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 This report contains the collective views of an international group of experts and does not necessarily represent the decisions
or the stated policy of the World Health Organization or the Food and Agriculture Organization of the United Nations

WHO Technical Report Series

900

EVALUATION OF CERTAIN
VETERINARY DRUG
RESIDUES IN FOOD

Fifty-fourth report of the

Joint FAO/WHO Expert Committee on
Food Additives

World Health Organization

Geneva 2001
WHO Library Cataloguing-in-Publication Data

Joint FAO/WHO Expert Committee on Food Additives (2000: Geneva, Switzerland)

Evaluation of certain veterinary drug residues in food : fifty-fourth report of the
Joint FAO/WHO Expert Committee on Food Additives.

(WHO technical report series ; 900)

1 .Veterinary drugs—toxicity 2.Drug residues—analysis 3.Drug residues—toxicity

4.Maximum allowable concentration—standards S.Food contamination I.Title
II.Series

ISBN 92 4 120900 3 (NLM classification: WA 712)

ISSN 0512-3054

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© World Health Organization 2001

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Typeset in Hong Kong
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2000/13520 — Best-set/Schuler — 6000
Contents

1. Introduction 1

2. General considerations 2
2.1 Modification of the agenda 2
2.2 Interpretation of data on inhibition of cholinesterase activity 2
2.3 Consideration of recommendations arising from an informal
meeting on harmonization with the Joint FAO/WHO Meeting on
Pesticide Residues 3

3. Comments on residues of specific veterinary drugs 7

3.1 Anthelminthic agent 7
3.1.1 Ivermectin 7
3.2 Antimicrobial agents 9
3.2.1 Flumequine 9
3.2.2 Lincomycin 13
3.2.3 Oxytetracycline 29
3.2.4 Tilmicosin 29
3.3 Insecticides 30
3.3.1 Cyhalothrin 30
3.3.2 Cypermethrin 41
3.3.3 a-Cypermethrin 42
3.3.4 Dicyclanil 43
3.3.5 Permethrin 52
3.3.6 Metrifonate (trichlorfon) 53
3.4 Production aid 64
3.4.1 Melengestrol acetate 64

4. Future work 80

5. Recommendations 80

Acknowledgement 81

References 81

Annex 1
Reports and other documents resulting from previous meetings of the
Joint FAO/WHO Expert Committee on Food Additives 83

Annex 2
Recommendations on compounds on the agenda and further information
required 91

Annex 3
Proposed draft definitions of commodities for Volume 3 of Residues of
veterinary drugs in foods 96

iii
Joint FAO/WHO Expert Committee on Food Additives
Geneva, 15-24 February 2000

Members
Professor A. Anadon, Department of Toxicology and Pharmacology, Faculty of
Veterinary Medicine, Universidad Complutense de Madrid, Madrid, Spain

Dr L.-E. Appelgren, Professor of Pharmacology, Department of Pharmacology and

Toxicology, Faculty of Veterinary Medicine, The Swedish University of Agricul-
tural Sciences, Uppsala, Sweden

Dr D. Arnold, Acting Director, Federal Institute for Health Protection of Consumers

and Veterinary Medicine, Berlin, Germany

Dr J. Boisseau, Director, National Agency for Veterinary Medicinal Products,

French Food Safety Agency, Fougeres, France (Vice-Chairman)

Professor A.R. Boobis, Section on Clinical Pharmacology, Division of Medicine,

Imperial College School of Medicine, Hammersmith Campus, London, England
(Joint Rapporteur)

Professor L.D.B. Kinabo, Department of Veterinary Physiology, Biochemistry, Phar-

macology and Toxicology, Sokoine University of Agriculture, Morogoro, United
Republic of Tanzania

Dr J.G. McLean, South Melbourne, Victoria, Australia (Chairman)

Dr J.D. MacNeil, Head, Centre for Veterinary Drug Residues, Health of Animals
Laboratory, Canadian Food Inspection Agency, Saskatoon, Saskatchewan,
Canada (Joint Rapporteur)
Dr M.A. Miller, Office of Women's Health, Food and Drug Administration, Rockville,
MD, USA

Professor J. Palermo-Neto, Applied Pharmacology and Toxicology Laboratory,

Department of Pathology, School of Veterinary Medicine, University of Sao
Paulo, Sao Paulo, Brazil

Dr J.L. Rojas Martinez, Chief, Toxicology and Residues Section, National Labora-
tory of Veterinary Sciences, Ministry of Agricultural and Animal Husbandry, San
Jose, Costa Rica

Dr S. Soback, Head, National Residue Laboratory, Kimron Veterinary Institute,

Ministry of Agriculture, Beit Dagan, Israel
Dr R.W. Stephany, Head, Laboratory for Residue Analysis, National Institute of
Public Health and the Environment, Bilthoven, Netherlands

Secretariat
Dr S.M.F. Calumpang, National Crop Protection Centre, University of the Philip-
pines, Los Banos, Laguna, Philippines (FAO Consultant)

Dr C.E. Cerniglia, Director, Division of Microbiology and Chemistry, National Cen-

ter for Toxicological Research, Food and Drug Administration, Jefferson, AR,
USA (WHO Temporary Adviser)

iv
Dr R.L. Ellis, Nutrition Officer, Food Quality Liaison Group, Food Quality and
Standards Service, Food and Nutrition Division, FAO, Rome, Italy (Joint
Secretary)

Dr L.G. Friedlander, Center for Veterinary Medicine, Food and Drug Administration,
Rockville, MD, USA (FAO Consultant)

Dr K. Greenlees, Center for Veterinary Medicine, Food and Drug Administration,

Rockville, MD, USA (WHO Temporary Adviser)

Mr D.J. Hamilton, Principal Scientific Officer, Animal and Plant Health Service,
Department of Primary Industries, Brisbane, Australia (FAO Consultant)

Dr R.J. Heitzman, Science Consultant, Newbury, Berkshire, England (FAO

Consultant)

Dr J.L. Herrman, Scientist, International Programme on Chemical Safety, WHO,

Geneva, Switzerland (Joint Secretary)

Dr R.C. Livingston, Center for Veterinary Medicine, Food and Drug Administration,
Rockville, MD, USA (FAO Consultant)

Dr K. Mitsumori, Chief, Third Section, Division of Pathology, Biological Safety

Research Centre, National Institute of Health Sciences, Tokyo, Japan (WHO
Temporary Adviser)

Professor M.R.A. Morgan, Procter Department of Food Science, University of

Leeds, Leeds, England (FAO Consultant)

Mrs M.E.J. Pronk, Engineer, Center for Substances and Risk Assessment, National
Institute of Public Health and the Environment, Bilthoven, Netherlands (WHO
Temporary Adviser)

Mr D. Renshaw, Principal Scientist, Veterinary Products, Department of Health

Food Standards and Safety Group, Department of Health, Joint Ministry of Food
and Fisheries, London, England (WHO Temporary Adviser)

Dr L. Ritter, Executive Director, Canadian Network of Toxicology Centres, Univer-

sity of Guelph, Guelph, Ontario, Canada (WHO Temporary Adviser)

Dr G. Roberts, Manager, Chemical Products Assessment Section, Therapeutic

Goods Administration, Commonwealth Department of Health and Aged Care,
Woden, Australian Capital Territory, Australia (WHO Temporary Adviser)

Dr B. Roestel, Head, International Affairs, National Agency for Veterinary Medicinal

Products, French Food Safety Agency, Fougeres, France (FAO Consultant)

Dr S. Sundlof, Director, Center for Veterinary Medicine, Food and Drug Administra-
tion, Rockville, MD, USA (WHO Temporary Adviser)

Dr A. Tejada, FAO Joint Secretary of the Joint FAO/WHO Meeting on Pesticide

Residues, Pesticide Management Group, Plant Production and Protection Divi-
sion, FAO, Rome, Italy

Professor F.R. Ungemach, Institute of Pharmacology, Pharmacy and Toxicology,

Faculty of Veterinary Medicine, University of Leipzig, Leipzig, Germany (WHO
Temporary Adviser)

v
 Dr R. Wells, Gordon, New South Wales, Australia (FAO Consultant)
Dr Y. Yamada, Food Standards Officer, Joint FAO/WHO Standards Programme,
Food and Nutrition Division, FAO, Rome, Italy
Professor J. Zmudzki, Head, Department of Pharmacology and Toxicology, Na-
tional Veterinary Research Institute, Pulawy, Poland (FAO Consultant)

vi
Monographs containing summaries of relevant data and toxicological evalu-
ations are available from WHO under the title:
Toxicological evaluation of certain veterinary drug residues in food. WHO
Food Additives Series, No. 45, 2000.
Residues monographs are issued separately by FAO under the title:
Residues of some veterinary drugs in animals and foods. FAO Food and
Nutrition Paper, No. 41/13, 2000.

INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

The preparatory work for toxicological evaluations of food additives and
contaminants by the Joint FAO/WHO Expert Committee on Food Addi-
tives (JECFA) is actively supported by certain of the Member States that
contribute to the work of the International Programme on Chemical
Safety (IPCS).
The International Programme on Chemical Safety (IPCS) is a joint ven-
ture of the United Nations Environment Programme, the International
Labour Organization, and the World Health Organization. One of the
main objectives of the IPCS is to carry out and disseminate evaluations
of the effects of chemicals on human health and the quality of the
environment.
This page intentionally left blank
1. Introduction
A meeting of the Joint FAO/WHO Expert Committee on Food Ad-
ditives was held at WHO Headquarters, Geneva, from 15 to 24 Feb-
ruary 2000. The meeting was opened by Dr T. Meredith, Coordinator,
Chemical Safety, Department of Protection of the Human Environ-
ment, WHO, on behalf of the Directors-General of the Food and
Agriculture Organization of the United Nations and the World
Health Organization.
Dr Meredith noted that many scientific principles and evaluation
procedures had been developed by the Committee over the past
13 years during its meetings on residues of veterinary drugs in food.
The Conference on International Food Trade Beyond the Year 2000,
held in Melbourne, Australia, in October 1999 (7), had recommended
that WHO update the scientific principles used by the Committee and
the Joint FAO/WHO Meeting on Pesticide Residues to evaluate resi-
dues of veterinary drugs and other chemicals in food (food additives,
pesticides and contaminants) and publish the principles in a single
document. Dr Meredith reported that WHO was considering ways
of responding to that recommendation. The FAO Secretariat had
already consolidated the principles for the evaluation of residues
of veterinary drugs in food, and the document that had been pro-
duced should be useful both to the Committee and to others
interested in understanding its evaluation procedures. The document
would also be useful to WHO when it critically reviews the principles
for evaluating veterinary drug residues in food and consolidates
them in response to the recommendation of the conference held in
Melbourne.
Twelve previous meetings of the Committee had been held to con-
sider veterinary drug residues in food (Annex 1, references 80,85,91,
97, 104, 110, 113, 119, 125, 128, 134 and 140) in response to the
recommendations of a Joint FAO/WHO Expert Consultation held in
1984 (2). The present meeting1 was convened in response to a recom-
mendation made at the fifty-second meeting of the Committee that
meetings on this subject should be held annually (Annex 1, reference
140). The Committee's purpose was to provide guidance to FAO and
WHO Member States and to the Codex Alimentarius Commission on
public health issues pertaining to residues of veterinary drugs in foods

1
As a result of the recommendations of the first Joint FAO/WHO Conference on Food
Additives held in 1955 (FAO Nutrition Meeting Report Series, No. 11, 1956; WHO
Technical Report Series, No. 107, 1956), there have been 53 previous meetings of the
Joint FAO/WHO Expert Committee on Food Additives (Annex 1).

1
 of animal origin. The specific tasks before the Committee were:
— to elaborate further principles for evaluating the safety of residues
of veterinary drugs in food, for establishing Acceptable Daily
Intakes (ADIs), and for recommending Maximum Residue Limits
(MRLs) for such residues when the drugs under consideration are
administered to food-producing animals in accordance with good
practice in the use of veterinary drugs (see section 2); and
— to evaluate the safety of residues of certain veterinary drugs (see
section 3 and Annex 2).

2 General considerations
2.1 Modification of the agenda
Temefos was removed from the agenda since no data had been
submitted.
2.2 Interpretation of data on inhibition of cholinesterase activity
The Committee considered the issue of interpreting data on inhibi-
tion of the activity of various cholinesterases, in particular brain and
erythrocyte acetylcholinesterase and plasma and brain butyrylcholine
esterase.
The Committee considered the report of a consultation on interpreta-
tion of inhibition of acetylcholinesterase activity (3) and the report of
the 1998 Joint FAO/WHO Meeting on Pesticide Residues (4), which
included a section on this topic. These reports were considered to
have been helpful, and the Committee agreed with their conclusions.
It particularly welcomed the guidance relating to methodological is-
sues in assessing cholinesterase activity provided in Annex 1 of the
report of the consultation.
The Committee agreed that inhibition of brain acetylcholinesterase
activity and clinical signs of neurobehavioural effects are the end-
points of greatest concern in toxicological studies of compounds that
inhibit acetylcholinesterase. The possibility of inhibition of acetylcho-
linesterase activity in the peripheral nervous system is also of concern,
but the Committee recognized that information suitable for assessing
such activity is not often available. It agreed that erythrocyte acetyl-
cholinesterase activity could serve as a surrogate for acetylcholin-
esterase activity in the peripheral nervous system and brain when
information on the latter was not available.
The Committee agreed that statistically significant inhibition of ace-
tylcholinesterase activity by 20% or more should be regarded as a
treatment-related effect and could form the basis for establishing an

2
 ADI if it was the most sensitive end-point, although each compound
should be considered on a case-by-case basis.
The Committee concluded that inhibition of brain and plasma buty-
rylcholinesterase activity is not of toxicological significance for
establishing an ADI, but that information on inhibition of these en-
zymes should nevertheless be provided, as it is a useful indicator of
absorption of a cholinesterase inhibitor and of occupational exposure.

2.3 Consideration of recommendations arising from an informal

meeting on harmonization with the Joint FAO/WHO Meeting
on Pesticide Residues
A meeting to harmonize the work of the Committee and the Joint
FAO/WHO Meeting on Pesticide Residues (JMPR) was held in
Rome in February 1999, at which issues relating to the evaluation of
chemicals used as both pesticides and veterinary drugs were dis-
cussed. It was noted that differences in the evaluation procedures
used by the Committee and JMPR had led to different approaches to
the definition of residues, estimation of dietary intake, description of
commodities for analysis and recommendations for MRLs. Other
topics discussed at the meeting included tissue matrices used for
the analysis of residues in meat/muscle, fat, milk and eggs, and risk
assessment.
At its present meeting, the Committee considered the recommenda-
tions summarized in the report of the informal meeting on harmoni-
zation (5) and addressed those directed to it, which are listed below
with comments.

Tissue
Clarification of the definition of muscle tissue (in Volume 3 of Residues of
veterinary drugs in foods [6]) is needed to establish the portion of the
commodity to which the MRL applies. Muscle tissue shall include interstitial
fat and exclude trimmable fat.
The Committee agreed with the recommendation to clarify the por-
tion of the commodity to which the MRL applies and will recommend
a revised definition.
For the determination of fat-soluble pesticide/veterinary drug residues in
meat/muscle for enforcement or monitoring purposes, laboratories are
advised to collect and to analyse trimmable fat and to report the residues on
a lipid basis, i.e. meat (fat) for JMPR and fat for JECFA. For meat without
trimmable fat, the entire commodity should be analysed as meat/muscle,
but only where the MRL has been set on a meat/muscle basis.
The Committee agreed that guidance for laboratories based on the
proposed definitions of muscle and fat that were drafted at its present

3
 meeting (see Annex 3) should be submitted to the Codex Committee
on Residues of Veterinary Drugs in Foods for consideration. It did,
however, note that such guidance is, in general, the responsibility of
national authorities.
For the determination of non-fat-soluble pesticides/veterinary drug residues
in meat/muscle, laboratories are advised to analyse meat/muscle with
trimmable fat removed, as far as is practical.

The Committee agreed that laboratories should analyse samples on

the basis of the proposed draft definitions of muscle and fat to be
submitted to the Codex Committee on Residues of Veterinary Drugs
in Foods. It did, however, note that such guidance is, in general, the
responsibility of national authorities.
Where JECFA and JMPR have recommended MRLs for the same chemical
with the same residue/marker definition for the same commodity, the higher
MRL shall prevail.

The Committee agreed that when two different MRLs exist, the
higher value should prevail, provided that it does not result in the
intake of residues exceeding the ADI. The risk assessment proce-
dures used by the Committee and JMPR are designed in such a way
that no adverse effects on public health would be expected to occur
when residues do not exceed the MRL.

Milk
For the determination of fat-soluble pesticide/veterinary drug residues in
milk, the milk fat portion of fresh milk should be analysed, and the results
expressed on a whole milk basis using 4% as the nominal fat content.

The Committee agreed with this recommendation.

For the determination of non-fat-soluble pesticide/veterinary drug residues
in milk, laboratories should analyse the whole milk and should report
residues on a whole milk basis.

The Committee agreed with this recommendation.

JECFA should consider expressing MRLs for milk on a weight (kg) basis
rather than the current volume (I) basis.

The Committee noted that this recommendation had already been

implemented. The reporting of residue concentrations on a weight-
per-weight basis is consistent with internationally recommended ana-
lytical protocols.

Eggs
JECFA should specify that the portion of the raw commodity "egg" (in shell)
to be analysed is the whole egg white and yolk combined after removal of
the shell.

4
The Committee agreed with the recommendation that the current
definition should be changed to avoid any ambiguity about the por-
tion of egg to be analysed for residues.

Harmonization
The working group noted the disparate residue definitions by the Codex
Committee on Pesticide Residues and the Codex Committee on Residues
of Veterinary Drugs in Foods for abamectin and recommended that the
Codex Committee on Residues of Veterinary Drugs in Foods and JECFA
consider expansion of its residue definition to include other isomers, such
as the photodegradation isomer of B1a.
The Committee carefully considered the toxicological and chemical
assessments of abamectin made by JMPR and concluded that inclu-
sion of the photodegradation isomer in the residue definition would
not be consistent with the assessment of abamectin as a veterinary
drug. Inclusion of other residues of abamectin would be reviewed at
a future meeting of the Committee.
Cypermethrin and a-cypermethrin should remain as the marker residue
definitions for their use as veterinary drugs for cypermethrin and a-
cypermethrin, respectively, and cypermethrin (sum of isomers) should
remain as the residue definition for the pesticide cypermethrin. Guidance
should be supplied to laboratories on the designation of the measured
residue as cypermethrin or a-cypermethrin based on the chromatography
of the test substance.
The Committee agreed with the recommendation on definition of
marker residues for the various uses. The Committee recognized that
the currently available analytical methods allow measurement of all
isomers of interest. Accordingly, guidance should be provided to
assist Member States in providing specific advice to their residue
control authorities.
Harmonization efforts should be undertaken on a case-by-case basis where
marker residue definition/residue definition differences occur between
JECFA and JMPR.
The Committee agreed with this recommendation and will coordinate
with the JMPR Secretariat when necessary.
JECFA should review the apparent anomaly of MRLs for both fat and
muscle for the fat-soluble drugs a-cypermethrin and cypermethrin. JECFA
should consider which sample tissues are to be analysed by the
enforcement laboratory.
The Committee agreed in principle with this recommendation, but
noted that the temporary MRLs for a-cypermethrin and cyper-
methrin were not extended at the present meeting because the re-
quired data were not provided. The Committee indicated that some
MRLs are recommended only for guidance; an example is the MRL
for metrifonate in the edible tissues of cattle treated in accordance

5
 with good practice in the use of veterinary drugs, where there are no
measurable residues. Such "guidance MRLs" should not be consid-
ered in calculations of the theoretical maximum intake of residues
and are not intended for residue control purposes.
For compounds that are common to both, JMPR and JECFA should use the
more specific animal commodity descriptions to enhance harmonization.
The Committee agreed with this recommendation and has routinely
noted the descriptions of specific animal commodities in recommend-
ing MRLs for residues of veterinary drugs in food. These descriptions
are useful to JMPR in their assessment of risks associated with
exposure to residues of pesticides.
Each expert panel needs a better understanding of the other's procedures
for food safety assessments for estimating MRLs and dietary exposure.
. . . JECFA will provide JMPR its guidance document describing the JECFA
evaluation procedures when the draft version is finalized.
The Committee agreed with this recommendation and has provided a
guidance document (7) to the JMPR Secretariat. The document
would also be made available to delegates at the Twelfth Session of
the Codex Committee on Residues of Veterinary Drugs in Foods, to
be held in March 2000.
The JECFA/JMPR group acknowledged the very different approaches used
for dietary exposure determinations. JMPR will provide JECFA with detailed
reports of its assessments, dietary intake calculations and per cent ADI
determinations for compounds of interest to JECFA. When the data are
available, JECFA will provide JMPR with median and upper limit animal
commodity residue values and dietary intake calculations/per cent ADI
determinations for compounds of interest to JMPR.
The Committee acknowledged that the way in which it calculates
dietary intake differs widely from that of JMPR. To facilitate intake
assessments by JMPR, the Committee would provide data on residues
of veterinary drugs of interest when they are available.
JECFA and JMPR should consider the exchange of one panel member
each for a portion of the expert panel meetings to facilitate the
harmonization of MRLs and risk assessment for substances used as
veterinary drugs and pesticides.
The Committee agreed with this recommendation. Two JMPR mem-
bers participated in the present meeting.
The FAO Joint Secretary for JMPR will attend the JECFA meeting and the
FAO Joint Secretary for JECFA will attend the JMPR meeting, particularly
when MRLs and risk assessments of substances used as both veterinary
drugs and pesticides are being considered.
The Committee agreed that both FAO Joint Secretaries should par-
ticipate in meetings at which a substance used as both a veterinary
drug and a pesticide is being considered.

6
 Joint meetings of JMPR and JECFA should be held on an ad hoc basis to
address issues of mutual interest (for example, how to address MRL and
ADI issues for classes of compounds with common modes of action).
The Committee agreed with this recommendation. Ad hoc meetings
of members of JECFA and JMPR and of the Joint Secretaries are
appropriate for resolving issues that might impede an assessment of
the risk posed by a compound or class of compounds, particularly on
general principles for risk assessments.
For compounds of mutual interest, JMPR and JECFA should have each
other's recommendations/reports available when conducting evaluations.
The Joint Secretaries will have the responsibility for obtaining and
distributing the documents and information, as appropriate.
The Committee agreed with this recommendation. The Joint Secre-
taries should ensure that for compounds already evaluated by the
other group, the evaluation report is provided to the person(s)
reviewing the compound, so that it is considered when the substance
is evaluated.
The proposed draft definitions of commodities for Volume 3 of Resi-
dues of veterinary drugs in foods (6) are listed in Annex 3.

