psychotrope de quelques
substances aromatiques utilisees en alimentation. Food Cosmet. Toxicol., 11, 1-9.
Magnan, V. (1869) Epilepsie alcoolique; action speciale de làbsinthe; epilepsie absinthique.
Comptes Rendus des Seances et Memoires de la Societe de Biologie, 5, 156-161.
Margaria, R. (1963) Acute and subacute toxicity study on thujone. Unpublished report oI the
Istituto di Fisiologia, Universita di Milano (cited Irom CoE Datasheet RD4.2/14-44, 1999).
Millet, Y., Jouglard J., Steinmetz M.D., Tognetti P., Joanny P. and Arditti J. (1981) Toxicity oI
some essential plant oils. Clinical and experimental study. Clin. Toxicol., 18 (12), 1485-
1498.
Mucciarelli, M., Caramiello R. and MaIIei M. (1995) Essential oils Irom some Artemisia
species growing spontaneously in North-West Italy. Flav. Frag. J., 10, 25-32.

148
NTP National Toxicology Program (2003) Testing status oI alpha-thujone and alpha/beta-thujone.
NTP homepage, 06/18/2003.
Opper, L. (1939) Pathologic picture oI thujone and monobrominated camphor convulsions:
comparison with pathologic picture oI human epilepsy. Arch. Neurol. Psych., 41, 460-470.
Pinto-Scognamiglio, W. (1967) Connaissances actuelles sur l'activite pharmacodynamique de la
thuyone aromatisant naturel d'un emploi etendu |Current knowledge on the
pharmacodynamic activity oI the prolonged administration oI thujone, a natural Ilavoring
agent|. Boll. Chim. Farm., 106, 292-300.
Pinto-Scognamiglio, W. (1968) EIIeti del tujone sullàttivita spontanea e sul comportamento
condizionato del ratto |EIIects oI thujone on spontaneous activity and on conditioned
behavior oI rats|. Boll. Chim. Farm., 107, 780-791.
Sampson, W.L. and Fernandez L. (1939) Experimental convulsions in the rat. J. Pharmacol.
Exp. Ther., 65, 275.
SCF (2003) Opinion oI the ScientiIic Committee on Food on Thujone. SCF 23 Add 2 Final, 1-
11.
Surber, W. (1962) Final report on subacute toxicity study oI thujone in rats. Report no. 143021
Institut Battelle, Geneve.

DATA BASES USED: Toxline (1969-2003), Biosis (1973-1990), Toxlit (1981-1990),
Cancerline (1981-2003), Biotechnologiy abstracts (1971-1990), Biobusiness (1983-1990),
Medline (1966-2003), Embase (1980-1990), Pascal (1983-1990), NTIS (1964-1990), Chemical
Abstracts (1967-2003), HSDB (-1990), CCRIS (-1990), RTECS (-1990). Keywords: thujone,
Artemisia.

149
APPENDIX:

Table I. Estimated consumption oI Ioods and alcoholic beverages containing thujone by adults
(Council oI Europe, 2000)
Foods / Beverages Consumers () Consumption (g/person/day)
Mean 97.5th percentile
Sage-Ilavoured sausages 31.0 17.5 53.7
StuIIing 13.6 7.4 20.4
Herb vinegar 6.5 2.5 17.4
Other meat dishes 17.0 38.9 114.8
Sage Derby` cheese * 0.1 12.9 12.9
Salad dressing 0.5 5.5 15.2
Vermouth 5.8 22.1 89.0
Liqueurs and bitters 3.4 10 47.4
Total (foods and beverages)
(excl. sweets)``
56.4 26.5 94.8
Sweets`` 11.5 3.9 18.0
(incl. sweets)``
61.0 25.2 92.8
Assumptions:
All categories oI pork sausages are considered to be sage-Ilavoured.
All types oI stuIIing are considered to be sage Ilavoured. StuIIing includes sausage and
sausagement stuIIings.
Other meat dishes` includes dishes known to contain sage or pork sausages, and other meat
products (meat loaI, chicken liver pate and liver sausage) that are considered to be sage-
Ilavoured.
All salad dressings (excluding salad cream and French dressing) are considered to contain sage.
All vinegar is assumed to be herb vinegar.
Vermouth includes both dry and sweet vermouth.
High and medium strength liqueurs are used Ior liqueurs and bitters.

* Only 1 consumer.
** Two intake estimates were made, including and excluding the contribution Irom sweets. Although the Committee is
proposing a maximum limit oI 50 mg/kg in sweets, the UK has only been able to identiIy one product, a herbal lozenge,
which Ialls into this category, as has previously been reported. However, as no consumption data Ior this speciIic product is
available, consumption data Ior all boiled sweets were used instead, which results in a very large over-estimate.

150
Table II. Estimated intake oI thujone by adults Irom sage-Ilavoured Ioods (Council oI Europe,
2000)
Intake
(micrograms/person/day)
Intake
(micrograms/kg bw/day)
Foods / Beverages Proposed
limit
(mg/kg)
mean 97.5th
percentile
mean 97.5th
percentile
Sage-Ilavoured sausages 10 175.3 536.6 2.5 7.7
StuIIing 10 74.0 204.3 1.1 2.9
Herb vinegar 5 12.6 86.9 0.2 1.1
Other meat dishes 10 389.3 1148 5.7 15.3
Sage Derby` cheese* 10 128.6 128.6 1.4 1.4
Salad dressings 10 55 152 0.9 2.5
Vermouth 2 44.2 177.9 0.7 2.3
Liqueurs & bitters 10 99.7 474 1.6 5.9
(excl. sweets)``
244.8 911.4 3.6 13.5
Sweets`` 50 195.8 900.2 3.0 13.4
(incl. sweets)``
263.5 984.7 3.9 14.2
Assumptions.
All categories oI pork sausages are considered to be sage-Ilavoured.
All types oI stuIIing are considered to be sage Ilavoured. StuIIing includes sausage and
sausagement stuIIings.
Other meat dishes` includes dishes known to contain sage or pork sausages, and other meat
products (meat loaI, chicken liver pate and liver sausage) that are considered to be sage-
Ilavoured.
All salad dressings (excluding salad cream and French dressing) are considered to contain sage.
All vinegar is assumed to be herb vinegar.
Vermouth includes both dry and sweet vermouth.
High and medium strength liqueurs are used Ior liqueurs and bitters.

Footnotes.
* Only 1 consumer.
** Two intake estimates were made, including and excluding the contribution Irom sweets. Although the
Committee is proposing a maximum limit oI 50 mg/kg in sweets, the UK has only been able to identiIy one
product, a herbal lozenge, which Ialls into this category, as has previously been reported. However, as no
consumption data Ior this speciIic product is available, consumption data Ior all boiled sweets were used
instead, which results in a very large over-estimate.

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