3 Comments on residues of specific veterinary

drugs
The Committee considered one antimicrobial agent, four insecticides
and one production aid for the first time. It reconsidered one anthel-
minthic agent, three antimicrobial agents and two insecticides. The
recommendations made with regard to these substances and details of
further studies and other information required are summarized in
Annex 2.
Toxicological monographs or monograph addenda were prepared on
all of the compounds on which toxicological evaluations were per-
formed. Residue monographs were prepared on all of the substances
reviewed.

3.1 Anthelminthic agent

3.1.1 Ivermectin
Ivermectin is widely used as a broad-spectrum antiparasitic drug
against nematodes and arthropods in food-producing animals. In hu-
man medicine, it is used mainly for the treatment of onchocerciasis.
Ivermectin is a mixture of two isomers. It contains 80% of the
isomer 22,23-dihydroavermectin Bla (ivermectin Bla) and =£20% of
the isomer 22,23-dihydroavermectin Blb (ivermectin Blb). Ivermectin

7
 was previously reviewed by the Committee at its thirty-sixth and
fortieth meetings (Annex 1, references 91 and 104). At the latter
meeting, the Committee allocated an ADI of 0-lmg/kg of body
weight and recommended MRLs in cattle of 100(mg/kg for liver and
40mg/kg for fat as ivermectin Bla. At its present meeting, the Commit-
tee reviewed a study in which the drug was applied topically to dairy
cows.
Residue data
Groups of six lactating Holstein-Friesian and six lactating Jersey
dairy cows were treated topically with a single dose of ivermectin
of approximately 0.58mg/kg of body weight (recommended dose =
0.5mg/kg of body weight). Animals of the two breeds differed in a
number of characteristics which might have influenced the depletion
of residues of ivermectin in milk. For example, the Holstein-Friesian
cows weighed 560-640 kg and were in about the middle of the second
to eighth lactation period, while the Jersey cows weighed 370-430 kg
and were at the beginning of the third or fourth lactation period.
During the first 220 h after treatment, the milk yield of the Holstein-
Friesian cows was 250-310 kg containing 9.9-12 kg of milk fat, while
that of the Jersey cows was 140-200 kg containing 7.4-13 kg of milk
fat. During that period, the total amount of residues secreted into the
milk was 0.6-1.0mg or 0.2-0.3% of the dose for the Holstein-Friesian
cows and 0.7-1.4 mg or 0.3-0.5% of the dose for the Jersey cows.
Milk samples from the two breeds were collected twice daily and
analysed by a method with a reported limit of detection of 1mg/kgfor
both ivermectin Bla and ivermectin B1b.
The concentrations of ivermectin Bla in the milk of the Holstein-
Friesian cows reached a maximum at the third or fourth milking after
treatment; and subsequently one or more, usually broader, maxima
were reached. The later maximum concentrations were typically
lower than the first one, except in milk obtained from one cow in
which the highest concentration was reached at the tenth milking,
about 130 h after treatment. In Jersey cows, the kinetics of depletion
of residues of ivermectin in milk was similar, but the maximum con-
centrations were generally lower.
In the Holstein-Friesian cows, the highest concentrations of ivermectin
Bla in the milk of individual animals during the period of observation
were 5-10(mg/kg. In the Jersey cows, the highest concentrations
of ivermectin Bla in milk were 10-18mg/kg. The contribution of resi-
dues of ivermectin B1b to the concentration of total residues was
insignificant in both studies and typically below the reported limit of
detection.

8
 Analytical methods
A method for the identification and quantification of ivermectin Bla
and ivermectin B1b residues in milk was available. It is a modification
of a published method considered by the Committee at its thirty-sixth
meeting (Annex 1, reference 97) and involves separation by high-
performance liquid chromatography (HPLC) and detection of deriva-
tives of the parent compounds by fluorescence. The method reviewed
by the Committee at its present meeting did not conform with Good
Laboratory Practice (GLP), and an incomplete set of data on its
validation was made available by the sponsor. Neither the limits of
detection nor the limits of quantification for the two compounds were
determined. The recovery of the method was estimated in tests with
milk containing ivermectin Bla at 5,25 and 50mg/kg and ivermectin B1b
at 2 and 4 mg/kg; however, many of the milk samples obtained in the
residue-depletion study contained lower concentrations.

Maximum Residue Limits

In reaching its decision on MRLs for ivermectin, the Committee took
the following factors into account:
• The ADI of 0-lmg/kg of body weight, which is equivalent to a
maximum ADI of 60mg for a 60-kg person.
• The previously recommended MRLs of 100 mg/kg for liver and
40mg/kg for fat in cattle account for 39.4mg of the maximum ADI,
leaving about 21 mg to be ingested from milk.
• A concentration of about 10 mg/kg of ivermectin Bla in whole milk
would result in an additional 15mg of residues, based on a daily
intake of 1.5kg of milk.
The Committee recommended a temporary MRL of 10mg/kg for
whole milk in cattle, expressed as ivermectin B1a.
The Committee was aware that with the formulation of ivermectin that
was reviewed, this temporary MRL would require the milk of up to 11
milkings to be discarded. The MRL could, however, serve as a basis for
the development of other formulations and/or other conditions of use.
The full set of data for validation of the analytical method and infor-
mation on other routes of application of ivermectin to cattle are
required to evaluate the residues in milk in 2002.

3.2 Antimicrobial agents

3.2.1 Flumequine
Flumequine is a fluoroquinolone antimicrobial agent predominantly
active against Gram-negative bacteria, which is used to control infec-
tions in beef cattle, sheep, pigs, chickens and farmed trout.

9
 Flumequine was previously considered by the Committee at its forty-
eighth meeting (Annex 1, reference 128), when it established an ADI
of 0-30mg/kg of body weight and recommended MRLs in cattle of
500mg/kg for muscle, 1000mg/kg for liver, 3000mg/kg for kidney and
1000mg/kg for fat, expressed as parent drug. These values would give
a theoretical maximum daily intake of residues of 1100mg, which is
within the maximum ADI of1800mgfor a 60-kg person.
In the absence of data on the contribution of parent drug to the total
residues in sheep, chickens and pigs, the Committee at its forty-eighth
meeting recommended temporary MRLs in these species of 500(mg/kg
for muscle, 1000mg/kg for liver, 3000mg/kg for kidney and 1000mg/kg
for fat, expressed as parent drug. The Committee also recommended
a temporary MRL of 500mg/kg for trout muscle, including skin in
normal proportions, expressed as parent drug.
At its forty-eighth meeting, the Committee requested that studies be
conducted with radiolabelled flumequine in pigs, sheep, chickens and
trout in order to estimate the proportion of the total residues
accounted for by the parent drug. The results of these studies were
required for evaluation in 2000.

Residue data
Cattle. Three male and three female beef cattle, weighing 125-135 kg,
were given [14C]flumequine for 5 consecutive days at a dose of
12mg/kg of body weight by subcutaneous injection into the neck. All
of the animals were killed 18 h after the final injection. Muscle, liver,
kidney, fat and tissue at the site of injection were analysed by HPLC
for determination of total radiolabelled residues.
About 17% of the radiolabel in liver at the time of slaughter was
associated with unmetabolized flumequine and the remainder with
metabolites or bound residues. Flumequine accounted for about 80%
of the total residues in muscle and kidney and for almost all of those
in fat and tissue at the site of injection.
Pigs. Three male and three female pigs weighing 45-49 kg were given
[14C]flumequine intramuscularly into the neck at an initial dose of
15mg/kg of body weight, followed by nine doses of 7.5mg/kg of body
weight at 12-hourly intervals. The animals were killed 16 h after the
final dose. Samples of muscle, liver, kidney, fat and tissue at the site of
injection were analysed by HPLC to determine the total radiolabelled
residues.
About 7% of the radiolabel in liver at the time of slaughter was
associated with unmetabolized flumequine and the remainder with

10
metabolites or bound residues. Flumequine accounted for about 75%
of the total residues in muscle and fat, and for 44% and 55% of those
in kidney and skin with adhering fat, respectively.
Sheep. Three male and three female sheep weighing 38-50 kg were
given [14C]flumequine intramuscularly into the neck at an initial dose
of 12mg/kg of body weight, followed by nine doses of 6mg/kg of body
weight at 12-hourly intervals. The animals were killed 16 h after the
final injection. Samples of muscle, liver, kidney, fat and tissue at the
site of injection were analysed by HPLC for determination of total
radiolabelled residues.
About 6% of the radiolabel in liver at the time of slaughter was
associated with unmetabolized flumequine and the remainder with
metabolites or bound residues. Unmetabolized flumequine accounted
for about 49% of the radiolabel in muscle and for 35% and 56% of
that in kidney and fat, respectively.
Chickens. Three male and three female broiler chickens weighing 2.2-
2.6kg were given [14C]flumequine by gavage into the crop at a dose of
18mg/kg of body weight for 5 consecutive days. All of the birds were
killed 12h after the final dose. Samples of muscle, liver, kidney, fat
and skin with adhering fat were analysed by HPLC for determination
of total radiolabelled residues.
Unmetabolized flumequine accounted for about 94% of the radiola-
bel in muscle and for about 70%, 76%, 100% and 77% of that in liver,
kidney, fat and skin with adhering fat, respectively.
Trout. Two groups of 20 rainbow trout with average weights of 90 g
and l00g were maintained in separate tanks with water temperatures
of 7°C and 16 °C, respectively. A single dose of [14C]flumequine was
administered by gavage as a 2% formulation in lactose enclosed in a
gelatine capsule at a dose of about 12mg/kg of body weight. The doses
received by individual fish were calculated from their weight at
slaughter. Five trout from the group maintained at 16 °C were slaugh-
tered 18 h after treatment and five 36 h after treatment, and five trout
from the group maintained at 7°C were slaughtered at 36 h and five at
96 h.
The results of HPLC and radiochemical analyses at the time of
slaughter showed no detectable metabolism of flumequine at any time
at either temperature.

Analytical methods
An HPLC method with fluorescence detection for determining
flumequine and its metabolite, 7-hydroxyflumequine, was used in

11
 the residue-depletion studies evaluated by the Committee at its
forty-eighth meeting. The method evaluated by the Committee at its
present meeting did not include an enzymatic hydrolysis step and had
only been validated for flumequine.
The flumequine residues were extracted with ethyl acetate, and iba-
floxacin was added to the sample extract after purification by liquid-
liquid extraction to check the retention time. An extra step was added
for all samples with a high fat content, in which the extract was parti-
tioned between acetonitrile and hexane. Separation and quantifica-
tion of flumequine were achieved by HPLC on a C18 column with
gradient elution involving various mixtures of acetonitrile and aque-
ous oxalic acid at a concentration of 2.7 xl0-3mol/l. Quantification
was achieved using a fluorescence detector and by comparison of the
results with a calibration curve constructed from data obtained by
analysis of tissue samples fortified with flumequine. The linearity,
accuracy, repeatability, and limits of detection and quantification of
the method were tested in a single laboratory and found to be accept-
able. The limit of quantification of the method was 50mg/kg for all
tissues, and the limit of detection was 10-25 mg/kg. The recovery was
greater than 75% for all tissues.
The Committee was aware that a number of suitable analytical meth-
ods are available for measuring flumequine residues in the edible
tissues of food-producing animals.
Maximum Residue Limits
In reaching its decision on MRLs for flumequine, the Committee took
the following factors into account:
• The ADI of 0-30 jig/kg, which corresponds to a maximum ADI of
1800 mg for a 60-kg person.
• On the basis of the residue-depletion study in cattle treated with
radiolabelled flumequine, the parent drug accounted for 79-100%
of the total residues in muscle, kidney and fat. These values are
somewhat greater than the value of 50% estimated from the studies
reviewed by the Committee at its forty-eighth meeting. The parent
drug also accounted for 17% of the total residues in liver, which is
lower than the value of 25% estimated from the studies reviewed at
the forty-eighth meeting.
• On the basis of the residue-depletion studies in sheep treated with
radiolabelled flumequine, the parent drug accounted for 40%
(range, 35-56%) of the total residues in muscle, kidney and fat, and
for 6% of those in liver.
• On the basis of the residue-depletion studies in pigs treated with
radiolabelled flumequine, the parent drug accounted for 59%

12
Table 1
Theoretical maximum daily intake of flumequine residues
Tissue Recommended Estimate of total Theoretical maximum daily intakeb
MRL (mg/kg)a residues (mg/kg) (mg flumequine equivalents)
Muscle 500 1250C 375
Liver 500 8300d 830
Kidney 3000 7500C 375
Fat 1000 2500C 125
Total 1705
a
Expressed as parent drug.
b
Based on a daily intake of 300g of muscle, 100g of liver, and 50g each of kidney and fat.
0
The marker residue accounted for 40% of the total residues in sheep muscle, kidney and fat.
d
The marker residue accounted for 6% of the total residues in sheep liver.

(range, 44-75%) of the total residues in muscle, kidney and fat, and
for 7% of those in liver.
• Flumequine undergoes less metabolism in chickens than in mam-
malian species. On the basis of the residue-depletion studies in
chickens treated with radiolabelled flumequine, the parent drug
accounted for 82% (range, 70-94%) of the total residues.
• There were no measurable residues of flumequine metabolites in
trout.
• Suitable analytical methods are available for measuring flumequine
residues.
The Committee recommended MRLs for flumequine of 500mg/kg for
muscle and liver, 3000 jig/kg for kidney and 1000 mg/kg for fat in cattle,
pigs, sheep and chickens, expressed as parent drug. The Committee
also recommended an MRL of 500mg/kgfor trout muscle with skin in
normal proportions.
From the values for the MRLs for sheep, the theoretical maximum
daily intake of flumequine residues would be approximately 1700mg
(Table 1). This would be within the maximum ADI of 1800mg for a
60-kg person.

3.2.2 Lincomycin
Lincomycin belongs to the galacto-octopyranoside class of antibio-
tics known as lincosaminides, which also includes pirlimycin and
clindamycin. It is mainly active against Gram-positive bacteria and
acts on the 50S subunit of the ribosome to inhibit RNA-dependent
protein synthesis. Lincomycin is used alone or in combination with
other antimicrobials such as spectinomycin, neomycin, sulfadiazine
and sulfadimidine. It can be given orally in feed or drinking-water, by
intramuscular injection, or as an intramammary infusion.

13
 The recommended doses are: 0.5 mg/kg of body weight in feed and 3-
50mg/kg of body weight in drinking-water for poultry; 0.2-13 mg/kg
of body weight in feed, 5-10 mg/kg of body weight in drinking-
water, and 5-10 mg/kg of body weight intramuscularly in pigs; 5 mg/kg
of body weight intramuscularly in calves and sheep; and 200-
330mg/quarter of the udder as an intramammary infusion, three times
after each milking, in dairy cows.
The Committee has not previously evaluated lincomycin.

Toxicological data
The Committee considered data from a range of toxicological studies
on lincomycin, including the results of studies on its pharmaco-
kinetics, metabolism, acute, short-term and long-term toxicity, car-
cinogenicity, genotoxicity, ototoxicity, immunotoxicity, reproductive
toxicity, developmental toxicity and microbiological safety. The
results of studies on the functionally and structurally related drug
clindamycin were considered in the assessment of the microbiological
safety of lincomycin. In all of the studies considered, the concentra-
tions of the compound were reported in terms of the activity of
lincomycin base. While many of the studies were conducted prior to
the development of GLP, all of the pivotal studies were carried out
according to appropriate standards for study protocol and conduct.
Dogs given lincomycin intramuscularly or orally showed rapid ab-
sorption, peak serum concentrations being achieved within 0.5 and
1.5h, respectively. In pigs given an oral dose, 53% (with a standard
deviation of 19%) was bioavailable, and 5-15% was bound to plasma
proteins. The peak concentrations in serum were reached within 3.6h
(standard deviation, 1.2h), with a half-life of 3.4h (standard deviation,
1.3h). Pig excreta contained little unchanged lincomycin: urine con-
tained 11-21% of the oral dose, half of which was unchanged parent
compound. Only trace amounts of N-desmethyllincomycin were
identified. The contents of the gastrointestinal tract accounted for 79-
86% of the excreted drug. In faecal samples, only 17% of the excreted
dose was unchanged parent compound, and the remainder consisted
of uncharacterized metabolites.
Lincomycin is well distributed in the human body. Significant concen-
trations were found in the bile, pleural fluid, brain, bone marrow,
synovial fluid, bone, joint capsules, eyes and peritoneal fluid following
oral administration. The distribution of the compound in cerebrospi-
nal fluid is generally poor, except in the presence of inflammation.
Lincomycin has been shown to cross the placenta, and peak concen-
trations of 0.2-3.8 mg/ml were found in amniotic fluid, which were

14
sustained for 52h after a single intramuscular injection of 600mg
to pregnant women. Lincomycin was present in the milk of these
women. The systemic oral bioavailability of lincomycin in persons
who had fasted was 25-50%, but this value can be as low as 5% in the
presence of food; 72% of the amount found in serum was bound to
serum proteins. Peak serum concentrations are usually reached
within 4h, with a half-life of 4.2-5.5 h.
Lincomycin is extensively metabolized, as less than 10% of the dose
was present as unchanged parent drug in animal tissues. The numer-
ous metabolites include N-desmethyllincomycin and lincomycin sulf-
oxide, which are reported to have 1-7% of the microbiological activity
of the parent compound.
Lincomycin has high acute toxicity only in rabbits. The LD50 in mice
and rats treated orally was 17-19 and 11-16g/kg of body weight
respectively, while the lethal dose in 9 of 15 rabbits was reported to be
50mg/kg of body weight.
Lincomycin was administered to mice for 90 days in the feed to
provide a dose of 0,10, 30,100, 300 or 3000mg/kg of body weight per
day. Animals given the two highest doses showed increased weight of
the intestine (and pancreas) and an increased incidence of luminal
distension and dilatation of the small and large intestine. The NOEL
was l00mg/kg of body weight per day.
In a 90-day toxicity study in dogs, groups of four males and four
females were given lincomycin orally at 0, 400 or 800mg/kg of body
weight per day. Transient increases in serum alanine aminotrans-
ferase activity were observed during the first month of treatment in all
dogs in the highest-dose group and in one dog in the group treated at
400mg/kg of body weight per day, but the activity had returned to
normal by the end of the study. No other treatment-related effects
were reported. The NOEL was 800mg/kg of body weight per day, the
highest dose tested.
Groups of 10 rats of each sex were treated for 1 year with lincomycin
at a dose of 0, 30, 100 or 300mg/kg of body weight per day by oral
gavage. No treatment-related effects were reported at any dose.
The NOEL was 300mg/kg of body weight per day, the highest dose
tested.
In a study that did not conform to GLP, lincomycin was administered
to groups of two male and two female dogs in capsules for 6 months
at a dose of 0,30,100 or 300mg/kg of body weight per day. The NOEL
was 300mg/kg of body weight per day, the highest dose tested. In a 1-
year study, groups of five male and five female dogs were given gelatin

15
 capsules containing premix-grade lincomycin at a dose of 0, 0.38, 0.75
or 1.5mg/kg of body weight per day or United States pharmacopoeia
(USP)-grade lincomycin at a dose of 1.5mg/kg of body weight per
day. The NOEL was 1.5mg/kg of body weight per day, the highest
dose tested.
In a long-term study of toxicity and carcinogenicity conducted accord-
ing to GLP, pregnant female rats and groups of 60 offspring of each
sex were given feed containing premix-grade lincomycin to provide a
dose of 0,0.38,0.75 or 1.5 mg/kg of body weight per day or USP-grade
lincomycin to provide a dose of 1.5 or 100 mg/kg of body weight per
day. Treatment of the offspring was continued for 26 months. No
treatment-related effects were reported. While lincomycin was not
carcinogenic under the conditions of the assay, the Committee
considered the administered dose to be insufficient for assessing the
carcinogenicity of lincomycin. The NOEL for non-neoplastic lesions
was 100 mg/kg of body weight per day, the highest dose tested.
Lincomycin was tested in vitro for its capacity to induce reverse
mutations in bacteria, gene mutations in Chinese hamster lung fibro-
blasts, unscheduled DNA synthesis in primary rat hepatocytes,
chromosomal aberrations in peripheral human lymphocytes, DNA
damage in Chinese hamster V79 cells and micronuclei in rat and
mouse bone marrow, and in vivo for its ability to induce sex-linked
recessive lethal mutations in Drosophila melanogaster. The only posi-
tive finding was the induction of unscheduled DNA synthesis in
primary rat hepatocytes, but this result could not be replicated.
Adequate studies of carcinogenicity were not available. However, the
weight of the evidence indicates that lincomycin is not genotoxic, and
lincomycin is not structurally similar to known carcinogens. The
Committee therefore concluded that the drug does not present a
carcinogenic risk, and further carcinogenic studies were deemed
unnecessary.
The reproductive and developmental toxicity of lincomycin was
evaluated in a three-generation study conducted prior to the formu-
lation of GLP. Groups of 30 male and 60 female F0 rats and 10 male
and 20 female Fl F2 and F3 animals were given diets containing
premix-grade lincomycin to provide a dose of 0, 0.38, 0.75 or
1.5 mg/kg of body weight per day or USP-grade lincomycin to provide
1.5 or 100 mg/kg of body weight per day, beginning with F0 weanling
rats and continuing through successive breeding of the F0, F1 and F2
progeny to weaning of the F3 litters. No treatment-related effects
were seen at any dose. The NOEL was 100 mg/kg of body weight per
day for the USP-grade lincomycin, the highest dose tested.

16
In a two-generation study that was conducted according to GLP,
lincomycin was administered to groups of 30 male and 30 female rats
by gavage at a dose of 0, 100, 300 or l000mg/kg of body weight per
day. No treatment-related effects were observed at any dose. The
NOEL was l000mg/kg of body weight per day, the highest dose
tested.
In a study of developmental toxicity which did not conform to GLP,
pregnant rats were given lincomycin by gastric gavage at a dose of 0,
10, 30 or l00mg/kg of body weight per day on days 6-15 of gestation.
An increased incidence of fetal resorptions was observed at the high-
est dose. The NOEL for embryotoxicity was 30mg/kg of body weight
per day.
The potential ototoxicity of lincomycin was tested in groups of three
cats that received intramuscular injections of 30 or 60mg/kg of body
weight per day for 2.5 months. Hearing and vestibular function were
evaluated. No treatment-related effects were reported.

Microbiological data
The Syrian hamster is used as an experimental model to evaluate
antibiotic-associated colitis. In studies in which lincomycin was
administered by various parenteral routes to hamsters, the NOEL for
antibiotic-associated colitis was 0.lmg/kg of body weight per day.
Administration of lincomycin at an oral therapeutic dose of 25-
66mg/kg of body weight daily to 12 patients for periods varying from
6 to 150 days caused antibiotic-associated colitis. Administration of
the structurally and functionally similar compound clindamycin to 10
patients for 7 days at a dose of l0mg/kg of body weight per day also
resulted in antibiotic-associated colitis. When clindamycin was ad-
ministered to 99 patients at doses of up to 2.5mg/kg of body weight
per day for up to 12 months, the NOEL for adverse effects on the
gastrointestinal microflora was 2.5mg/kg of body weight per day.
The ability of lincomycin to affect faecal excretion of pathogens was
investigated in a study of 32 pigs aged 4-5 weeks that were given the
drug for periods of up to 45 days at a concentration of l00g/t of
feed, equal to a dose of 5.6mg/kg of body weight per day. Comparison
of these animals with pigs that received the vehicle alone showed that
treatment had no effect on the faecal excretion of Salmonella
typhimurium, or on its susceptibility to lincomycin.
The sensitivity of staphylococci isolated from pigs and poultry to
lincomycin was investigated in a 10-year study that ended in 1980. No
change in the susceptibility of Staphylococcus aureus strains isolated
was observed.

17
 A study was conducted between 1971 and 1982 to examine the
patterns of susceptibility to antibiotics of more than 5 million bacte-
rial strains from hospitals across the USA. The susceptibility of
Gram-positive aerobic and anaerobic bacteria to lincomycin changed
little during the survey period. In a separate study, the median
microbiological inhibitory concentration (MIC50) for representative
bacteria from the human gut was reported for lincomycin. The
no-observed-effect concentration (NOEC) for the effect of lin-
comycin on Fusobacterium, the most sensitive representative species,
was 0.2mg/ml.
Clindamycin was tested at a concentration of 0, 0.26, 2.6, 25 or
260mg/ml of culture for 7 days in semi-continuous cultures of compo-
site faecal samples from humans. During these treatments and for 7-8
days thereafter, Clostridium difficile was added daily at a concentration
of 103 cells/ml. The NOEL for clindamycin was 2.6mg/mlon the basis of
overgrowth of C. difficile, changes in pH, and changes in the concentra-
tion of volatile fatty acids in the culture medium.
A decision-tree for evaluating the potential of veterinary drug resi-
dues to affect the human intestinal microflora was developed by the
Committee at its fifty-second meeting (Annex 1, reference 140;
Fig. 1). At its present meeting, the Committee used the decision-tree
to evaluate the potential of lincomycin residues to affect the human
intestinal flora:
• Does the ingested residue have antimicrobial properties?
Yes.
• Does the drug residue enter the lower bowel?
Yes.
• Is the ingested residue transformed irreversibly to inactive metabo-
lites by chemical transformation, metabolism mediated by the host,
or by the intestinal microflora in the bowel and/or by binding to
intestinal contents?
Yes, but microbiologically active residues remain.
• Do data on the effects of the drug on the colonic microflora provide
a basis to conclude that the ADI derived from toxicological data is
sufficiently low to protect the intestinal microflora?
No.
• Do clinical data from therapeutic use of the class of drugs in humans
or from in vitro or in vivo model systems indicate that effects could
occur in the gastrointestinal microflora?
Yes.

18
• Determine the most sensitive adverse effect(s) of the drug on the
human intestinal microflora.
The available data indicate that disruption of the barrier to
colonization of the human gastrointestinal microflora is the
major concern, rather than the emergence of drug-resistant
populations. Lincosaminides are used widely in human medicine
and have been shown to disrupt the intestinal microflora. In a
study of the magnitude of and trends in the development of
bacterial resistance to lincosaminides, the pattern of suscep-
tibility of human isolates of Gram-positive aerobic and anaerobic
bacteria changed little over a 12-year period (1971-83). Resis-
tance has been shown to develop in staphylococci isolated from
humans or animals, but most isolates remain susceptible to
lincomycin.
While no data were available on the effects of lincomycin on the
metabolic activity of the intestinal microflora, the Committee con-
cluded that disruption of the barrier to colonization of the gas-
trointestinal microflora is the most appropriate microbiological
end-point for determining an ADI for lincomycin.
• If disruption of the colonization barrier is the concern, determine the
MIC of the drug against 100 strains of the predominant intestinal
flora and take the geometric mean MIC of the most sensitive genus or
genera to derive an ADI using the equation discussed at the forty-
seventh meeting (Annex 1, reference 125). Other model systems may
be used to establish a NOEC or a NOEL to derive an ADI.
Disruption of the barrier to colonization of the human gastrointes-
tinal microflora is the microbiological end-point of concern with
lincomycin. No studies suitable for deriving a NOEL for the effects
of lincomycin on the gastrointestinal microflora in humans were
available. However, clindamycin has the same spectrum of activity
and the same reported spectrum of adverse effects in humans
as lincomycin, and is generally considered to be a more potent
antibacterial agent than lincomycin. Data were available to show
that the availability to the colon of orally administered clindamycin
is one-tenth of that of lincomycin. The Committee concluded that
the study of the clinical effects of orally administered clindamycin
in humans is the most appropriate one for determining the micro-
biological safety of lincomycin.
The Committee could have established an ADI of 0-300 mg/kg of
body weight for lincomycin, based on the NOEL of 30mg/kg of body
weight per day for embryotoxicity in rats and a safety factor of 100.
The Committee noted, however, that lincomycin belongs to a group

19
Figure 1
Decision-tree for determining the potential adverse effects of residues of veterinary antimicrobial drugs on the human intestinal
microflora

Assess the effects of veterinary drug residues, including metabolites, on the human intestinal microflora.

yes Does the ingested residue have antimicrobial properties (recommendation 1(a))? No
y y

No Does the drug residue enter the lower bowel (e.g. with the food Yes Conclude that the drug residue will not affect the intestinal
— bolus, by biliary circulation and/or by mucosal secretion) microflora and use toxicological data to derive the ADI
(recommendation 1(b))? (recommendation 1(a)).
V V

Conclude that the drug residue will not affect the No is the ingested residue transformed irreversibly to inactive metabolites by chemical yes
intestinal microflora and use toxicological data to — transformation, metabolism mediated by the host or by the intestinal microflora in the —
derive the ADI (recommendation 1(b)). bowel and/or by binding to intestinal contents (recommendations 1(b)-1(d))?

y y

Do data on the effects of the drug on the colonic Conclude that the drug residue will not affect the intestinal microflora and use
Yes microflora provide a basis to conclude that the toxicological data to derive the ADI (recommendations 1(c) and 1(d)).
ADI derived from toxicological data is sufficiently
low to protect the intestinal microflora No
(recommendation 1(e))?
y y

Conclude that the drug residue will not affect the Yes Do clinical data from therapeutic use of the class of drugs in humans or No
gastrointestinal microflora and use toxicological data to data from in vitro or in vivo model systems indicate that effects could —
derive the ADI (recommendation 1(e)). occur in the gastrointestinal microflora (recommendation 1(f))?
v y
Determine the most sensitive adverse effect(s) of the Conclude that the drug residue will not affect the gastrointestinal
drug on the human intestinal microflora, including microflora and use toxicological data to derive the ADI.
selection of drug-resistant populations, disruption of
the barrier to colonization or changes in the
metabolic activity of the microflora in the
gastrointestinal tract that have been specifically
linked to adverse effects on human health.
 T T i
If emergence of antimicrobial If disruption of the colonization barrier is the concern, determine the If the concern is change in a
resistance is the concern, MIC of the drug against 100 strains of the predominant intestinal flora specific enzymatic activity that is
conduct tests in vitro (continuous and take the geometric mean MIC of the most sensitive genus or directly linked to adverse effects
culture of faecal inocula) or in genera to derive an ADI using the equationa discussed at the forty- on human health, conduct tests in
vivo (rodents inoculated with seventh meeting of the Committee (Annex 1, reference 125). vitro (continuous culture of faecal
human flora); challenge the Other model systems may be used to establish a no-observed-effect inocula) or in vivo (rodents
model system with an antibiotic- concentration (NOEC) to derive an ADI (recommendation 2(b)). inoculated with human flora) to
resistant species and determine A more realistic ADI can be derived by conducting tests in vitro determine the concentration of the
the concentration of the drug that (continuous culture of faecal inocula) or in vivo (rodents inoculated drug that does not alter that
does not select for resistance or with human flora). Challenge the model systems with appropriate specific enzymatic activity when
the antibiotic-resistant strain when species (e.g. Clostridium difficile, Salmonella spp., Enterococcus spp., compared with absence of the
compared with absence of the Escherichia coli) and determine the concentration of the drug that drug. Use the dose of the drug
drug. Use the dose of the drug does not alter the shedding characteristics of the organisms when that has no effect to derive an ADI
that has no effect to derive an compared with absence of the drug. Use the dose of the drug that has (recommendation 2(e)).
ADI (recommendation 2(d)). no effect to derive an ADI (recommendation 2(c)). '

a
The equation is as follows:
M MIC(mg/g) x Mass of colonic contents (g)
Upper limit of ADI (mg/kg of body weight) =
Fraction of oral dose bioavailable x Safety factor x Weight of human (kg)
where:
MIC50 = Minimum concentration of an antimicrobial drug completely inhibiting the growth of 50% of the cultures of a particular microorganism, as judged by
the naked eye, after a given period of incubation. For the purpose of the evaluation, the MIC50 value is the mean MIC50 for the strain(s) of the
relevant species tested. Alternatively, the lowest MIC50 value for the most sensitive species can be used.
Although MIC50 values are usually expressed in |4.mg/ml, they are expressed as mg/g in this equation so that the ADI will be in (mg/kg. When the MIC50 value is
converted to these units, it is assumed that the density of the experimental medium is 1 g/ml.
A value of 220g is used for the mass of the colonic contents and a value of 60kg is used for the weight of an adult. The safety factor used to take
account of uncertainty about the amount and relevance of data available for review may range from 1 to 10. A value of 1 is used when extensive relevant
microbiological data are provided.
 of lincosaminides that is active against Gram-positive bacteria and
that the human gastrointestinal flora are sensitive to therapeutic
doses of this group of compounds. Because this is the most sensitive
end-point, the Committee established an ADI of 0-30mg/kg of body
weight for lincomycin, based on the NOEL of 2.5mg/kg of body
weight per day for the effects of clindamycin on the gastrointestinal
microflora1 and a safety factor of 100.2 The Committee rounded the
value of the ADI to one significant figure, in accordance with usual
practice.

Pharmacokinetic and metabolism data

The Committee considered a number of studies on the metabolism
and pharmacokinetics of lincomycin, including several studies in pigs
in which the drug was administered by intravenous, oral and intra-
muscular routes.

Lincomycin is rapidly absorbed after parenteral or oral administra-

tion in all food animal species and is widely distributed to tissues. It is
extensively metabolized to compounds that are reported to have no
or minimal antimicrobial activity.

In a study in pigs, seven animals were treated intravenously with

lincomycin hydrochloride at l0mg/kg of body weight, followed 7 days
later by the same dose given orally. After the intravenous treatment,
elimination of the drug followed a biphasic, two-compartment model
with a mean half-life of 2h. After the oral treatment, 53% ± 19% of
the dose was absorbed and 5-15% was estimated to be bound to the
plasma proteins at blood concentrations of 0.5-20 mg/kg. The absorp-
tion and bioavailability of the drug after oral administration followed
first-order kinetics, and the distribution and excretion followed a one-
compartment model and first-order elimination with a mean half-life
of 3.4 h. The concentration of lincomycin in the blood reached a
maximum of 1.45 mg/kg at 3.6 h after oral treatment. The wide varia-
tion found in the maximum concentration was attributed to the effect
of different food intakes.

1
The observed NOEL was 2.5 mg/kg of body weight per day. While this was the highest
dose administered, a separate trial showed an effect on the gastrointestinal microflora of
clindamycin at 10 mg/kg of body weight per day and of lincomycin at 25 mg/kg of body
weight per day.
2
The overall safety factor was 100, based on a safety factor of 10 to address variation
among individuals and a correction factor of 10 to address the higher oral availability to
the colon of lincomycin compared to clindamycin.

22
In a study in which [14C]lincomycin hydrochloride was administered
orally to pigs, the highest concentrations of radiolabel were found in
liver and kidney, with much lower concentrations in muscle and fat. In
two groups of pigs which received unlabelled lincomycin hydrochlo-
ride intramuscularly at 1 mg/kg of body weight per day for either 3 or
7 days, the highest concentrations were found in urine, consistent with
very rapid excretion. The tissue concentrations were highest at the
injection sites, followed by kidney, liver, muscle and fat.
In dairy cattle given lincomycin intravenously at 5.5 or 11 mg/kg of
body weight, analysis of blood, milk and urine samples showed that
the elimination of the drug followed first-order kinetics, with about
32% of the dose excreted in the urine. However, whereas only about
1.5% of an intravenous dose was excreted into the milk, analysis of
blood samples from one cow given an intramammary infusion at
11 mg/kg of body weight showed that 85% of the dose was absorbed
into the blood. Approximately 65% of the dose was metabolized to
inactive metabolites, regardless of the route of administration.
In a pharmacokinetic study in chickens, eight birds received
unlabelled lincomycin hydrochloride in the diet at 10 mg/kg of feed
for 36 days, followed by [14C]lincomycin hydrochloride at 0.47-
0.76 mg/kg of body weight orally twice daily for 12 days. During
treatment, 90% of the radiolabel was found in excreta. The half-life in
bile and offal was 8.3 and 11.3h, respectively. Only liver samples
collected at 1 h after treatment contained detectable residues (limit of
detection, 0.1 mg/kg) and these were biologically inactive.
In pigs, lincomycin was metabolized rapidly and extensively; 26 me-
tabolites were found in liver. Except for the parent compound, none
of the metabolites was characterized, and none accounted for more
than 10% of the total radiolabelled residues. In a comparative study
of a microbiological method and a gas chromatography-mass spec-
trometry (GC-MS) method, lincomycin appeared to account for all of
the microbiologically active residues in pig liver and kidney.
In comparative studies in pigs and rats and in chickens and rats, the
metabolism of lincomycin was found to be qualitatively similar, al-
though the metabolites were not identified.
In chickens given a dose of about 7 mg/kg of body weight per day in
drinking-water for 7 days, the liver and kidney contained the high-
est concentrations of total residues. In liver immediately after with-
drawal of the medicated drinking-water, lincomycin accounted for 20%
of the total residues and lincomycin sulfoxide, N-desmethyllincomycin
and N-desmethyllincomycin sulf oxide accounted for 40%, 5% and

23
 10%, respectively. The remaining residues were not identified. In
muscle, lincomycin accounted for 16% of the total residues and an
unidentified metabolite for 37 %. In skin with adhering fat immediately
after withdrawal of the drug, lincomycin accounted for 18% of the total
residues and the same unidentified metabolite for 11%. In excreta,
lincomycin accounted for 60-85% of the total residues during treat-
ment and 50-55% of the total residues 4 days after treatment. Of the
remaining residues found in excreta during treatment, 6-10% were
identified as lincomycin sulfoxide, 3-6% as N-methyllincomycin, and
10% as an unknown metabolite.

Residue data
Cattle. Three studies were conducted according to GLP in which
lincomycin was administered by intramammary infusion. One study
in which the highest recommended dose was used involved 24 cows
that received three consecutive doses of 330 mg of lincomycin into
each of the four quarters of the udder at 12-h intervals. Pooled milk
samples were taken at 12-h intervals for eight milkings after the last
treatment and analysed by GC-MS. The mean concentrations of
lincomycin in milk were 53mg/kg at 12 h, 7.0mg/kg at 24 h, 0.7mg/kg
at 36 h, 0.2mg/kg at 48 h, 0.04mg/kg at 60 h, and below the limit of
quantification (0.015 mg/kg) at all other times.
In another study that complied with GLP, 16 cows were given three
consecutive intramammary infusions of 330 mg of lincomycin into
each of the four quarters of the udder at 12-h intervals. Tissue
samples from groups of four cows killed 1, 7, 14 and 21 days after
treatment were analysed by GC-MS. The mean concentrations
of lincomycin residues in liver were 0.23 mg/kg on day 1 and
0.06 mg/kg on day 7, and ranged from 0.02 to 0.04 mg/kg on day
14 and from below the limit of quantification (0.02 mg/kg) to
0.05 mg/kg on day 21. Residues were found in muscle and kidney
only on day 1, and no residues were found in fat.
In a study conducted prior to the development of GLP, five lactating
cows received three consecutive infusions of 200 mg of lincomycin
into one quarter of the udder at 12-h intervals. Milk samples taken
during treatment and at 12-h intervals for 10 milkings after treatment
were analysed by a microbiological assay. The mean concentrations of
residues in milk decreased from 115 mg/kg at 12 h to 18 mg/kg at 24 h,
1.4 mg/kg at 36 h, and below the limit of quantification (0.2 mg/kg) at
48 h.
Veal calves. In a study conducted according to GLP, four groups of five
veal calves weighing 60-80 kg received daily intramuscular injections of

24
5 mg of lincomycin for 5 days, two doses being given on day 1 on both
sides of the neck. Animals were killed after 8 h and on days 7,14 and 21
after the last treatment, and tissue samples were taken and analysed by
GC-MS. At 8h, the highest mean concentrations of residues were
found in kidney (3.3mg/kg) and at the site of the last injection (2.4 mg/
kg). The mean concentration of residues in muscle was 0.72mg/kg. The
concentrations ranged from below the limit of quantification (0.02 mg/
kg) to 0.14mg/kg in liver and from below the limit of quantification to
0.26mg/kg in fat at 8h. The only other sample in which residues were
detected was a liver sample taken on day 14; a concentration of
0.072mg/kg was found.
Pigs. Several residue-depletion studies in pigs treated orally or intra-
muscularly with lincomycin were evaluated. In a study conducted prior
to the development of GLP, four groups of six pigs were fed diets
containing [14C]lincomycin at concentrations equivalent to 1.2,2.0,6.0-
7.0 or 10-12 mg/kg of body weight per day for 3 days and were killed 12 h
after the last treatment. An additional group of six pigs received 10-
12 mg/kg of body weight per day for 3 days and was killed 48 h after
treatment. The mean concentrations of total residues in tissue samples
from the four treated groups at 12h were 0.01,0.02,0.05 and 0.15mg/kg
in muscle; 0.40,0.64,1.6 and 3.4mg/kg in liver; 0.22,0.41,1.2 and 3.1 mg/
kg in kidney; and 0.02,0.02,0.13 and 0.35 mg/kg in fat, respectively. At
12 h, the mean concentrations of microbiologically active residues in
liver and kidney samples from these animals were 0.01 mg/kg and
0.42 mg/kg, respectively.
For the pigs dosed at 10-12 mg/kg of body weight and killed at 48 h
after withdrawal, the mean concentrations of total residues were
0.09mg/kg in muscle, 0.82mg/kg in liver, 0.64mg/kg in kidney and
0.10 mg/kg in fat. When the liver samples from these animals were
reanalysed using an improved microbiological assay and by GC-MS,
lincomycin accounted for 6% of the total residues at 12 h and 25% at
48 h after treatment.
In a study conducted according to GLP, 12 pigs received an intramus-
cular dose of 11 mg/kg of body weight of [14C]lincomycin daily for 3 days.
Two groups of three pigs were killed at 12 and 24 h and six pigs were
killed at 48 h after treatment. A total of 78-85% of the administered
radiolabel was recovered. The mean concentrations of total residues
were highest in liver at 12h (17 mg/kg), followed by kidney (12 mg/kg),
the injection site (l.0mg/kg), fat (0.59mg/kg) and muscle (0.39mg/kg).
By 48 h, the mean concentrations of total residues had declined to
3.8mg/kg in liver, 3.1 mg/kg in kidney, 0.58mg/kg at the injection site,
0.20mg/kg in fat and 0.14 mg/kg in muscle. When the liver and kidney

25
 samples were analysed using an improved microbiological assay and by
GC-MS, the parent drug accounted for 14% of the total residues at 12h,
3%at24handl.6%at48hinliver,and55%atl2h,20%at24hand7%
at 48 h in kidney.
The most relevant residue-depletion study, which was conducted
according to GLP, involved two groups of 24 pigs given the maximum
recommended intramuscular dose (11mg/kg of body weight) of one
of two formulations of lincomycin for 3 days. The animals were killed
3, 6, 12, 24, 48 and 144 h after treatment. Samples of muscle, liver,
kidney, fat and tissue from the site of injection were taken from the
pigs in each group and analysed by GC-MS. The mean concentrations
of residues decreased rapidly between 3 and 48 h after administration
of the two formulations, from 6.4 and 4.7mg/kg to 0.06 and 0.07mg/kg
in liver and from 29 and 21mg/kg to 0.17 and 0.24mg/kg in kidney,
respectively. In both tissues, the concentrations were below the limit of
quantification (0.02mg/kg) at 144 h. In muscle, the mean concentra-
tions were 3.6 and 2.6mg/kg at 3h, 0.06 and 0.09mg/kg at 24h, and
below the limit of quantification at all other times. In fat, the mean
concentrations were 0.47 and 0.47mg/kg at 3h, 0.02 and 0.03mg/kg
at 24 h, and below the limit of quantification at all other times. In
tissue from the injection site, the mean concentrations were 115 and
250mg/kg at 3h, 0.02 and 0.03mg/kg at 48 h, and below the limit of
quantification at 144 h.
Sheep. In a study conducted according to GLP, four groups of five
sheep received daily intramuscular doses of lincomycin at 5mg/kg
of body weight for 3 days. Animals were killed at 8h and at days
7, 14 and 21 after treatment, and samples of muscle, liver, kidney
and tissue at the site of injection were analysed by GC-MS. At 8h
after the last treatment, the mean concentrations of lincomycin
were highest at the site of injection (14mg/kg), followed by kidney
(9.0mg/kg) and liver (4.3mg/kg); the lowest concentration was
found in muscle (0.95mg/kg). By day 7, only two of five samples of
liver contained concentrations of residues above the limit of
quantification of the method.
Chickens. In a study conducted according to GLP, groups of 21
female and 21 male 35-day-old broiler chickens were given
[14C]lincomycin at doses of 5.1-6.6mg/kg of body weight per day in
their drinking-water for 7 days. Total residues were measured in
muscle, liver, kidney and skin with adhering fat immediately and 0.5,
1, 2, 4 and 7 days after withdrawal of the medicated water. Liver and
kidney contained the highest concentrations, but these decreased
between day 0 and day 7 from 1.6 mg/kg to the limit of quantifica-
tion of the analytical method (0.02 mg/kg) in liver and from 1.3 to

26
0.01 mg/kg in kidney. Lincomycin accounted for 20% of the total
radiolabel in liver at Oh, 12% at 12h, 8% at 24h, 2% at 48h, and 5%
at 96 h. The mean concentrations in muscle decreased from 0.05 mg/kg
at Oh to below 0.005mg/kg at day 2, and those in skin with adhering
fat from 0.13mg/kg at Oh to below 0.005mg/kg at day 7. Immediately
after the last treatment, the radiolabel in muscle accounted for 16% of
the administered dose and that in skin with adhering fat for 18%.
In a study conducted according to GLP, 18 laying hens were given
gelatin capsules containing [14C]lincomycin at a dose equivalent to
0.5 mg/kg of body weight per day twice daily for 12 days. Eggs were
collected on days 1-3 of treatment, and tissues were collected from six
birds 4, 28 and 76 h after treatment. The mean concentration of total
residues in eggs rose from 0.002 mg/kg at day 1 to 0.008 mg/kg at day
10 during treatment and decreased to 0.005 mg/kg by day 2 after
treatment. The mean concentrations of total residues were highest
in kidney (0.15 mg/kg) and liver (0.14 mg/kg) and at the limit of
quantification (0.02 mg/kg) in muscle and skin with adhering fat
4h after treatment. The concentrations of residues in all tissues
were below 0.01 mg/kg 76 h after treatment.

Effect of lincomycin on starter cultures in milk processing

The effect of lincomycin on the performance of bacterial starter cul-
tures used for the production of Italian cheese, yoghurt, buttermilk
and sour cream was investigated. The four-parameter Weibull growth
curve was used to model the change in pH as a function of time. No
significant effects were observed with concentrations of lincomycin of
up to 0.16 mg/kg.

Analytical methods
Several methods have been used at different times to determine the
concentrations of lincomycin residues in animal tissues and food
products. The methods include a microbiological assay, thin-layer
chromatography-bioautography, gas chromatography with an alka-
line flame detector and GC-MS.
A microbiological method with Micrococcus luteus has been
described for monitoring the presence of lincomycin in animal
tissues and milk. The limit of quantification was reported to be
0.02-0.2 mg/kg.
GC-MS methods have been used to study the depletion of lincomycin
residues from the tissues of cattle, pigs and sheep, and data on valida-
tion of these methods were provided for these applications and for
monitoring lincomycin residues in chicken tissues. These methods

27
 involve extensive sample purification, followed by derivatization and
GC-MS analysis in the electron impact mode, with detection of the ion
fragment at mlz 126 atomic mass units. The recovery of lincomycin by
these methods was 84-102%, depending on the tissue, with a limit of
quantification of 0.02-0.06 mg/kg of tissue from cattle, pigs, sheep and
chickens. The coefficient of variation of the method was consistently
less than 10%.
For confirmation of lincomycin residues in pig liver, GC-MS is used in
the chemical ionization mode with methane as the reactant gas. The
ions analysed are the derivatized molecular ion fragment at mlz 575
atomic mass units and three fragments at mlz 126,515 and 527 atomic
mass units.
Maximum Residue Limits
In recommending MRLs for lincomycin, the Committee took the
following factors into account:
• An ADI of 0-30 mg/kg of body weight was established by the Com-
mittee on the basis of a microbiological end-point. This would
result in a maximum ADI of 1800 mg of the parent drug and/or its
equivalents for a 60-kg person.
• In pig tissues, lincomycin is the major microbiologically active
residue.
• In milk, lincomycin accounts for 90% of the total residues.
• There was insufficient evidence that lincomycin is the major micro-
biologically active residue in cattle and sheep tissues and in hens'
eggs.
• The parent drug is the marker residue.
• Kidney and liver contain the highest concentrations of residues.
• A validated GC-MS analytical method is available which could be
used routinely in many laboratories. It has a limit of quantification
of 0.02-0.06 mg/kg in tissues of cattle, calves, pigs, sheep and chickens.
• Lincomycin has no effect on bacterial starter cultures used in the
production of milk products at concentrations below 0.16 mg/kg.
• Lincomycin is a drug with a long history of use.
• A comparison of the microbiological assay and GC-MS with pig
liver samples showed that the two assays give similar results, with a
correlation coefficient greater than 0.98. The methods appear to be
suitable for monitoring lincomycin at concentrations within the
recommended MRLs.
The Committee recommended MRLs for lincomycin in pigs of l00mg/
kg for muscle and fat, 500 jig/kg for liver and 1500(mg/kg for kidney,
expressed as parent drug. The Committee also recommended the
same MRLs for lincomycin in the edible tissues of cattle, sheep and

28
 chickens, but made them temporary, in accordance with its policy on
the evaluation of veterinary drugs with a long history of use (Annex
1, reference 104). The Committee also recommended an MRL of
150mg/kg for cows' milk, expressed as parent drug.
The MRLs recommended above would result in a theoretical maxi-
mum daily intake of 385 mg, based on a daily food intake of 300 g of
muscle, 100 g of liver, 50 g each of kidney and fat, and 1.5kg of milk.
The Committee was unable to recommend an MRL for chickens' eggs.
The Committee required the following information for evaluation in
2002:
• Data from residue-depletion studies in cattle, sheep and chickens
which show that lincomycin is the major microbiologically active
residue in the edible tissues.
• Data from residue-depletion studies showing that lincomycin is the
major microbiologically active residue in chickens' eggs.
• The results of a residue-depletion study in which the GC-MS
method is used to analyse residues in chickens' eggs.
3.2.3 Oxytetracycline
Oxytetracycline was last evaluated by the Committee at its fiftieth
meeting (Annex 1, reference 134), when it established an ADI of
0-30mg/kg of body weight for Oxytetracycline, tetracycline and
chlortetracycline, alone or in combination. At that meeting, it rec-
ommended MRLs of 200mg/kg for muscle, 600mg/kg for liver and
1200 jig/kg for kidney, expressed as parent drug, alone or in combi-
nation with chlortetracycline and tetracycline, in cattle, pigs, sheep
and poultry. The Committee also recommended MRLs of l00mg/kg
for milk in cattle and sheep, and 400mg/kg for poultry eggs, expressed
as parent drug, alone or in combination with chlortetracycline and
tetracycline, and 200mg/kg for muscle in giant tiger prawns (Penaeus
monodon) and fish, expressed as parent drug alone. The MRL for
fish muscle was designated as temporary, pending evaluation of the
pattern of use of Oxytetracycline in aquaculture.
The requested data were not submitted for consideration at the
present meeting; however, the Committee decided to extend the tem-
porary MRL for fish muscle until 2002, pending the results of a
residue-depletion study and information on the validation of the
analytical method for fish.
3.2.4 Tilmicosin
Tilmicosin was previously evaluated by the Committee at its forty-
seventh meeting (Annex 1, reference 125), when it established an

29
 ADI of 0-40mg/kg of body weight and recommended MRLs of 100mg/
kg for muscle and fat, 1000mg/kg for liver and 300(mg/kg for kidney in
both cattle and sheep, as well as 50mg/kg for ewes' milk, expressed as
parent drug. The recommended MRL for ewes' milk was designated
as temporary, pending the results of a study with radiolabelled drug in
lactating ewes, to determine the relationship between total residues
and the parent drug in milk. The Committee also recommended
MRLs of l00 mg/kg for muscle and fat, 1500 mg/kg for liver and
l000mg/kg for kidney, expressed as parent drug, in pigs.
The requested information was not submitted for consideration at the
present meeting. The Committee therefore decided not to extend the
temporary MRL for ewes' milk. The MRLs for muscle, liver, kidney
and fat of cattle, pigs and sheep were retained.

3.3 Insecticides
3.3.1 Cyhalothrin
Cyhalothrin is a type II pyrethroid insecticide and acaricide. Technical-
grade cyhalothrin (about 98% pure), which was the material used in
most of the toxicological studies, consists mainly of four of the possible
16 isomers. These four isomers comprise two pairs of enantiomers, A
and B, in a ratio of 60:40. Within each pair, the enantiomers are
present in equal amounts. A related product, l-cyhalothrin, contains
only the B pair of enantiomers.
Cyhalothrin is used predominantly on cattle and sheep, and to a
lesser extent on pigs and goats, for the control of a broad range of
ectoparasites, including flies, lice and ticks. Cyhalothrin is applied
topically as a pour-on formulation to cattle at a dose of up to 60ml
(1.2g) for ticks and 10ml (0.2 g) for lice or flies, and to sheep and
pigs at a dose of 5ml (0.lg) for all applications. Cyhalothrin is also
available as a 20% (w/v) emulsifiable concentrate for use as a
spray or a dip, prepared by dilution to 0.002-0.2%. It is applied at
a dose of 0.1-4 litre per animal. In general, the more dilute the
spray or dip, the greater the volume applied.
The Committee has not previously evaluated cyhalothrin. It was
evaluated toxicologically by the 1984 Joint FAO/WHO Meeting on
Pesticide Residues (8), when an ADI of 0-20mg/kg of body weight
was established. Residues of cyhalothrin were evaluated by the 1984,
1986 and 1988 Joint FAO/WHO Meetings on Pesticide Residues (8-
10), which recommended MRLs for cabbage, cottonseed, cottonseed
oil (crude and edible), pome fruits and potato, expressed as the sum
of the cyhalothrin isomers.

30
Toxicological data
The Committee considered the results of studies on the pharmaco-
kinetics, metabolism, acute, short-term and long-term toxicity, carci-
nogenicity, genotoxicity, reproductive toxicity, neurotoxicity and
neurobehavioural effects of cyhalothrin and effects in humans ex-
posed to the drug. Although some of the studies were not fully com-
pliant with GLP, all of the pivotal studies were carried out according
to appropriate standards for study protocol and conduct.
Oral doses of cyhalothrin were readily but incompletely absorbed by
the experimental species studied (rats and dogs) and the subsequent
metabolism was similar, involving initial cleavage of the molecule at
the ester bond, presumably resulting in detoxification. The metabo-
lites were rapidly excreted, some as conjugates, whereas small
amounts of unchanged cyhalothrin persisted as residues in fatty tis-
sues. The results were similar to those in food-producing animals.
Serum and urine from exposed workers contained the metabolites
3-(2-chloro-3,3,3-trifluoroprop-l-enyl)-2,2-dimethylcyclopropane-
carboxylic acid, 3-phenoxybenzoic acid and 3-(4'-hydroxyphenoxy)-
benzoic acid. These metabolites were products of cleavage of the
cyhalothrin molecule at the ester bond, and their presence suggests that
the initial metabolism of this compound in humans is similar to that in
the animal species that have been investigated.
No toxicological studies on metabolites of cyhalothrin were available,
but the Committee considered it likely that the metabolites would be
less toxic than cyhalothrin, as none contains an intact pyrethroid
structure.
The acute toxicity of cyhalothrin is characterized by effects on the
nervous system, principally the central nervous system. The signs of
toxicity are typical of type II pyrethroids and comprise writhing,
salivation, exaggerated jaw opening, increasing extensor tone in the
hind legs causing a rolling gait, poor coordination progressing to
coarse tremor, tonic seizures and apnoea. The LD50 values after oral
administration depended on the vehicle used, but varied from 37 to
62mg/kg of body weight in mice and from 51 to 240mg/kg of body
weight in rats. Higher values were found in other species or after
administration by other routes.
In mice that received a dose of 0, 5, 10, 20, 40 or 80mg/kg of body
weight per day orally for 5 days, ataxia and convulsions were seen in
those given the two highest doses. The dose of 20mg/kg of body
weight per day caused body-weight loss, ataxia and roughness of the
coat. At 5 mg/kg of body weight per day, the only adverse effect seen
was a rough coat.

31
 In a 28-day study in mice, cyhalothrin was added to the diet at a
concentration of 0, 5, 25,100, 500 or 2000mg/kg of feed. Atrophy of
the red pulp of the spleen and an increased mortality rate were seen
at the highest dose. Adaptive liver changes, including enlarged liver,
hypertrophy of the centrilobular hepatocytes, increased activity of
aminopyrine N-demethylase, and proliferation of the smooth endo-
plasmic reticulum were seen at concentrations of l00mg/kg of feed
and above; lowered lymphocyte counts were also seen at these doses.
At 25mg/kg of feed, the only effect seen was piloerection. The NOEL
was 5 mg/kg of feed, equal to 0.65 mg/kg of body weight per day, on
the basis of piloerection at higher doses.
The main effects in five toxicity studies lasting 10-90 days in rats were
adaptive changes in the liver similar to those seen in mice. In a 28-day
range-finding study in which rats were fed diets containing
cyhalothrin at a concentration of 0,5,10,20 or 250mg/kg of feed, only
the highest concentration caused adverse effects. These effects were
characterized by hepatocellular hypertrophy, increased activity of
aminopyrine N-demethylase in the liver, and proliferation of hepatic
smooth endoplasmic reticulum. The NOEL was 20 mg/kg of feed,
equivalent to 2 mg/kg of body weight per day. In a 28-day study in rats
given 0, 1, 5, 10, 20 or 250 mg/kg of feed, increased activity of ami-
nopyrine N-demethylase and proliferation of smooth endoplasmic
reticulum in the liver were seen at the highest dose only. No such
effect was seen in the liver at doses equivalent to 2 mg/kg of body
weight per day or less. Females given 10 mg/kg of feed (equivalent to
1 mg/kg of body weight per day) or more had decreased body-weight
gain. The NOEL was 5 mg/kg of feed, equivalent to 0.5 mg/kg of body
weight per day, which is lower than the levels found in other short-
term studies in rats. A third 28-day study in rats was inadequately
reported, but showed that administration of cyhalothrin at 20 mg/kg
of feed (equivalent to 2 mg/kg of body weight per day) caused pro-
liferation of hepatic smooth endoplasmic reticulum; a NOEL was not
identified in this study.
A 90-day toxicity study was performed in which rats were given
cyhalothrin at 0, 10, 50 or 250 mg/kg of feed. Males given the
two highest doses showed increased activity of aminopyrine N-
demethylase and proliferation of smooth endoplasmic reticulum in
the liver. Females given the highest dose showed increased activity
of hepatic aminopyrine N-demethylase activity. The NOEL was
10 mg/kg of feed, equal to 0.56 mg/kg of body weight per day.
Groups of dogs received cyhalothrin at a dose of 0,2.5,10 or 30 mg/kg
of body weight per day for 4 weeks or a dose of 0,1.0,2.5 or 10 mg/kg
of body weight per day for 26 weeks. In the 4-week study, the two

32
highest doses caused an increased incidence of vomiting, and the
highest dose caused body-weight loss, unsteady gait and increased
serum activities of alanine and aspartate aminotransferases. Muscular
trembling was seen in dogs in all groups, including the controls, in the
4-week study. In the 26-week study, doses of l0mg/kg of body weight
per day or more caused clinical signs of toxicity, including vomiting,
salivation, lack of coordination, unsteadiness, collapse, muscular
spasms and convulsions. As treatment at all doses in both studies
increased the prevalence of liquid faeces, a NOEL was not identified.
The Committee considered it possible that the liquid faeces were a
consequence of the neurological effects of cyhalothrin. The lowest-
observed-effect level (LOEL) was l.0mg/kg of body weight per day.
In a long-term study of toxicity and carcinogenicity in mice,
cyhalothrin was given in the diet at a concentration of 0, 20, 100 or
500mg/kg of feed for 104 weeks. An increased incidence of mammary
adenocarcinomas was seen in treated females at the two highest
doses. The highest incidence (14%) was only slightly greater than
the upper limit of the range in historical controls (2-12%). The Com-
mittee could not exclude the possibility that the adenocarcinomas
seen in the groups given 100 or 500mg/kg of feed were caused by
cyhalothrin. Clinical signs of toxicity (piloerection and hunched pos-
ture) and increased serum activities of aspartate and alanine
aminotransferases were seen at these doses. The NOEL was 20mg/kg
of feed, equal to 1.9mg/kg of body weight per day, on the basis of
these effects.
In a long-term study of toxicity and carcinogenicity in rats,
cyhalothrin was given in the diet at a concentration of 0, 10, 50 or
250mg/kg of feed for 104 weeks. There was no treatment-related
increase in the incidence of any type of tumour. Adverse effects found
at the highest dose included decreased body weight, altered blood
biochemistry and increased relative weight of the liver in both sexes
and of the adrenals in females. The NOEL was 50mg/kg of feed,
equal to 2.3mg/kg of body weight per day, on the basis of these
effects.
Cyhalothrin was not genotoxic in a range of studies, including a test
for reverse mutation in Salmonella typhimurium, a test for cytoge-
netic effects in the bone marrow of rats treated in vivo, a test for
dominant lethal mutation in mice, and an assay of cell transformation
in vitro. The Committee concluded that cyhalothrin is not genotoxic.
Furthermore, the Committee considered it likely that the mammary
adenocarcinomas found in the long-term toxicity/carcinogenicity
study in mice were due to a non-genotoxic mechanism.

33
 A three-generation study of reproductive toxicity was performed in
rats given cyhalothrin in the diet at a concentration of 0, 10, 30 or
l00mg/kg of feed. Adverse effects, including reduced parental body-
weight gain and reduced litter size, were found only at the highest
dose. The NOEL for these effects was 30mg/kg of feed, equal to
1.7mg/kg of body weight per day.
In a study of developmental toxicity, rats received a dose of 0,5,10 or
15mg/kg of body weight per day by gavage on days 6-15 of gestation.
Maternal toxicity, characterized by body-weight loss and poor coordi-
nation, was seen at the highest dose. Embryotoxicity also occurred
at this dose. Abnormalities were seen in 5 of 17 fetuses in one litter
from a dam at l0mg/kg of body weight per day, but the effect was
considered not to be due to treatment with cyhalothrin as no fetotoxi-
city was seen at the higher dose. The NOEL for maternal toxicity was
l0mg/kg of body weight per day, and that for developmental toxicity
was 15mg/kg of body weight per day, the highest dose tested.
Two studies of developmental toxicity in rats treated by dermal ad-
ministration provided some evidence that cyhalothrin can delay fetal
development at doses lower than those administered orally in the
study described above. However, the Committee considered that oral
administration is a more relevant route and that the NOEL in the
study in which this route was used was the appropriate one for evalu-
ating developmental toxicity in rats.
A study of developmental toxicity was conducted in rabbits, which
received cyhalothrin at a dose of 0, 3, 10 or 30mg/kg of body weight
per day by gavage. The highest dose caused initial body-weight
loss in the does, which was followed by reduced body-weight gain.
There was no significant effect on development at any dose. The
NOEL for maternal toxicity was l0mg/kg of body weight per day,
and that for developmental toxicity was 30mg/kg of body weight
per day, the highest dose tested.
Single oral doses of up to l0g/kg of body weight did not induce
clinical or histopathological signs of neurotoxicity in hens.
Various studies of neurobehavioural effects induced by cyhalothrin
have been conducted in rats, including a range-finding test of perfor-
mance on an inclined plane, a test for acute auditory startle response
and tests of inhibitory avoidance. In the inclined plane test, rats were
given a single oral dose of 0, 15, 25, 30, 40, 50, 60, 75, 100 or
200mg/kg of body weight. All of these doses caused soft faeces in at
least some of the rats, and doses of 25 mg/kg of body weight or more
caused clinical signs of neurotoxicity. The results were, however,

34
variable, and no conclusion could be reached about the neurotoxic
effects of the compound.
In the test for acute auditory startle response, in which a single oral
dose of 0, 5, 15 or 75mg/kg of body weight was given, the highest
dose resulted in reduced body-weight gain, transient clinical signs of
toxicity and a reduced amplitude of the mean response in animals of
both sexes. Statistically significant, but not dose-related, reductions
in amplitude of the mean startle response were also seen at 5 and
15mg/kg of body weight in females only. The Committee was aware
that a biphasic auditory startle response had been reported with some
other type II pyrethroids, with a reduced response at high doses and an
increased response at lower doses. A NOEL for the startle response
was not identified in this study.
In the inhibitory avoidance tests, rats were exposed either in utero
(dams were given 200mg/l in drinking-water) or during lactation
(dams were given 1ml of a 0.018% solution dermally); however, the
doses received by the animals could not be estimated reliably. The
rats exposed in utero showed a decreased motivational response
when tested as adults, but no effects were seen on inhibitory avoid-
ance behaviour or locomotor frequency. Animals exposed during
lactation had a shorter latency in learning avoidance behaviour.
There was no NOEL in either study, as adverse effects were seen at
the only dose tested in each study and the doses received by the
animals were not known precisely.
Observations and case reports in humans provided little information
relevant to the establishment of an ADI for cyhalothrin. In most
instances, no information on doses was available, the route of expo-
sure was dermal, and some studies were of exposure to l-cyhalothrin
rather than cyhalothrin.
The Committee established a temporary ADI of 0-2mg/kg of body
weight for cyhalothrin by applying a 500-fold safety factor to the
LOEL of l.0mg/kg of body weight per day for induction of liquid
faeces in dogs in the 26-week study. The Committee considered it
possible that the liquid faeces were a consequence of the neurological
effects of cyhalothrin. The Committee applied the 500-fold safety
factor to account for the absence of a NOEL for liquid faeces in dogs
and for the absence of a NOEL for neurobehavioural effects. The
ADI was made temporary because cyhalothrin belongs to a class of
substances that are characterized by their toxicity to the central ner-
vous system, and therefore neurobehavioural effects may be the most
sensitive indicator of the toxicity of this compound. There was an
adequate margin of safety between the ADI and the NOELs identified

35
 in other studies on cyhalothrin, including the NOEL of 0.65mg/kg of
body weight per day for piloerection in the 28-day study in mice.
The results of studies appropriate for identifying a NOEL for
neurobehavioural effects in laboratory animals are required for
evaluation in 2002.
The Committee noted that the NOEL for toxicological effects other
than the neurotoxicity related to the pyrethroid structure was 2.3 mg/
kg of body weight per day for adaptive changes in the livers of rats in
the long-term toxicity/carcinogenicity study. This suggests that the
toxicity of the metabolites (none of which has a pyrethroid structure)
is no greater than 50% of that of the parent drug.

Pharmacokinetic data
The results of studies in laboratory and food-producing animals show
consistently that more than 90% of a dose of cyhalothrin is eliminated
rapidly in the urine and faeces. The tissue in which residues are found
primarily is fat, and the residues occur as the isomers of cyhalothrin,
although in proportions different from those in formulated products.
Cyhalothrin is eliminated in milk as the isomers found in the original
product.
Cattle. In a study conducted according to GLP, [14C]cyhalothrin was
given orally twice daily at a dose of 1 mg/kg of body weight for 7 days to
two cows. The first cow received [14C]cyhalothrin labelled in the Cl-
position of the cyclopropane ring (cyclopropyl label), while the second
received [14C]cyhalothrin labelled at the a-carbon of the benzyl group
(benzyl label). When the cows were killed 16 h after the final treatment,
most of the radiolabel had been eliminated in faeces (49%) and urine
(27 %). The major metabolites identified in urine, kidney and liver from
the cow given cyclopropyl-labelled cyhalothrin were l6-(lRS)-cis-3-
(Z-2-chloro-3,3,3-trifluoroprop-l-enyl)-2,2-dimethylcyclopropane-
carboxylic acid (CPA) and its conjugates, while the glutamic acid
derivative of 3-phenoxybenzoic acid was the major urinary metabolite
identified after administration of benzyl-labelled cyhalothrin. The
parent compound was the major component of the total residues in
faeces and in muscle, fat and milk; it accounted for 10% or more of the
total residues in kidney, but less than 5% of those in liver. Confirma-
tion was provided by studies conducted with [14C]l,-cyhalothrin, which
contains only the B-enantiomer pair of the isomeric mixture.

Residue data
All of the studies summarized in this section were conducted in com-
pliance with GLP, and the cyhalothrin residues in the studies of
unlabelled compound were measured as the sum of the isomers.

36
Cattle. In a study in which one cow received [14C-l-cyclo-
propyl] cyhalothrin and a second cow received the benzyl-labelled
compound orally twice daily at 1mg/kg of body weight for 7 days, the
concentrations of total residues were higher in the tissues of the
animal treated with the cyclopropyl-labelled drug. This indicates
greater retention of the CPA fragment and its conjugates than of the
fragments from the remainder of the cyhalothrin molecule after
cleavage at the ester bond. The total concentrations of radiolabel in
tissues were highest in perirenal fat (2.7mg/kg), followed by sub-
cutaneous fat (1.6mg/kg), liver (1.3mg/kg), kidney (0.60mg/kg) and
muscle (0.19mg/kg). In milk, the highest concentration measured was
0.59mg/kg, and this was associated with the cream fraction. The usual
60:40 ratio of A: B isomers in the formulated product was essentially
reversed in perirenal fat and in milk, suggesting that the B isomers are
more persistent than the A isomers. Confirmatory data on the distri-
bution of cyhalothrin in tissues was provided by studies conducted
with [14C]l-cyhalothrin, which contains the more toxic B-enantiomer
pair.
Groups of five calves weighing 105-165 kg received cyhalothrin from
two tapes attached to an ear tag and from seven spray treatments (at
a dose equivalent to 2mg/kg of body weight) at 14-day intervals. The
tag tapes were an experimental product that is not currently recom-
mended for use, and the recommended time between spray applica-
tions is 2-4 weeks. The animals were slaughtered in groups of five at
0, 3, 7 and 14 days after the last spray treatment. The highest concen-
trations of residues were found in perirenal fat and decreased from
0.32mg/kg (standard deviation, 0.13mg/kg) immediately after the end
of treatment to 0.16mg/kg (standard deviation, 0.03mg/kg) 14 days
after treatment. The concentrations of residues were consistently 10-
25% lower in subcutaneous fat than in perirenal fat. Immediately
after the end of treatment, the concentrations were 0.005 mg/kg in
liver and muscle and 0.010 mg/kg in kidney. The treatment schedule
was considered to represent the most extreme use of cyhalothrin as
the spray product.
Four applications of a pour-on formulation of cyhalothrin were ap-
plied topically at weekly intervals to cattle weighing 250-350 kg at a
dose equivalent to 2 mg/kg of body weight. Although more frequent
application may be required for treatment of ticks, the recommended
treatment interval for the pour-on preparation is 4-8 weeks. The
highest concentration of residues in animals slaughtered 7h after the
final application was found in perirenal fat (0.91 mg/kg; standard de-
viation, 0.37mg/kg). In animals slaughtered 14 days after the final
treatment, the concentrations were 0.91 mg/kg (standard deviation,

37
 0.55mg/kg) in perirenal fat and 0.35mg/kg (standard deviation,
0.17mg/kg) in subcutaneous fat. The concentrations in all liver
samples were at or below the limit of quantification of the analytical
method (O.Olmg/kg). Samples of other tissues were not analysed in
this study.
After a single pour-on application of cyhalothrin solution to five dairy
cows at a dose equivalent to 0.4mg/kg of body weight, the highest
concentration of residues found in milk was 0.04mg/kg (standard
deviation, O.Olmg/kg) at the fifth milking (day 3); the value had
decreased to less than O.Olmg/kg by day 7.
Five dairy cows received seven spray treatments with cyhalothrin at
14-day intervals, each at a dose of about 2mg/kg of body weight. Most
of the 150 milk samples collected and analysed during treatment
contained concentrations of residues in excess of 0.005 mg/kg, exceed-
ing O.Olmg/kg in only 15 samples. The highest concentrations were
found in milk samples taken 2-3 days after each application, and the
highest mean concentration was 0.012 mg/kg (standard deviation,
0.005 mg/kg) in milk collected 2 days after the third spraying. In a
subsequent study, five cows received four repeated spray treatments
at a dose of approximately 2.9 mg/kg of body weight with a 7-day
interval between treatments. The concentration of residues in
milk typically reached a maximum 3-4 days after spraying, with a
maximum mean concentration of 0.013 mg/kg (standard deviation,
0.004 mg/kg) at the sixth milking, 3 days after the third spray applica-
tion. In animals slaughtered 16 h after the final spraying, the concen-
trations of residues in tissues were O.lOmg/kg (standard deviation,
0.03 mg/kg) in perirenal fat, 0.16 mg/kg (standard deviation, 0.22 mg/
kg) in subcutaneous fat, and below O.Olmg/kg in liver, kidney and
muscle.
Four dairy cows weighing 480-535 kg received four applications at
7-day intervals of a pour-on formulation of cyhalothrin at a dose
equivalent to 1.2 mg/kg of body weight. Four cows in a second group
each received a single application of 3.6mg/kg of body weight, fol-
lowed by three weekly applications of 2.4 mg/kg of body weight. After
the dose of 1.2 mg/kg of body weight, the highest concentrations of
residues were found at the third to fifth milkings after the initial
application, with a maximum of 0.18mg/kg (standard deviation,
0.11 mg/kg). The mean concentrations were below 0.005mg/kg by the
time of the seventh milking after the final application. In the cows that
received the dose of 3.6mg/kg of body weight, the highest concentra-
tion of residues found in milk was 0.47 mg/kg on day 3 after the first
application, while the highest concentration found after the first appli-
cation of 2.4 mg/kg of body weight was 0.30 mg/kg at the third milking

38
of one cow. The highest concentrations were found in milk from the
third to seventh milkings after the initial treatment, and the concen-
tration was not consistently below 0.005 mg/kg until 16 milkings after
the final treatment. The limit of quantification of the analytical
method was 0.01 mg/kg. The recommended interval between applica-
tions of the pour-on product in cattle is 4-8 weeks.
Pigs. Groups of four pigs weighing 39-50 kg received a single applica-
tion of 5 ml of a pour-on formulation of 2% cyhalothrin (equivalent to
3 mg/kg of body weight) and were killed 3, 7 or 14 days later. The
concentrations of residues were highest in skin at the site of applica-
tion, decreasing from 0.82 mg/kg (standard deviation, 0.44 mg/kg) at
day 3 to 0.33 mg/kg (standard deviation, 0.13 mg/kg) at day 14. In skin
remote from the site of application, the concentrations decreased
from 0.23 mg/kg (standard deviation, 0.04 mg/kg) at day 3 to 0.09 mg/
kg (standard deviation, 0.08 mg/kg) at day 14. The concentrations in
abdominal and subcutaneous fat were 0.08 mg/kg (standard deviation,
0.10 mg/kg) and 0.08 mg/kg (standard deviation, 0.05 mg/kg) at day 3
and 0.04 mg/kg (standard deviation, 0.01 mg/kg) and below 0.01 mg/kg
at day 14, respectively. No residues of cyhalothrin were detected in
liver, kidney or muscle at day 3.
Sheep. Four groups of three sheep weighing 28-36 kg received
three 5-ml applications of a 2% pour-on formulation of cyhalothrin
(equivalent to 2.2 mg/kg of body weight) from a syringe directly onto
the skin at 14-day intervals. The groups were killed at 16 h and 3,7 and
14 days after the final treatment. The concentrations of residues were
similar in subcutaneous and perirenal fat (0.03-0.13 mg/kg) taken at
16 h and 3 and 7 days. No residues were detected in subcutaneous fat
(limit of detection, 0.01 mg/kg) at day 14, but the concentration in
perirenal fat at this time was 0.04-0.10mg/kg. No residues were de-
tected in liver (limit of detection, 0.05 mg/kg) or in kidney or muscle
(limit of detection, 0.01 mg/kg) samples at any time after treatment.

Analytical methods
The method used to measure residue depletion is based on gas chro-
matography with detection by electron capture (GC-ECD). Valida-
tion was provided for the analysis of cyhalothrin residues in edible
tissues from cattle, pigs and sheep and in cows' milk. The method
involves extraction of the sample, followed by solvent partitioning
and solid-phase extraction for purification of the extracts. The choice
of cartridge depends on the sample matrix and the detection system.
The analytical recovery was 74-100%, depending on the sample ma-
trix. The limit of detection for the assay is 0.001-0.005 mg/kg, and the
limit of quantification is 0.01 mg/kg for all tissues (except sheep liver,

39
 0.05mg/kg) and milk. The isomers appear as a single chromatographic
peak in this method. No interference was found from the matrix or
from the related synthetic pyrethroids deltamethrin, cypermethrin,
permethrin, fenvalerate, cyfluthrin or flumethrin. An additional
method was provided in which gas chromatography is used, with
detection by mass spectrometry using chemical ionization in the nega-
tive ion mode. The limits of detection and of quantification are some-
what higher than those of the GC-ECD method, and the isomers are
partially resolved in the chromatographic separation. The GC-ECD
method has been used successfully in several laboratories since the
1990s and in a number of trials.
Maximum Residue Limits
In recommending MRLs for cyhalothrin, the Committee took the
following factors into account:
• A temporary ADI of 0-2 mg/kg of body weight was established by
the Committee on the basis of a toxicological end-point, which
would permit a maximum ADI of 120mgfor a 60-kg person.
• The sum of the isomers of cyhalothrin is the appropriate marker
residue, as previously established by the 1984 Joint FAO/WHO
Meeting on Pesticide Residues (8).
• Cyhalothrin isomers account for less than 5 % of the total residues
in cattle liver and 10% or more of those in kidney. The metabolites
were considered to be half as toxic as the parent compound. The
marker residue accounted for about 6% of the total residues in liver
and 20% of those in kidney.
• The residues found in muscle, fat and milk consisted of the parent
compound.
• Weekly application of the pour-on formulation to cattle and the use
of more than 10ml of this product result in much higher concentra-
tions of residues in fat and milk than are found with less frequent
application of smaller volumes. While such usage may be within the
range of the recommended applications of cyhalothrin, it was con-
sidered to be an unsuitable basis for establishing MRLs as it re-
presents extreme usage. Use of the pour-on product can result in
residue concentrations in excess of the MRL for milk.
• The maximum intakes allowed by the 1988 Joint FAO/WHO Meet-
ing on Pesticide Residues (10) for horticultural use account for 10%
or less of the temporary ADI established by the Committee.
• A suitable analytical method is available for analysis of cyhalothrin
residues in edible tissues and milk.
• The recommended MRLs for liver, kidney and muscle are based on
twice the limit of quantification of the analytical method as vali-
dated for tissues and harmonized for cattle, pigs and sheep.

40
Table 2
Theoretical maximum daily intake of cyhalothrin residues

Tissue Recommended Estimate of total Theoretical maximum daily intakeb

MRL (mg/kg)a residues (mg/kg) (mg cyhalothrin equivalents)
Muscle 20 20 6
Liver 20 333C 33
Kidney 20 100d 5
Fat 400 400 20
Milk 30 30 45
Total 109
a
Expressed as parent drug.
b
Based on a daily intake of 300g of muscle, 100g of liver, 50g each of kidney and fat and
1.5kg of milk.
c
The marker residue accounted for 6% of the total residues in liver.
d
The marker residue accounted for 20% of the total residues in kidney.

• The recommended MRLs for fat are based on the upper confidence
limit of the highest mean concentration of residues as determined
in residue-depletion studies in which the product was used in accor-
dance with good practice in the use of veterinary drugs. The MRL
recommended for milk is based on the upper confidence limit of the
highest mean concentration of residues, as determined in residue-
depletion studies in which treatment with the spray formulation
was given in accordance with good practice in the use of veterinary
drugs.
The Committee recommended temporary MRLs in cattle, pigs and
sheep of 20mg/kg for muscle, liver and kidney and 400mg/kg for fat,
expressed as parent drug. The Committee also recommended a tem-
porary MRL of 30 mg/kg for cows' milk.
From these MRLs, the theoretical maximum daily intake of
cyhalothrin residues due to veterinary use would be 109mg(Table 2).
Since the maximum ADI is 120mg,this gives a margin of safety for the
possible ingestion of residues from other uses.
Data on the validation of the analytical method for sheep liver, to
confirm the limit of quantification of l0mg/kg, are required for evalu-
ation in 2002.

3.3.2 Cypermethrin
Cypermethrin was previously evaluated by the Committee at its forty-
seventh meeting (Annex 1, reference 725), when it established an
ADI of 0-50 jig/kg of body weight and recommended temporary
MRLs of 200mg/kg for muscle, liver and kidney and l000mg/kg for
fat, expressed as parent drug, in cattle, sheep and chickens. It also

41
 recommended temporary MRLs of 50 mg/kg for milk (whole) and
100mg/kg for eggs, expressed as parent drug, in cattle and chickens,
respectively.
At its forty-seventh meeting, the Committee required the following
information for evaluation in 2000:
1. The results of residue-depletion studies with radiolabelled
cypermethrin that extend beyond the recommended withdrawal
times for the drug in its topical formulation. The depletion of the
total residues and of the parent drug should be determined.
2. Evidence that interconversion of the isomeric forms does not occur
during metabolism in the target species.
3. Further information on the validation of the analytical methods,
particularly data on the derivation of the limits of detection and
quantification.
At the present meeting, the Committee noted that no information
had been provided to answer the first and second requests and that
there was no indication that the sponsors would provide this informa-
tion in the near future.
A study in sheep treated orally with an isomeric mixture of radiolabelled
cypermethrin was available. This study did not address topical admin-
istration of the drug, and the ratio of cis: trans isomers (80:20) was
different from that of the isomeric mixture of cis: trans cypermethrin
(45:55) which had been evaluated at the forty-seventh meeting.
In response to the third request, a suitable analytical method was
provided for quantifying the total residues resulting from the use of
the cypermethrin formulation containing an 80:20 mixture of the cis-
and trans-isomers. Since the method uses gas chromatography, in
which all the possible isomers co-elute, it can be used to quantify total
residues resulting from the use of cypermethrin containing any ratio
of cis- and trans-isomers.
Since the Committee did not receive the required information and
there was no indication that it would be provided in the future, the
temporary MRLs recommended at the forty-seventh meeting were
not extended.
The Committee also noted that no information was available for a
toxicological evaluation of the 80:20 cis:trans isomeric mixture of
cypermethrin.
3.3.3 a-Cypermethrin
a-Cypermethrin was previously evaluated by the Committee at its
forty-seventh meeting (Annex 1, reference 725), when it established

42
 an ADI of 0-20mg/kg of body weight and recommended temporary
MRLs of l00mg/kg for muscle, liver and kidney and 500mg/kg for fat,
expressed as parent drug, in cattle, sheep and chickens. It also recom-
mended temporary MRLs of 25 jig/kg for whole milk and 50 mg/kg for
eggs, expressed as parent drug, in cattle and chickens, respectively.
At its forty-seventh meeting, the Committee required the following
information for evaluation in 2000:
1. The results of residue-depletion studies with radiolabelled a-
cypermethrin in sheep and chickens that extend beyond the recom-
mended withdrawal times for the drug in its topical formulation.
The depletion of the total residues and of the parent drug should
be determined.
2. The residue-depletion study with radiolabelled a-cypermethrin in
cattle that was submitted should be reassessed to determine the
depletion of the total residues and of the parent drug.
3. Evidence that the interconversion of isomeric forms of a-
cypermethrin does not occur during metabolism in the target
species.
4. Further information on the validation of the analytical methods,
particularly data on the derivation of the limits of detection and
quantification.
Since the Committee did not receive the required information and
there was no indication that it would be provided in the future, the
temporary MRLs recommended at the forty-seventh meeting were
not extended.

3.3.4 Dicyclanil
Dicyclanil has not previously been evaluated by the Committee. It is
a pyrimidine-derived insect growth regulator used for the topical
treatment of sheep to prevent larval infestation by the blowfly
(Lucilia cuprina). It is applied as a pour-on formulation containing
5% (w/v) of the drug.
At the present meeting, data were provided on the use of dicyclanil
applied as a pour-on formulation to sheep at a maximum dose of
0.lg/kg of body weight.

Toxicological data
The Committee considered the results of studies on the pharmacoki-
netics, metabolism, acute, short-term and long-term toxicity, carcino-
genicity, genotoxicity, reproductive toxicity and pharmacology of
dicyclanil. All of the pivotal studies were carried out according to
appropriate standards for study protocol and conduct.

43
 After repeated oral administration of radiolabelled dicyclanil to rats,
the radiolabel was rapidly and almost completely absorbed and dis-
tributed to the major organs and tissues. Elimination was rapid (93%
or more of the total dose within 24 h) and was virtually complete
within 3 days. The major route of elimination was the urine (79-83%
of the total dose within 24h), while the faecal route was of minor
importance (6-12% of the total dose within 24h). Biotransfor-
mation in the rat involves oxidative opening of the cyclopropyl ring
at various positions, followed by further oxidation and cleavage of
the cyclopropyl-N bond (i.e. dealkylation). The pathways by which
dicyclanil is metabolized in sheep treated topically are essentially the
same as those in rats.

After oral administration of dicyclanil to rats, the LD50 values were

560mg/kg of body weight in males and 500mg/kg of body weight in
females. Dicyclanil is moderately hazardous when given as a single
oral dose.

In a 3-month study of toxicity, rats received dicyclanil in the diet at

a concentration of 0, 5, 25, 125 or 500mg/kg of feed. Males showed
decreased food consumption and body-weight gain and slightly de-
creased plasma glucose concentrations at 125 and 500mg/kg of feed;
the relative weights of the kidneys, brain and testes were increased in
the highest-dose group. Females in the highest-dose group showed
decreased food consumption, body-weight gain and plasma glucose
concentrations and increased relative weights of the liver and brain;
plasma glucose concentrations were also reduced at 125mg/kg of
feed. The NOEL was 25mg/kg of feed, equal to 1.6mg/kg of body
weight per day, on the basis of the reduction in body-weight gain.

In a 3-month study of toxicity, dogs received dicyclanil in the diet at

a concentration of 0, 20,100, 500 or 1500mg/kg of feed. Clinical signs
of toxicity, including signs of neurotoxicity, decreased food consump-
tion and body-weight gain, and changes in clinical chemistry and
erythrocyte parameters were observed mainly at the highest concen-
tration. Plasma cholesterol and phospholipid concentrations were
increased in animals at concentrations of l00mg/kg of feed and
above. The weights of the spleen (males and females) and thymus and
testes (males only) were decreased at 1500mg/kg of feed. Atrophy
of the spleen in males and females and atrophy of the thymus,
mesenteric lymph node, testes and prostate in males were observed at
concentrations of lOOmg/kg of feed and above. The weights of the
liver, adrenals and kidneys were increased in animals in the highest-
dose group; in females, the liver weights were also increased at lower

44
doses. Inflammatory changes were seen in the urinary bladder of
females at concentrations of l00mg/kg of feed and above and in the
liver of males and females at 1500mg/kg of feed. Hepatocyte oedema
was observed in females in all treatment groups, but hepatocel-
lular damage was not seen in any of the treatment groups. As the
Committee considered hepatocyte oedema to be without toxicologi-
cal significance in the absence of hepatocellular damage, the NOEL
was 20mg/kg of feed, equal to 0.61mg/kg of body weight per day, on
the basis of increased plasma cholesterol concentrations and histo-
pathological alterations of the prostate and urinary bladder.
In a 1-year study of toxicity, groups of four male and four female dogs
received dicyclanil in the diet at a concentration of 0, 5, 25, 150 or
750mg/kg of feed. Males showed slightly decreased plasma calcium
concentrations and increased absolute and relative liver weights at
750mg/kg of feed. Plasma cholesterol concentrations were increased
in males at 150 and 750mg/kg of feed, and this effect was not reversed
after a 4-week recovery period. Females receiving the highest dose
vomited and had slightly reduced food consumption and body-weight
gain, slightly increased plasma cholesterol concentrations, increased
absolute and relative liver weights, and decreased absolute and rela-
tive heart weights. Macroscopic and microscopic findings consisting of
necrosis of the liver and tubular lesions in the kidneys were found in
one male and one female in the highest-dose group that died prema-
turely; atrophy of the testes and prostate (in the male) and vascular
thrombus (in the female) were also observed. These animals suffered
from acute, severe liver failure, cardiovascular disturbances and stress
due to weight loss. Comparable acute, severe liver toxicity was not
observed in the 3-month study of toxicity in dogs given dicyclanil in
the diet at concentrations of up to 1500mg/kg of feed. The findings in
the two animals that died prematurely were therefore considered to
be incidental. The NOEL was 25mg/kg of feed, equal to 0.71 mg/kg of
body weight per day, on the basis of increased plasma cholesterol
concentrations in male dogs. This NOEL is supported by the NOEL
in the 3-month toxicity study in dogs. It should be noted that the
histopathological alterations observed in the 3-month study were not
seen among the surviving animals in the 1-year study.
In a study of carcinogenicity, mice received diets containing dicyclanil
at a concentration of 0, 10, 100, 500 or 1500 mg/kg of feed for 18
months. The animals in the highest-dose group were killed during
weeks 58-59 because of self-inflicted injuries and poor health. On the
basis of significant reductions in body-weight gain, the concentrations
of 500 and 1500 mg/kg of feed in females and 1500 mg/kg of feed
in males were considered to exceed the maximum tolerated dose.

45
 The liver was the main target organ in both male and female mice.
The effects included pigmentation of the Kupffer cells (with
haemosiderin) and hepatocellular necrosis in males at concentrations
of l00mg/kg of feed and above, an increased incidence of hepatocel-
lular adenomas in females at 500 and 1500mg/kg of feed, and an
increased incidence of hepatocellular carcinomas in females at
1500mg/kg of feed. The Committee noted that these liver tumours
were observed only at doses that exceeded the maximum tolerated
dose and that there were signs of hepatocellular proliferation in these
animals, which might have been involved in the hepatic carcinogenesis
observed. Pigmentation of the olfactory epithelium (with oxidized
lipofuscins) was observed in both sexes at 100 and 500mg/kg of feed;
in males, this effect was accompanied by an increased incidence of
inflammatory cell infiltration in the underlying Bowman glands. Males
and females in the group treated at 500mg/kg of feed also showed
pigmentation of the adrenal glands (with partly oxidized lipofuscins)
and hypercellularity of the bone marrow. As the Committee consid-
ered the effects on the olfactory epithelium to be of no biological
significance, the NOEL was l0mg/kg of feed, equal to l.lmg/kg of
body weight per day, on the basis of the effects on the liver.
In a 2-year study of carcinogenicity and toxicity, rats received diets
containing dicyclanil at concentrations of 0, 5, 25,125 or 500mg/kg of
feed. Animals in the highest-dose group showed decreased food con-
sumption and body-weight gain and increased relative weights of
almost all organs. Treatment-related histopathological alterations
were observed in the exocrine pancreas (hyperplasia) of males and in
the liver (biliary cysts) of females at the highest dose, and in the
olfactory epithelium (pigmentation resulting from accumulation of
oxidized lipofuscins) of males at concentrations of 25 mg/kg of feed
and above and of females at concentrations of 125 mg/kg of feed and
above. Although the alterations in the olfactory epithelium represent
enhancement of a naturally occurring age-related process, treatment
had no effect on survival, behaviour or general well-being, and there
were no other morphological changes in the olfactory mucosa. The
Committee therefore concluded that the effect on the olfactory epi-
thelium was of no biological significance. Dicyclanil did not affect the
incidence of tumours. The NOEL was 125 mg/kg of feed, equal to
22 mg/kg of body weight per day, on the basis of changes in body
weight and histopathological changes in the liver and pancreas.
Dicyclanil has been tested in vitro for its ability to induce reverse
mutations in Salmonella typhimurium and Escherichia coli, gene mu-
tations in Chinese hamster lung cells, chromosomal aberrations in
Chinese hamster ovary cells and unscheduled DNA synthesis in pri-

46
mary rat hepatocytes. It has also been tested in vivo for its ability to
induce micronuclei in bone-marrow cells of mice treated orally. The
results of all of these tests were negative. On the basis of these data,
the Committee concluded that dicyclanil is not genotoxic.
The Committee concluded that dicyclanil does not represent a carci-
nogenic risk for humans, as the liver tumours observed in female mice
occurred in only one tissue of animals of one sex and one species and
at levels that were above the maximum tolerated dose.
In a two-generation study of reproductive toxicity, with two litters per
generation, rats were given dicyclanil in the diet at a concentration of
0, 5, 30,200 or 500mg/kg of feed. Treatment reduced the body-weight
gain of the parental animals at 500mg/kg of feed and, marginally, at
200mg/kg of feed. Secondary to this effect, dicyclanil increased the
relative weights of most organs in animals at 500mg/kg of feed and
of the brain (males and females) and kidneys and testes (males) at
200mg/kg of feed. Reproductive parameters were not affected. The
only effect of dicyclanil on pups was to reduce their weight gain from
day 4 postpartum onwards. The NOEL for parental toxicity was
30mg/kg of feed, equal to 2mg/kg of body weight per day, on the basis
of reduced body-weight gain. The NOEL for reproductive toxicity
was 500mg/kg of feed, equal to 24mg/kg of body weight per day, the
highest dose tested. The NOEL for toxicity to the pups was 200mg/kg
of feed, equal to 21mg/kg of body weight per day, on the basis of
reduced body-weight gain.
In a study of developmental toxicity in rats given dicyclanil at a dose
of 0,1,5,25 or 75mg/kg of body weight per day orally on days 6-15 of
gestation, the highest dose was toxic to the dams, as seen by reduc-
tions in body-weight gain, food consumption and the absolute weight
of the gravid uterus. Marginal reductions in body-weight gain and
food consumption were also observed at 25 mg/kg of body weight per
day. The effects on the fetuses, observed only at the highest dose,
were reduced weight, a slightly increased incidence of dilatation of
the renal pelvis, and a number of mainly sternebral defects and varia-
tions due to poor or absent ossification. There was no evidence of
teratogenicity. The NOEL for maternal toxicity was 5 mg/kg of body
weight per day, on the basis of the reduction in body-weight gain. The
NOEL for developmental toxicity was 25 mg/kg of body weight per
day, on the basis of reduced fetal weight, increased dilatation of the
renal pelvis, and increased skeletal anomalies and variations consis-
tent with a slight delay in skeletal maturation.
In a study of developmental toxicity in rabbits given dicyclanil at a
dose of 0, 1, 3,10 or 30mg/kg of body weight per day orally on days

47
 7-19 of gestation, dams in the highest-dose group showed reduced
food consumption and body-weight gain; reduced body-weight gain
was also observed in dams at l0mg/kg of body weight per day. The
fetuses of dams in the highest-dose group had lower body weights
than controls and an increased incidence of skeletal variations indica-
tive of a slight delay in ossification. There was no evidence of terato-
genicity. The NOEL for maternal toxicity was 3 mg/kg of body weight
per day, on the basis of reduced body-weight gain. The NOEL for
developmental toxicity was 10 mg/kg of body weight per day, on the
basis of reduced fetal weight and skeletal variations consistent with
delayed ossification.
In pharmacological tests in vitro, dicyclanil had no effect on the
skeletal neuromuscular junction at doses of up to 3mmol/l. At con-
centrations of 0.3mmol/l and higher, it had slightly antagonistic
effects on smooth muscle contractions induced by agonists. In mice
and rats given a single oral dose of 0, 1, 10, 50 or 100 mg/kg of body
weight, the highest dose affected general behaviour, locomotor activ-
ity, motor coordination, heart rate, and tidal and minute lung volume.
Locomotor activity was also affected at 10 mg/kg of body weight and,
very slightly, at 1 mg/kg of body weight. Treatment had no effect on
body temperature, hypnotic potentiation, gastrointestinal motility,
blood pressure, heart beat or respiratory rate.
The Committee established an ADI of 0-7mg/kg of body weight for
dicyclanil, based on the NOEL of 0.71 mg/kg of body weight per day
for increased plasma cholesterol concentrations in the 1-year study of
toxicity in dogs and a safety factor of 100.

Pharmacokinetic and metabolism data

Rats. Studies in rats showed that the major metabolites of dicyclanil
in urine, faeces and tissues were parent dicyclanil, 2,4,6-
triaminopyrimidine-5-carbonitrile (descyclopropyl dicyclanil), N-
(4,6-diamino-5-cyanopyrimidin-2-yl)propionamide, 2-(4,6-diamino-
5-cyanopyrimidin-2-ylamino)-3-hydroxypropionic acid, and 3-(4,6-
diamino-5-cyanopyrimidin-2-ylamino)propionic acid. Thin-layer
chromatography (TLC) and HPLC were used to separate 12 metabo-
lite fractions from urine, faeces and tissues. The pattern did not vary
by sex or dose. The major urinary metabolite, N-(4,6-diamino-5-
cyanopyrimidin-2-yl)propionamide, represented 50% of the total
dose.
Sheep. Three studies were available in which sheep were treated with
a [14C]dicyclanil formulation typical of those used in current veteri-
nary applications. In two studies, the pour-on formulation was used,

48
and this resulted in greater dermal absorption than a spray technique
("jetting"), although the maximum concentration of radiolabel in
blood was reached more slowly. A biphasic curve characterized
depletion over the first 5 days after treatment, and a half-life of 2 days
was found. Continuous dermal absorption was observed after pour-
on application. A small portion of the administered dose was recov-
ered in urine and faeces.
The metabolic pathways in sheep were similar to those in rats. Meta-
bolic conversion proceeds through opening of the cyclopropyl ring
and oxidation of the a-carbon to a secondary propionic acid amide
(N-[4,6-diamino-5-cyanopyrimidin-2-yl]propionamide), or opening
of the cyclopropyl ring and oxidation to a b-alanine derivative
(3-[4,6-diamino-5-cyanopyrimidin-2-ylamino]propionic acid) and
dealkylation to descyclopropyl dicyclanil. The metabolism in sheep
was determined by analysis of samples obtained from animals treated
with a pour-on formulation containing [14C-pyrimidine]dicyclanil.
Pooled extracts of urine, faeces, bile, excess material that had run off
the animals during treatment, wool, fat, muscle, liver and kidney were
analysed by one- and two-dimensional TLC and HPLC. In the two
studies in which pour-on administration was used, 4-10% and
40-60% of the administered dose was recovered, respectively, in the
run-off during the first hour after administration. In one study, about
4% of the dose retained on the skin was recovered in urine and
faeces during the 7 days after dosing, while in the other study 0.6%
of the retained dose was recovered within 48 h of administration. In
urine, five fractions were identified, consisting of parent dicyclanil,
N-(4,6-diamino-5-cyanopyrimidin-2-yl)propionamide, descyclopropyl
dicyclanil, 3- (4,6-diamino-5-cyanopyrimidin-2-ylamino)propionic acid,
and an unidentified polar fraction from which descyclopropyl dicy-
clanil was released by microwave extraction. Dicyclanil and N-(4,6-
diamino-5-cyanopyrimidin-2-yl)propionamide accounted for 63%
and 6% of the total residues in urine, respectively.
Only 1 % of the total administered radiolabel was present in pooled
faeces collected during 48 h after administration. Exhaustive extrac-
tion allowed recovery of 91% of the radiolabel in faeces, of which
72% was dicyclanil and 2% was descyclopropyl dicyclanil. In bile,
most of the radiolabel appeared to be associated with strongly polar
metabolites, although unchanged parent drug and descyclopropyl
dicyclanil were also found.

Residue data
Sheep. In the studies in which [14C]dicyclanil was tested, high concen-
trations of radiolabel were extracted from muscle and fat at all times

49
 after dosing, and less was extracted from liver and kidney. The main
residue in muscle and fat tissues was the parent compound, which
accounted for over 90% of the total residues. Dicyclanil and one
unidentified metabolite were extracted from liver and kidney; kidney
also contained descyclopropyl dicyclanil. Most of the extractable
radiolabelled residues in liver and kidney were not characterized
because of the extremely low levels of radioactivity. The radiolabel
was widely distributed throughout the body, the concentration being
highest in subcutaneous fat under the application area. Between day
7 and day 21 after treatment, the concentration of radiolabel de-
creased most sharply in blood and muscle and least in omental and
subcutaneous fat.
The extractability of radiolabel from kidney and liver decreased as a
function of time; for instance, 90% of the radiolabel in kidney was
recovered on day 1 but only 50% on day 14. In the liver, 40-60% of
the radiolabel was extractable, and an additional 20% could be ex-
tracted under harsh conditions. The extractable metabolites had a
half-life of about 24 hours. The concentration of non-extractable
residues in kidney, as dicyclanil equivalents, decreased from 0.009 to
0.006 mg/kg during the 3 days after administration. In liver, the con-
centration decreased from 0.07 to 0.02 mg/kg within 14 days after
dosing. Overall, the parent drug accounted for about 15% of the total
residues in liver and about 25% of those in kidney. In some samples,
small amounts of descyclopropyl dicyclanil were found in kidney.
Almost 100% of the radiolabel was extracted from adipose and
muscle tissue and found to consist mainly of unchanged dicyclanil,
with small amounts of descyclopropyl dicyclanil. A small amount of
N-(4,6-diamino-5-cyanopyrimidin-2-yl)propionamide was found in
muscle.
The effects of application technique, wool length, dose, breed, age
and sex on residue depletion were studied with unlabelled dicyclanil
in a total of 340 sheep and lambs in eight studies. Only the five studies
that complied with GLP were considered relevant for the evaluation,
as the others involved use of analytical methods with which only
dicyclanil or both dicyclanil and descyclopropyl dicyclanil were deter-
mined. In the five studies used for the assessment, tissues were
analysed for the presence of both dicyclanil and descyclopropyl
dicyclanil. The tissue samples were collected at 2, 3, 7,11,14, 21, 28,
35,42,56,58 and 84 days after administration in all the studies except
one, in which they were collected at 7, 28, 56, 84 and 120 days after
administration.
The recommended dose of the pour-on formulation, 0.1 g/kg of body
weight, was used in only one study, while the other studies used 2-4

50
times the recommended dose. In the most comprehensive study, tissue
samples were collected 7, 14, 21 and 35 days after dosing. The com-
bined mean concentrations of dicyclanil and descyclopropyl dicyclanil
residues were expressed as dicyclanil equivalents and corrected for
recovery and for the proportion of the total residues accounted for
by the marker residue, determined in the studies with radiolabelled
drug. The highest combined mean concentrations of dicyclanil and
descyclopropyl dicyclanil, expressed as dicyclanil equivalents, were
0.36mg/kg in muscle, 6.3mg/kg in liver, 3.3mg/kg in kidney and
0.25mg/kg in fat, measured 7 days after administration of the com-
pound at the recommended dose. The respective values 35 days after
dosing were 0.03, 0.38, 0.23 and 0.06mg/kg.
The maximum concentrations of dicyclanil in the various tissues were
generally found 3-14 days after administration, except in one study in
which maximum concentrations of only 0.02-0.03 mg/kg were mea-
sured 56 days after administration. Concentrations of dicyclanil resi-
dues exceeding 0.1 mg/kg were found after administration at the
recommended dose in only one study; however, the mean residue
concentrations in most tissues were below 0.1 mg/kg 14 days after
administration, although that in subcutaneous fat was 0.12 mg/kg 28
days after treatment.

Analytical methods
The concentrations of dicyclanil residues in muscle, liver, kidney and
fat were determined by a fully validated HPLC method which allows
separation of dicyclanil from its metabolite descyclopropyl dicyclanil.
Aqueous acetonitrile is used for the initial extraction and is followed
by filtration. Separation of the lipids and successive purification steps
are accomplished on various solid-phase extraction cartridges. Sepa-
ration using HPLC is obtained by using a strong cation-exchange
column with a mobile phase consisting of acetonitrile, sodium per-
chlorate and perchloric acid. Dicyclanil and descyclopropyl dicyclanil
elute at different times and are detected by their absorption of ultra-
violet light at 270 nm. The limit of quantification for each compound
is 0.01 mg/kg.

Maximum Residue Limits

In recommending MRLs for dicyclanil, the Committee took the
following factors into account:
• An ADI of 0-7mg/kg of body weight, based on a toxicological end-
point, was established. This corresponds to a maximum ADI of
420 mg for a 60-kg person.
• The parent drug is the marker residue.

51
Table 3
Theoretical maximum daily intake of dicyclanil residues
Tissue Recommended Estimate of total Theoretical maximum daily intakeb
MRL (mg/kg)a residues (mg/kg) (mg dicyclanil equivalents)
Muscle 200 200 60
Liver 400 2666C 267
Kidney 400 1600d 80
Fat 150 150 8
Total 415
Expressed as parent drug.
Based on a daily intake of 300g of muscle, 100g of liver, and 50g each of kidney and fat.
The marker residue accounted for 15% of the total residues in liver.
The marker residue accounted for 25% of the total residues in kidney.

• On the basis of the studies with radiolabelled cyhalothrin in sheep,

the parent drug was estimated to account for 15% of the total
residues in liver and 25% of those in kidney.
• An HPLC method for the detection of dicyclanil residues is avail-
able, which is suitable to meet regulatory needs, with a limit of
quantification of l0mg/kg.
The Committee recommended MRLs in sheep of 200mg/kg for
muscle, 400mg/kg for liver and kidney, and 150 mg/kg for fat, ex-
pressed as parent drug.
From these values, the theoretical maximum daily intake of residues
as dicyclanil equivalents from veterinary use is 415 mg (Table 3). The
Committee did not recommend an MRL for ewes' milk as it did not
consider the use of dicyclanil in lactating sheep.

3.3.5 Permethrin
Permethrin has not been evaluated previously by the Committee. At
its present meeting, the Committee considered information on an
80:20 cis: trans isomeric mixture of permethrin which is used as an
ectoparasiticide on cattle. The mixture is applied as a pour-on formu-
lation at a dose rate of 4mg/kg of body weight up to a maximum of
1.6g per animal.
Information was provided on the depletion of residues of the 80:20
cis: trans isomeric mixture of permethrin after topical application at
the recommended dose of 4mg/kg of body weight to calves and lactat-
ing cattle. Information was also provided on a proposed analytical
method for regulatory purposes. The Committee was informed of an
ongoing residue-depletion study in which the radiolabelled isomeric
mixture was applied topically to cattle, which will provide information
on the depletion of total residues and the parent drug. The biotrans-

52
 formation of permethrin will also be examined in that study. The
Committee noted that additional validation of the proposed analyti-
cal method is required.
The 40:60 cis: trans isomeric mixture of permethrin was evaluated by
the 1982 Joint FAO/WHO Meeting on Pesticide Residues (11), which
noted that different isomeric mixtures would require independent
evaluation. The 1999 Joint FAO/WHO Meeting on Pesticide Resi-
dues (12} established an ADI of 0-0.05 mg/kg of body weight for
technical-grade permethrin containing the cis- and trans-isomers in
ratios of between 25:75 and 40:60.
At its present meeting, the Committee did not receive data to permit
an evaluation of the toxicity of the 80:20 cis: trans isomeric mixture of
permethrin intended for veterinary use. The Committee was aware
that the ds-isomer is substantially more toxic than the trans-isomer
when given as a single dose.
The Committee concluded that the available toxicological database
was inadequate to allow assessment of the toxicity of the 80:20
cis: trans isomeric mixture proposed for use as a veterinary drug. In
the absence of an ADI, the Committee was unable to recommend
MRLs for this isomeric mixture of permethrin.

3.3.6 Metrifonate (trichlorfon)

Metrifonate (trichlorfon) is an organophosphonate pesticide with in-
secticidal, acaricidal and anthelminthic properties. It is used as an
insecticide on food crops and forests. It is given orally, topically or
parenterally for the control of endo- and ectoparasites in and on
animals of various species. The recommended dose for treatment of
cattle orally or topically with pour-on, wash or spray formulations is
50-75 mg/kg of body weight. Repeated dosing may be necessary. The
preparations for use on horses are similar, but the recommended oral
dose is 35 mg/kg of body weight. One topical formulation for use on
horses also contains febantel. The drug is also given orally to humans
for infestation with Schistosoma haematobium, and has been studied
for use in the treatment of Alzheimer disease.
Metrifonate has not previously been evaluated by the Committee. It
was evaluated under the name "trichlorfon" on three occasions by the
Joint FAO/WHO Meeting on Pesticide Residues (13-15), which
established an ADI of 0-0.01 mg/kg of body weight in 1978 (15).

Toxicological data
The Committee considered the results of studies on the
toxicokinetics, metabolism, acute, short-term and long-term toxicity,

53
 genotoxicity, reproductive and developmental toxicity and neurotox-
icity of metrifonate. It also considered studies of immune function
and studies in humans. Most of the available studies were relatively
old and many were published reports which lacked raw data or data
on individual animals. Such studies are difficult to evaluate, and most
could not be assessed independently. Unpublished reports of studies
conducted during the 1980s and 1990s contained sufficient detail and
were carried out according to appropriate standards for study proto-
col and conduct.
The absorption of metrifonate is rapid in all species tested, including
humans, irrespective of the route of administration. Peak blood con-
centrations were achieved within 1-2 h but decreased quickly thereaf-
ter; the half-life of metrifonate in human plasma was approximately
2h. The drug is widely distributed. Metrifonate was detected in the
milk of lactating cows, and the compound and its metabolites were
found in fetal tissue in treated guinea-pigs. Metrifonate undergoes
conversion to dichlorvos via a dehydrochlorination reaction that
occurs spontaneously at pH values above 5.5. Although little dichlor-
vos was recovered, it is generally believed to be responsible for the
anticholinesterase effects of metrifonate. In mammals metrifonate is
also metabolized via O-demethylation and cleavage of phosphorus-
carbon bonds. The major metabolites are desmethyl metrifonate,
desmethyl dichlorvos, dimethyl hydrogen phosphate, methyl di-
hydrogen phosphate and phosphoric acid. Excretion of metrifonate
and its metabolites occurs primarily via the urine.
Metrifonate is moderately toxic when given as a single oral dose, the
LD50 values being 620-950 mg/kg of body weight in mice, 140-660 mg/
kg of body weight in rats, 160 mg/kg of body weight in rabbits, and
300-420 mg/kg of body weight in dogs. The signs of toxicity were
indicative of inhibition of acetylcholinesterase activity, and the cause
of death was usually respiratory failure. The rapid recovery observed
after sublethal doses suggests that metrifonate is readily detoxified.
Metrifonate was given orally to mice at doses of 10-750 mg/kg of body
weight per day, to rats at doses of 0.05-250 mg/kg of body weight per
day, and to dogs at doses of 0.5-45 mg/kg of body weight per day for
periods of up to 16 weeks. The weights of the liver, kidney and spleen
were increased at doses of 250 mg/kg of body weight per day and
above in mice and at doses of 50 mg/kg of body weight per day and
above in rats, but these changes were usually not associated with
other evidence of toxicity, except in one study in rats in which hyper-
trophy of centrilobular hepatocytes and biochemical changes sugges-
tive of altered lipid metabolism were seen. These effects occurred at

54
doses which also induced significant signs of cholinergic intoxication.
Slight anaemia was observed in one study in rats at l00 mg/kg of body
weight per day and in one study in dogs at 45mg/kg of body weight
per day. In one of three dogs given metrifonate at 45mg/kg of body
weight per day, the prostate and testes were small and immature, and
oligospermia was observed, but the animal had also lost weight and
appeared to be severely stressed by the treatment. The NOELs for
inhibition of brain and erythrocyte acetylcholinesterase activity were
15mg/kg of body weight per day in mice and 5mg/kg of body weight
per day in rats; the NOEL for inhibition of erythrocyte acetylcho-
linesterase activity in dogs was 5 mg/kg of body weight per day.
In two studies of up to 2 years' duration, mice were given metrifonate
orally at 15-750mg/kg of body weight per day. Body-weight gain was
impaired at doses of 30 mg/kg of body weight per day and above. The
mean weight of the liver was increased at a dose of 240 mg/kg of body
weight per day in one study, but no pathological alterations were
found. The NOEL for inhibition of brain and erythrocyte acetylcho-
linesterase activity was 49 mg/kg of body weight per day. The inci-
dences of tumours were unaffected by treatment. Neither study was
suitable for identifying a NOEL for establishing an ADI.
Six studies of up to 2 years' duration were conducted in which
rats were given metrifonate in the diet at 2.5-160 mg/kg of body
weight per day. The weights of the liver and spleen were increased in
three studies at 50 mg/kg of body weight per day and above.
Hypercholesterolaemia, duodenal hyperplasia, and gastritis of the
non-glandular stomach were seen at 13 mg/kg of body weight per day
and above. Anaemia, enhancement of age-related nephropathy, in-
creased hyperplasia of the renal tubules, degeneration of the stomach
tunica muscularis, and inflammation and hyperplasia of the lungs
were observed at 76 mg/kg of body weight per day and above. Other
observations included ovarian atrophy at 20 mg/kg of body weight per
day and above, depression of spermatogenesis at 50 mg/kg of body
weight per day, and a slight increase in the incidence of benign mam-
mary tumours in females with a reduction in the time to appearance
at 20 mg/kg of body weight per day and above. The NOEL for inhi-
bition of brain and erythrocyte acetylcholinesterase activity was
13 mg/kg of body weight per day. The incidence of mammary tumours
was slightly increased in female Sprague-Dawley rats in two studies.
However, the Committee concluded that metrifonate does not have
carcinogenic potential in rats, as the tumours were benign, this strain
of rats has a relatively high and variable background incidence of
mammary tumours, and the finding was not replicated in several other
studies in various species. The overall NOEL in rats was 4.5 mg/kg of

55
 body weight per day, on the basis of the hypercholesterolaemia and
pathological changes to the gastrointestinal tract.
In two studies of 1 and 4 years' duration, dogs were given metrifonate
orally at doses of 1.3-80mg/kg of body weight per day. Deaths
occurred at doses of 20mg/kg of body weight per day and above, and
signs of hepatocellular damage and increased severity of inflamma-
tion were seen in the liver at doses of 25 mg/kg of body weight per day
and above. In animals given 20-25 mg/kg of body weight per day, the
weight of the spleen was increased, with atrophy of lymphoid tissue;
small ovaries, reduced testis weight, and decreased spermatogenesis
were also observed. "Blood cholinesterase" activity was inhibited
at doses of 5 mg/kg of body weight per day and above. The overall
NOEL in dogs was 1.3 mg/kg of body weight per day, on the basis of
inhibition of cholinesterase activity.
Metrifonate was administered orally to rhesus monkeys at a dose of 0,
0.2, 1 or 5 mg/kg of body weight per day for 10 years. Inhibition of
plasma, erythrocyte and brain cholinesterase activities was seen at 1
and 5 mg/kg of body weight per day; animals in the highest-dose group
also showed clinical signs of toxicity indicative of cholinesterase inhi-
bition, reduced body-weight gain and anaemia. In a separate 6-month
study in rhesus monkeys, erythrocyte acetylcholinesterase activity
was not depressed at 0.2 mg/kg of body weight per day, the highest
dose tested. The NOEL in monkeys was 0.2 mg/kg of body weight per
day, on the basis of inhibition of brain and erythrocyte acetylcho-
linesterase activity.
Metrifonate has been tested in a wide range of assays for genotoxicity
and chromosomal-damaging activity, with considerable variation in
the results. Metrifonate induced point mutations in microorganisms
and cultured mammalian cells, DNA damage in microorganisms, and
chromosomal aberrations and sister chromosome exchanges in cul-
tured mammalian cells. The results of tests for point mutation in
Drosophila melanogaster and for sister chromatid exchange in bone
marrow were negative. Although positive results were obtained in a
few assays for chromosomal damage in bone marrow and the germ
cells of male animals exposed to near-lethal doses, the results of most
studies of micronucleus formation, metaphase alterations, and domi-
nant lethal mutations were negative. Since the tests conducted in vivo
produced mostly negative results when metrifonate was administered
orally, the Committee considered that the weight of evidence indi-
cates that metrifonate is unlikely to have genotoxic potential.
Metrifonate was given in the diet to rats at doses equivalent to 0,10,
30, 100 or 300 mg/kg of body weight per day in a three-generation

56
study of reproductive toxicity, with two litters per generation. The
body-weight gain of F0 dams at 100 and 300mg/kg of body weight per
day was depressed. The slight effects on the gonads reported in the
studies of general toxicity were not consistently reflected in this study.
The pregnancy rate of rats of the Flb generation at 100 and 300mg/kg
of body weight per day was reduced, but the fertility of other genera-
tions was unaffected. The litter size of dams in the highest-dose group
was reduced and all of the offspring died, but reproductive parameters
were unaffected at lower doses. The NOEL was 30mg/kg of body
weight per day in adults, on the basis of reduced body-weight gain, and
l00mg/kg of body weight per day for reproductive effects, on the basis
of reduced litter size and the death of offspring.
Two studies of developmental toxicity were conducted in mice given
metrifonate by gavage at 200-600 mg/kg of body weight per day.
Maternal toxicity in the form of reduced body-weight gain was
observed in all treated groups; animals dosed at 300mg/kg of body
weight per day and above also showed an increased mortality rate.
The effects of these maternally toxic doses included increased
embryotoxicity, decreased fetal weight, and delayed fetal develop-
ment. The incidence of cleft palate was slightly increased at 500 and
600 mg/kg of body weight per day. NOELs were not identified for
maternal toxicity, embryotoxicity or fetotoxicity.
Studies of developmental toxicity were conducted in rats given
metrifonate in the diet or by gavage at doses of 8-520 mg/kg of body
weight per day. In three studies, maternal toxicity in the form of
reduced body-weight gain and/or an increased mortality rate was
observed at doses of 150 mg/kg of body weight per day and above. The
NOEL for maternal toxicity was 75 mg/kg of body weight per day.
Fetotoxic effects included reduced fetal body weight at 75 mg/kg of
body weight per day, minor skeletal changes at 380 mg/kg of body
weight per day, and an increased incidence of fetal malformations
such as morphological alterations of the skull, central nervous system
and limbs, micrognathia, cleft palate, facial haematomas, generalized
oedema, and defects in major blood vessels at doses of 430mg/kg of
body weight per day and above. In three other studies, the incidence
of fetal malformations was unaffected. The NOEL for developmental
effects was 50 mg/kg of body weight per day, on the basis of
fetotoxicity.
In a study in hamsters, metrifonate was given by gavage at a dose of
0, 200, 300 or 400 mg/kg of body weight per day during pregnancy.
Signs of cholinesterase inhibition were observed in all treated groups;
those dosed at 300 and 400 mg/kg of body weight per day also had

57
 depressed body-weight gain. The NOEL for maternal toxicity was
l00mg/kg of body weight per day. Fetal body weight was decreased at
300 and 400mg/kg of body weight per day, and an increased incidence
of fetal stunting was seen at the highest dose. The NOEL for
developmental effects was 200mg/kg of body weight per day.
Guinea-pigs given metrifonate by gavage at a dose of l00mg/kg of
body weight per day for 6 days during gestation had abortions and
stillborn fetuses, and the offspring had reduced body weight and brain
weight, locomotor disturbances, and morphological and biochemical
alterations in the brain.
In two studies of developmental toxicity in rabbits, metrifonate was
given by gavage at doses of 5-110mg/kg of body weight per day.
Overt toxicity and inhibition of erythrocyte acetylcholinesterase ac-
tivity were observed in dams given doses of 35 mg/kg of body weight
per day and above, and body-weight gain was affected at all doses.
The rate of fetal resorption was slightly increased and fetal develop-
ment retarded at the highest dose, but no fetal abnormalities were
found. A NOEL for maternal toxicity was not identified. The NOEL
for developmental effects was 45 mg/kg of body weight per day.
Several outbreaks of congenital tremor with cerebellar hypoplasia
were reported in piglets of sows that had been treated with
metrifonate, and the syndrome was subsequently reproduced experi-
mentally. The piglets of sows treated with metrifonate in the diet at
50-100 mg/kg of body weight per day 55-98 days after conception
showed neurological signs and hypoplasia and loss of Purkinje cells in
the cerebellum.
Rats given metrifonate in the diet at a dose equivalent to 200 mg/kg
of body weight per day for 13 weeks showed decreased locomotor
activity and loss of coordination. In another feeding study in rats, a
dose equivalent to 30 mg/kg of body weight per day for 3 weeks
increased locomotor activity and impaired learning ability and nerve
conduction.
The results of numerous studies in hens consistently demonstrate the
acute neurotoxicity of metrifonate. Delayed neurotoxicity, associated
with degeneration of nervous tissue and marked inhibition of neuro-
pathy target esterase, was not seen in hens. However, a monkey
(species unspecified) given a single oral dose of 250 mg/kg of body
weight showed impaired nerve conduction 4 weeks after treatment
and histological evidence of demyelination and axonal degeneration.
Metrifonate has been used as an anthelminthic agent in humans. Oral
doses of up to 10 mg/kg of body weight given on 2-4 occasions were

58
well tolerated, with few clinical symptoms of toxicity. Oral admini-
stration of a dose of 24mg/kg of body weight caused cholinergic
symptoms, but the effects were not cumulative. In a few cases, sper-
matogenesis appeared to have been impaired. The results of clinical
trials with metrifonate in the treatment of Alzheimer disease showed
dose-related but reversible muscular weakness. The recommended
dose of 0.5-0.9 mg/kg of body weight resulted in a small increase in
the frequency of generalized weakness and fatigue, while doses of
1.25 mg/kg of body weight and higher produced significant weakness.
Dose-related inhibition of erythrocyte acetylcholinesterase activity
was also observed in these studies.
In 121 subjects, an initial oral dose of 0.5 mg/kg of body weight per day
for 2 weeks inhibited erythrocyte acetylcholinesterase activity by
29%. Subsequent administration of an oral dose of 0.2 mg/kg of body
weight per day for 10 weeks maintained the level of inhibition of
erythrocyte acetylcholinesterase activity at 30-37%, with no increase
with increased duration of dosing. Since this dose enhanced the inhi-
bition caused by the initial dose by only 8%, a non-significant change,
the Committee concluded that the NOEL was 0.2 mg/kg of body
weight per day.
In some cases of severe human poisoning with metrifonate, weakness
and loss of feeling in the extremities, difficulty in walking, muscular
atrophy, and motor nerve damage have been observed. In many of
these cases, the doses might have been lethal in the absence of medi-
cal intervention. The Committee concluded that extremely high doses
of metrifonate would be required to achieve the level of inhibition of
neuropathy target esterase associated with delayed neurotoxicity.
The Committee concluded that inhibition of acetylcholinesterase
activity was the most relevant end-point for establishing an ADI for
metrifonate. The most appropriate NOEL was 0.2mg/kg of body
weight per day for inhibition of erythrocyte acetylcholinesterase
activity in humans treated orally. A safety factor of 10 was applied
to this figure, giving an ADI of 0-20mg/kg of body weight.

Pharmacokinetic data
Cattle. Metrifonate labelled with 14C on the a-carbon of the
trichloroethyl group was applied to the backs of eight calves at a
target dose of 40 mg/kg of body weight, and one male and one female
were slaughtered on each of days 1, 2, 3 and 5 after treatment.
Between 14 and 49% of the dose was not available for absorption
because of run-off from the backs of the treated animals. When the
animals were washed after slaughter, the radiolabel in the washings

59
 represented 4-16% of the administered dose. The amount remaining
on the skin represented 8-28% of the dose and would have continued
to contribute to the tissue residues while it was absorbed. Only 2 and
6% of the initial dose was estimated to have been absorbed by the two
calves slaughtered 5 days after dosing.
The pharmacokinetics of metrifonate in plasma, urine and faeces was
measured in the two calves slaughtered 5 days after treatment. Ab-
sorption was rapid; the maximum concentration of total radiolabel in
plasma (Cmax), expressed as metrifonate equivalents, was l.0mg/kg at
4h after treatment in the male and 0.36mg/kg at 6h in the female.
Thereafter, the Cmax declined in a biphasic manner, rapidly up to 24 h
and more slowly between 24 and 120 h after dosing. The half-lives for
elimination of total radiolabel into the plasma of the two calves were
124 and 258 h, respectively. Relatively little of the radiolabel was
excreted: 2.8 and 1.6% of the administered dose was excreted in
urine and 3.3 and 0.3% in the faeces of the two calves, respectively. If
account is taken of the significant portion of the administered dose
that was not available for absorption, the amount excreted is higher,
expressed as a percentage of the retained dose. Over the 5 days after
treatment, 14 and 5.2% of the retained dose was excreted in urine and
16 and 1.0% in the faeces of the two calves, respectively.
The metabolic profiles of topically administered metrifonate were
investigated in tissues from the two calves slaughtered on day 1 after
dosing. Extensive extraction procedures resulted in extraction of
83-97% of the total residues in muscle, liver and kidney and 73-85%
of those in fat. Identification of metabolites in the extractable
fractions proved to be difficult, because of the complex nature of the
residues. In all samples of fat taken on day 1 after dosing, metrifonate
and dichloroacetic acid were identified as the major metabolites.
Dichlorvos was found in one sample. Liver and kidney contained
mainly polar and other unknown metabolites. Muscle contained
metrifonate, but the amounts were not quantified. Since neither
metrifonate nor its major metabolites were quantified in any tissue,
the proportion of the total residues represented by a single compound
such as metrifonate could not be calculated. A similar pattern of
biotransformation was found in tissues from the other six calves.
In a study conducted in 1952, a lactating dairy cow infested with
"grubs" (not specified) was given [32P]metrifonate orally at a dose of
25mg/kg of body weight. Samples of blood, urine, faeces and milk
were collected, and the concentrations of total radiolabel and that
associated with metrifonate and dichlorvos were measured. The Cmax
in blood, 15mg/kg, expressed as metrifonate equivalents, was attained

60
2h after dosing; 7.5% was accounted for by metrifonate. Most (66%)
of the radiolabel was eliminated in urine within 12 h, but only 0.26%
of the excreted radiolabelled residues was associated with metri-
fonate. A major metabolite, which accounted for 73% of the excreted
radiolabelled residues, was tentatively identified as a dehydrochlori-
nation product of the two possible glucuronides of metrifonate. Less
than 3% of the radiolabel was excreted in faeces. Dichlorvos was not
detected in any of the samples.
Goats. The metabolic fate of [14C]metrifonate given orally at a dose of
8.6mg/kg of body weight per day on 3 consecutive days was studied in
two lactating goats. The goats were killed 4h after the last dose, and
samples of tissues and milk were collected and analysed for metabo-
lites. Unmetabolized metrifonate accounted for 6-7% of the total
residues in muscle and kidney, but was not present in liver, fat or milk.
A large proportion of the radiolabel was incorporated into tissue
proteins and sugars and accounted for 38% of the total residues in
muscle, 52% in liver, and 23% in kidney. The conjugates of
dichloroacetic acid accounted for 43% of the total residues in muscle,
11% in liver, 44% in kidney, and 70% in fat. Other metabolites,
including desmethylmetrifonate, desmethyldichlorvos and glucuronyl
metrifonate, were identified at low levels.

Residue data
Cattle. In the study described above in which [14C]metrifonate was
applied topically to the backs of eight calves at a target dose of
40mg/kg of body weight, samples of fat, muscle, and skin close to and
distant from the application site were collected at slaughter. The
concentration of radiolabel in plasma and in tissues distant from the
treated area appeared to reach a maximum at 3 days and declined
thereafter. The concentrations of total residues in the tissues of
animals slaughtered 1, 2, 3 and 5 days after dosing, expressed as the
mean metrifonate equivalents in two samples of each tissue, were
respectively 100, 220,180 and 92mg/kg in muscle; 590,1300, 2000 and
940mg/kg in liver; and 470, 730, 1200 and 560mg/kg in kidney. The
concentrations of total residues in various fats 1 day after treatment,
expressed as metrifonate equivalents, were 22 mg/kg in renal fat, 42 fig/
kg in omental fat, and 970 and 110 mg/kg in subcutaneous fat close to
and distant from the application site, respectively. At days 2, 3 and 5,
the concentrations of total residues in fat (combined) were 810, 950
and 200mg/kg, respectively.
Two new studies that complied with GLP were evaluated in which the
residues of metrifonate and dichlorvos were measured in the tissues
and milk of cattle after topical application of unlabelled metrifonate.

61
 Adult cattle were treated with a single spray of a 5% solution
of metrifonate at a dose of 40mg/kg of body weight, and killed in
groups (two males and two females) 12 h and 1, 3 and 7 days after
treatment. Samples of liver and kidney, and of muscle and fat close to
and distant from the area of application were collected and analysed
for residues of both metrifonate and dichlorvos by a validated liquid
chromatography-tandem MS method. No residues of metrifonate
were detected (limit of quantification, 50mg/kg) at any time in muscle
or fat distant from the area of application or in liver or kidney.
However, 12 h after dosing, residues of metrifonate were found at
concentrations of 51-170 mg/kg in subcutaneous fat close to the treated
area in samples from all four animals. Residues of dichlorvos were
found at a concentration of 140mg/kg only in fat tissue close to the
treated site in a sample taken from one animal on day 1, and residues
of metrifonate were found in samples of muscle (at 90 mg/kg) and fat
(at 2350mg/kg) from this animal.
In the second study, eight dairy cows were treated once with a spray
of a 5% solution of metrifonate at a dose of 40mg/kg of body weight,
and the concentration of metrifonate in milk was measured by a
validated liquid chromatography-tandem MS method. Most of the
metrifonate was excreted in milk during the first 12 h after treatment.
The mean concentrations (± standard deviation) were 79 ± 57mg/kg
6h after dosing and 61 ± 84(mg/kg 12 h after dosing; the highest concen-
tration was 200mg/kg in the milk from one cow 6h after dosing. The
concentrations of residues were just above the limit of quantification
(25mg/kg) in one cow (28mg/kg) at 24 h and in another cow (29mg/kg)
at 36 h, but thereafter no residues were detected (limit of detection,
2.5mg/kg).
Three horses were given single oral doses of a paste containing
both metrifonate and febantel at 35 and 6mg/kg of body weight,
respectively. The horses were killed 14 days later, and samples of
fat and muscle were assayed for the sum of metrifonate and dichlor-
vos measured as the common breakdown product, dimethyl phos-
phite, by gas chromatography. No residues were detected at
concentrations above the limit of quantification of the analytical
method (50mg/kg).

Analytical methods
A validated GC-MS method has been reported for the identification
and quantification of metrifonate residues in liver, kidney, muscle and
fat of cattle, using d6-metrifonate as an internal standard. A modifica-
tion of this method has been validated for the analysis of metrifonate
residues in the edible tissues of cattle and horses and in cows' milk.

62
In the modified method, residues of metrifonate are extracted
from tissues and milk using acetonitrile containing 0.1% formic
acid. Samples are purified by applying the extracts to a silica car-
tridge, washing the cartridge with ethyl acetate: heptane (1:9),
and eluting the metrifonate fraction with ethyl acetate. GC-MS is
performed with either an internal standard (d6-metrifonate) or an
external standard. Selective ion monitoring is then used to detect
metrifonate and d6-metrifonate at m/z 109 and 115 atomic mass units,
respectively.
The method has been validated for use over the linear range of the
detector response, which is 0.01-1 mg/kg for tissues and 0.05-2 mg/kg
for milk. Using the detector in the selective ion-monitoring mode
with an established gas chromatography retention time makes it
highly likely that the method is specific. The recovery of metri-
fonate in all tissues is 70-112%. The limit of detection of the
method in cattle is 9mg/kg for liver and muscle, 6mg/kg for kidney and
fat, and 2mg/kg for milk. In horses, the limit of detection is 8mg/kg
for muscle, 20mg/kg for liver, 16mg/kg for kidney, and 9 jig/kg for
fat. The limit of quantification is 50mg/kg for all tissues and 25(mg/kg
for milk.
No interference from the matrix has been reported in this method,
which was proposed by the sponsor for use in routine surveillance. As
the internal standard is not available commercially, either external
standardization should be used or the introduction of a suitable surro-
gate standard should be investigated.

Maximum Residue Limits

In reaching its decision on MRLs for metrifonate, the Committee
took the following factors into account:
• The ADI is 0-20 mg/kg of body weight, which is equivalent to a
maximum ADI of 1200 m,g for a 60-kg person.
• Only metrifonate and dichlorvos are of toxicological concern.
Metrifonate is a pro-drug, and dichlorvos is the only metabolite
with effective insecticidal action.
• Dichlorvos is highly unstable and is not present in the edible tissues
or milk of food-producing animals.
• The metabolism of metrifonate is broadly similar in target and
laboratory animals.
• The parent drug is the marker residue.
• Metrifonate is metabolized so extensively that the proportion of
the total residues represented by the residue marker cannot be
determined.

63
 • There is a suitable analytical method available for routine determi-
nation of metrifonate, with a limit of quantification of 50mg/kg for
muscle, liver, kidney and fat and 25m.g/kg for milk.
• The concentrations of metrifonate in muscle and fat samples distant
from the site of application were below the limit of quantification at
all times. Within 1 day of administration of a pour-on preparation,
the concentrations of residues in muscle and fat samples collected
close to the site of application were above the limit of quantification
in only a few animals. However, no residues were present in fat or
muscle close to the site of administration by 3 days after treatment.
• Residues of metrifonate were found at concentrations above the
limit of quantification in milk (25mg/kg) from the first three
milkings after treatment, but thereafter the concentrations were
below the limit of quantification. No residues of dichlorvos were
found at concentrations greater than the limit of quantification
(25 mg/kg) in any sample of milk.
Residues of metrifonate were not detected in the residue-depletion
studies reviewed by the Committee. The Committee concluded that
residues of metrifonate would not be found in muscle, liver, kidney or
fat at the limit of quantification of the available analytical methods.
Therefore, the Committee recommended that MRLs should not be
allocated for muscle, liver, kidney and fat, since no detectable resi-
dues should be present in tissues from animals treated with
metrifonate in accordance with good practice in the use of veterinary
drugs. The limit of quantification may be used by national govern-
ments as a guide for the maximum concentrations in muscle, liver,
kidney and fat of cattle. The guidance values are 50mg/kg for muscle,
liver, kidney and fat in cattle, expressed as parent drug. Insufficient
information was available to extend these MRLs to horses.
The Committee recommended an MRL of 50mg/kg for cows' milk,
expressed as parent drug.
From this MRL, the theoretical maximum daily intake of metrifonate
residues would be 75mg, based on a daily consumption of 1.5kg of
milk. This is equivalent to only 6% of the maximum ADI.

3.4 Production aid

3.4.1 Melengestrol acetate
Melengestrol acetate is a synthetic progestogen which is active after
oral administration. It is used to improve the efficiency of feed conver-
sion, promote growth, and suppress estrus in female beef cattle. The
range of approved doses is 0.25-0.50 mg/heifer per day. The drug can
be administered alone or in combination with other growth-promoting

64
drugs. Melengestrol acetate is fed for the duration of the fattening and
finishing period, usually for 90-150 days.
Melengestrol acetate has not previously been evaluated by the
Committee.

Toxicological data
The Committee considered information from a range of studies on
melengestrol acetate, including studies on its pharmacokinetics,
biotransformation, acute, short-term and long-term toxicity, carcino-
genicity, genotoxicity, and reproductive and developmental toxicity.
It also considered the results of studies in humans. Most of the studies
were conducted before 1979 according to the standards in existence
at that time and were not carried out in compliance with GLP. More
recent studies were conducted according to the appropriate standards
for study protocol and conduct.
The results of limited studies of the pharmacokinetics of melengestrol
acetate in rabbits and humans have been reported. The bioavailability
of melengestrol acetate after oral administration and its kinetics in
plasma have not been determined. In studies in which radiolabelled
melengestrol acetate was used, 3H or 14C was inserted at the 6-methyl
position. In rabbits, 59% of an orally administered dose of
[14C]melengestrol was excreted within 7 days in urine and faeces at
a ratio of about 1:3, with a peak elimination rate on the first day.
In women, the excretion of [14C]melengestrol acetate was complete
within 10 days, and 74% of the radiolabel was recovered in urine and
faeces. The half-life estimated from the data on excretion was 3-5 days.
Limited information was available on the biotransformation of
melengestrol acetate in cattle, rabbits and humans in vivo and in cattle
and rat liver microsomes in vitro. Melengestrol acetate is extensively
metabolized, with the formation of numerous metabolites. The me-
tabolites have been neither adequately identified nor characterized
with respect to their biological activity. In cattle, intact melengestrol
acetate accounted for up to 86% and 29% of the total radiolabel in
fat and liver, respectively. In cattle and rat liver microsomes, several
mono- and dihydroxylated metabolites were identified. In the urine of
rabbits, two-thirds of the radiolabel was found as glucuronides. The 6-
methyl-hydroxylated metabolite was identified in the free and conju-
gated forms as one of the major metabolites. In humans, 68% of the
radiolabel in urine was associated with conjugates, whereas faeces
contained more unconjugated compounds. Peaks representing 13
metabolites with an intact steroid nucleus were detected, one of which
was identified as 2a-monohydroxylated melengestrol acetate.

65
 Melengestrol acetate has little toxicity after a single dose, although
the studies of acute toxicity were limited, since a large volume of the
vehicle had to be administered. The LD50 values after intraperitoneal
injection were >2.5g/kg of body weight in mice and >2g/kg of body
weight in rats. No deaths were observed among rats given doses of
8 g/kg of body weight orally or 5 g/kg of body weight subcutaneously.
Dermal application to the intact or abraded skin of rabbits at the
maximum achievable dose of 22mg/kg of body weight caused no toxic
reaction.
Short-term tests of the toxicity of melengestrol acetate have been
performed in mice, rats, rabbits, dogs and monkeys. Melengestrol
acetate had a greater effect in females than in males, with hormonal
(progestational and corticosteroidal) effects as the most sensitive
end-points.
In TUC/ICR mice of each sex that received melengestrol acetate
orally at a dose of 0, 1, 3, 10 or 30mg/kg of body weight per day for
30 days, the body weights were slightly increased at 3 mg/kg of body
weight per day but were decreased at higher doses. Changes in female
reproductive organs, such as decreased weights of the ovaries and
uteri at the highest dose and the absence of corpora lutea at doses of
3 mg/kg of body weight per day and above were considered to be
progestational changes. The NOEL for hormonal effects was 1 mg/kg
of body weight per day.
In a 21-day study, puberal female C3Han/f mice received melengestrol
acetate in the diet at concentrations equal to 0,0.05,0.25,0.5,1.5, 2.5,
5 or 25 mg/kg of body weight per day. Body weight was significantly
increased at doses of 2.5 mg/kg of body weight per day and above, and
the serum concentration of prolactin and the weight of the uterus but
not the ovaries were increased at the highest dose. The NOEL was
1.5 mg/kg of body weight per day.
In mature female ICR and C3Han/f mice given an oral dose of 0,0.25,
0.5,2.5,5,10,15,20,25 or 40mg/kg of body weight per day for 20 days,
melengestrol acetate caused a significant, dose-related increase in
mammary duct proliferation in C3Han/f mice at doses of 15 mg/kg of
body weight per day and above. The drug had no effect on mammary
duct proliferation in ICR mice.
In order to elucidate the contribution of increased serum prolactin
concentration to melengestrol acetate-induced mammary duct prolif-
eration, groups of weanling female C3Han/f mice were given diets
containing melengestrol acetate at concentrations providing doses
of 0, 0.5, 1.5, 2.5, 5, 10 or 25 mg/kg of body weight per day for 20

66
days with or without the prolactin inhibitor 6-methyl-8|3-ergoline-
acetonitrile. The serum prolactin concentration and mammary duct
proliferation were enhanced at all doses, and the effects were partially
inhibited by 6-methyl-8b-ergoline-acetonitrile. There was no statisti-
cally significant association between mammary duct proliferation and
serum prolactin concentration. A NOEL could not be identified.
In juvenile rats given melengestrol acetate for 28 days by gavage at a
dose of 0,1, 3 or l0mg/kg of body weight per day, food consumption
and body weight were reduced in all treated animals. Haematological
changes were also seen, which included a dose-related increase in the
erythrocyte volume fraction and a decreased leukocyte count in ani-
mals at the highest dose. In females, the weights of the adrenals,
uterus and ovaries were reduced at all doses, and this effect was
associated with atrophy of these organs and the absence of corpora
lutea in most animals. In males, atrophy of the adrenal and accessory
sex glands was observed only at 3 and l0mg/kg of body weight per
day. The effects reported are consistent with progestational and
corticosteroidal activity. A NOEL could not be identified.
In a 90-day study of toxicity, rats received melengestrol acetate in
their diet at concentrations providing 0, 0.015, 0.15 or 0.3mg/kg
of body weight per day. The serum cholesterol concentration was
increased in females at 0.15 and 0.3mg/kg of body weight per day.
Changes characteristic of the hormonal effects of melengestrol
acetate were observed, such as: decreased weights of the adrenals,
ovaries and uterus at 0.3 mg/kg of body weight per day; hyperplasia
of the mammary glands and endometrium, agenesis of the corpora
lutea, and bone-marrow hypoplasia at 0.15 and 0.3 mg/kg of body
weight per day; and enlargement of the mammary glands at
0.015 mg/kg of body weight per day. Other effects observed at the
lowest dose, although not statistically significant, were consistent with
the changes seen at higher doses. The Committee concluded that
0.015 mg/kg of body weight per day was the minimally effective dose.
In another 90-day study, weanling rats were fed diets containing
melengestrol acetate at 0 or 0.055 mg/kg of body weight per day. The
treatment-related effects observed were slight increases in erythro-
cyte volume fraction, erythrocyte count and haemoglobin concentra-
tion, and significantly lower adrenal, ovarian and testicular weights. A
NOEL could not be identified.
Rabbits were injected intramuscularly with melengestrol acetate at
20mg/kg of body weight every second day for 22 days. All animals
lost weight and had diarrhoea. Haematological evaluation revealed

67
 decreased leukocyte counts and impaired platelet function. All four
males died during the last week of treatment from thoracic bleeding
after blood sampling. At termination of the study, serum cholesterol
concentrations and the activities of aspartate aminotransferase, lac-
tate dehydrogenase and alkaline phosphatase were increased in the
four surviving females. At necropsy, these animals were found to have
muscular atrophy, small adrenals, and enlarged livers with swollen
hepatocytes containing glycogen deposits.
Groups of two male and two female beagle dogs were given
melengestrol acetate in gelatin capsules orally at a dose of 0, 1, 3 or
lOmg/kg of body weight per day for 29 days. Treatment at 3 and
l0mg/kg of body weight per day induced slight-to-moderate diuresis,
with urine of decreased specific gravity. Body weight was slightly
decreased and food consumption increased in all treated animals.
Small increases were observed in the activity of serum alkaline phos-
phatase at 3 and l0mg/kg of body weight per day and of serum
alanine aminotransferase at l0mg/kg of body weight per day. A dose-
related decrease in adrenal weight and increase in liver weight were
seen, with histopathological changes indicative of glycogen deposi-
tion. A NOEL was not identified.
Groups of eight adult female rhesus monkeys were treated orally with
melengestrol acetate at a dose of 0, 1.5, 15, 75 or 150 mg/kg of body
weight per day for one menstrual cycle. Ovulation was monitored by
measuring the periovulatory surge of luteinizing hormone and the
decrease in estrogen concentration and confirmed by laparoscopy.
The number of monkeys that ovulated decreased significantly during
treatment, from 88% in controls and the lowest-dose group, to 38, 25
and 12% at 15, 75 and 150 ug/kg of body weight per day, respectively.
The menstrual cycle was prolonged at 75 and 150mg/kgof body weight
per day, but melengestrol acetate had no significant effect on the
serum concentrations of progesterone and estrogens (estradiol-17b
and estrone). Changes in the periovulatory surge of luteinizing hor-
mone and the suppression of ovulation were the most sensitive end-
points in this study. The NOEL for suppression of ovulation was
1.5mg/kg of body weight per day.
In a range-finding study for hormonal effects, groups of six female
cynomolgus monkeys were treated orally with melengestrol acetate
by nasogastric intubation at a dose of 0, 2.5, 5 or 10 mg/kg of body
weight per day for one menstrual cycle. One monkey in the lowest-
dose group and one in the highest-dose group were withdrawn from
the study because they showed anorexia. One monkey from each of
the groups dosed at 5 and 10mg/kgof body weight per day failed to

68
ovulate during treatment. Two monkeys from each of the groups
dosed at 2.5 and 10 mg/kg of body weight per day had prolonged
menstrual cycles; one animal from the group dosed at 5 mg/kg of body
weight per day and one control were also affected. No consistent
dose-response relationship was seen for effects on hormone concen-
trations. The serum concentration of estradiol was decreased during
the luteal phase of the menstrual cycle in animals at 5 and 10 mg/kg
of body weight per day, and that of luteinizing hormone was sup-
pressed at 2.5 and 5 mg/kg of body weight per day. Melengestrol
acetate had no consistent effect on the serum concentrations of pro-
gesterone and follicle-stimulating hormone. The authors concluded
that "melengestrol acetate may have exerted subtle effects on the
menstrual cycle of cynomolgus monkeys".
In a follow-up study, female cynomolgus monkeys were given
melengestrol acetate at a dose of 0, 5, 10 or 25 mg/kg of body weight
per day for three consecutive menstrual cycles, up to a maximum of
105 days. Groups of eight animals were observed for three consecu-
tive menstrual cycles before treatment. Two animals (one from each
of the groups dosed at 5 and 10 mg/kg of body weight per day) were
not included in the final evaluation because they had abnormal cycles
before treatment. The occurrence of ovulation was determined by
observing the periovulatory surge of luteinizing hormone, the peak of
estradiol, and the increase in progesterone concentration in the luteal
phase. The hormonal and menstrual cycle variables showed the
changes that would be expected to be induced by a progestogen, such
as significantly decreased serum concentrations of luteinizing hor-
mone and estradiol at 10 and 25 mg/kg of body weight per day and of
progesterone at 25 mg/kg of body weight per day. Significantly fewer
animals in the highest-dose group menstruated and ovulated, and
significantly more animals in the groups dosed at 10 and 25mg/kgof
body weight per day had changed cycles. In the remaining animals,
the dose-related prolongation of the first menstrual cycle did not
reach statistical significance. The serum concentrations of follicle-
stimulating hormone and cortisol were not affected by melengestrol
acetate. The effects at 5 mg/kg of body weight per day, although not
statistically significant, were consistent with the hormonal response
seen at higher doses. The Committee considered that 5 mg/kg of body
weight per day was the minimally effective dose and was close to the
NOEL for hormonal effects.
In heifers fed melengestrol acetate at a dose equal to 0.16 mg/kg of
body weight per day for 15-116 days after estrus, treatment reduced
the number of animals in estrus by 40%, and doses equal to 0.7 and
1.1mg/kgof body weight per day consistently suppressed estrus in all

69
 animals. Melengestrol acetate was also fed to heifers at a dose equal
to 1.8mg/kg of body weight per day from 2.5 to 11.3 months of age.
When the animals reached maturity, the serum concentrations of
estradiol-17b and estrone were significantly increased over those in
controls, and that of progesterone was suppressed to values similar to
those occurring in the proestrus period. The serum concentrations of
cortisol and corticosterone were depressed to about 50% of those in
untreated animals. A NOEL could not be identified for the progesta-
tional and corticosteroidal activity of melengestrol in cattle.
In a study of carcinogenicity, ICR mice received diets containing
melengestrol acetate at concentrations providing doses equal to 0,
0.017 or 17mg/kg of body weight per day for up to 24.5 months. The
animals in the highest-dose group weighed more than controls
throughout the study, and their survival rate was significantly lower.
These effects were attributed to the stress of obesity caused by
melengestrol acetate. The incidence of benign and malignant tumours
was reduced in treated females but not males. A slight, non-significant
increase in the incidence of mammary adenocarcinomas was observed
in animals in the highest-dose group. No firm conclusion could be
drawn about the carcinogenic potential of melengestrol acetate in
ICR mice.
In a similar study, prepuberal C3Han/f mice, which were previously
shown to be more sensitive than ICR mice to the effects of
melengestrol acetate on mammary duct proliferation, were given
diets containing melengestrol acetate at concentrations providing
doses equal to 0, 0.017 or 17mg/kg of body weight per day for up
to 33 months. Females in the highest-dose group had an increased
incidence of malignant tumours, consisting mainly of mammary
adenocarcinomas. This increase was assumed to be due not to a direct
carcinogenic effect of melengestrol acetate but to the promoting
effect of increased prolactin concentrations.
In another study, five groups of mature C3Han/f mice aged 63-84,77-
91, 84-105, 98-112 and 119-126 days were used to assess the effect of
age on the development of melengestrol acetate-induced mammary
tumours. The animals received a diet containing melengestrol acetate
at concentrations providing doses equivalent to 0,0.5,1,1.5,2.5,5,10,
15 or 25mg/kg of body weight per day. The study was terminated
after 27 months, when the mortality rate reached 90%. Age had a
significant effect on the development of mammary tumours in both
treated and control mice, with the greatest incidence in the youngest
group. Except for a lower incidence of mammary tumours in the
group that received lOmg/kg of body weight per day, the incidence

70
increased in a dose-related manner from 1.5mg/kg of body weight per
day. Melengestrol acetate had no effect on the time at which tumours
were first detected. The treatment-related non-neoplastic lesions that
were observed consisted of progestational effects, such as increased
cystic endometrial hyperplasia at doses of 5 nig/kg of body weight per
day and above. On the basis of the finding of a higher incidence of
mammary tumours in younger animals, which are more sensitive to
prolactin, it has been postulated that melengestrol acetate causes
tumours indirectly in C3Han/f mice, by increasing the release of pro-
lactin. The NOEL for induction of mammary tumours was 1 mg/kg of
body weight per day.
In a study to investigate the relationship between long-term adminis-
tration of melengestrol acetate, serum prolactin concentration and
mammary duct proliferation, female C3Han/f mice aged 44 days were
fed melengestrol acetate in the diet at concentrations providing doses
equivalent to 0, 0.5,1.5, 2.5, 5,10 or 25 mg/kg of body weight per day
for 1 year. Additional groups were also given a daily subcutaneous
injection of the prolactin inhibitor 6-methyl-8b-ergoline-acetonitrile,
but the dose of this compound was found to be too low and these
groups were not evaluated further. Body weights were increased in
animals in the highest-dose group. The serum prolactin concentra-
tion, which was determined only at termination of the study, was
increased in all the treatment groups; the increase was significant in
those dosed at 10 mg/kg of body weight per day and above. An
increasing trend in the incidence of animals with exacerbated mam-
mary duct proliferation was observed at doses of 2.5 mg/kg of body
weight per day and above; the incidence was significantly increased at
doses of 5 mg/kg of body weight per day and above. The NOEL for
hormonal effects was close to 0.5 mg/kg of body weight per day.
In a follow-up study, female C3Han/f mice of 44 days of age were fed
diets containing melengestrol acetate at concentrations providing
doses equivalent to 0,0.5,1.5,2.5,5,10 or 25 mg/kg of body weight per
day for a maximum of about 29 months. Additional groups of animals
receiving 0, 5,10 or 25mg/kg of body weight per day were also given
a daily subcutaneous injection of 100 mg of the prolactin inhibitor 6-
methyl-Sp-ergoline-acetonitrile. Mice in all the treatment groups
showed more rapid weight gain than controls during the first year but
decreased body-weight gain during the second year. 6-Methyl-8b-
ergoline-acetonitrile did not significantly affect the melengestrol
acetate-induced changes in body weight. The survival rate decreased
with increasing dose of melengestrol acetate, attaining significance at
5 mg/kg of body weight per day and above. Mice in which prolactin
was inhibited survived significantly longer than those in matched

71
 groups without prolactin inhibition. The only treatment-related non-
neoplastic lesions that were observed consisted of increased inci-
dences of endometrial hyperplasia, uterine adenomyosis and acute
metritis (at the highest dose), and decreased numbers of cystic ovaries
and cystic endometrial glands. 6-Methyl-8p-ergoline-acetonitrile did
not prevent these effects. In the mammary glands of treated mice,
adenocarcinomas and occasional benign adenomas were identified; a
dose-related increase in the incidence of mammary tumours was ob-
served, and the incidence of adenocarcinomas in animals dosed at
1.5mg/kg of body weight per day and above was significantly higher
than in controls. 6-Methyl-8b-ergoline-acetonitrile partially inhibited
mammary tumour development in both control and melengestrol-
treated groups. Examination by electron microscopy of the mammary
tumours from selected animals at each dose and from controls re-
vealed viral particles commonly associated with the murine mammary
tumour virus. Melengestrol acetate decreased the incidence of ovarian
tubular adenomas in animals dosed at 5mg/kg of body weight per
day and above. The incidence of hepatocellular adenomas was
significantly increased in animals dosed at 5mg/kg of body weight
per day and above, including those treated with 6-methyl-8b-
ergoline-acetonitrile, but the dose-response relationship was not con-
sistent up to this dose. There was no treatment-related effect on the
incidence of hepatocellular hyperplastic nodules or hepatocellular
carcinoma. The Committee concluded that melengestrol acetate
indirectly modulates mammary tumorigenesis in female C3Han/f
mice, possibly by stimulating the secretion of prolactin. The NOEL for
mammary tumorigenesis was 0.5mg/kg of body weight per day.
A NOEL could not be identified for the hormonal effects of
melengestrol acetate on the ovaries and uterus. The minimally effec-
tive dose for increasing the incidence of hepatocellular adenomas
was 5 mg/kg of body weight per day.

Melengestrol acetate was administered orally to male and female

beagle dogs at a dose of 0, 1 or 2mg/kg of body weight per day for
2 years, or at 8mg/kg of body weight per day for 1 year followed
by 4 jig/kg of body weight per day for another year. Females in
the highest-dose groups showed clinical signs of the progestational
activity of melengestrol acetate, such as pyometra and dystocia,
during the second year. These animals also showed increased serum
alkaline phosphatase activity and, after 18 months, an increased
total leukocyte count and reduced erythrocyte count, haemoglobin
concentration and erythrocyte volume fraction. Most of these changes
occurred in females with abnormalities of the reproductive tract.
Females in the highest-dose groups had alterations of the endome-

72
trium characteristic of progestational activity. No neoplastic changes
were seen in the mammary gland at any dose. The Committee con-
cluded that progestational effects were the most sensitive end-
point. The NOEL for hormonal effects was 1 (mg/kg of body weight
per day.
Melengestrol acetate has been tested for genotoxicity in a range
of assays in vitro and in vivo. Gene mutations were not induced in
Salmonella typhimurlum or mammalian cells. Unscheduled DNA
synthesis was not observed in rat primary hepatocytes or in the alka-
line elution assay in Chinese hamster V79 cells. Melengestrol acetate
did not induce micronuclei in the bone marrow of mice exposed in
vivo by intraperitoneal injection. The Committee concluded that
melengestrol acetate is not genotoxic.
In a one-generation study of reproductive toxicity in rats, melen-
gestrol acetate was administered in the diet at concentrations equiva-
lent to 0, 0.03, 0.06, 0.13, 0.25 or 1 mg/kg of body weight per
day. Melengestrol acetate suppressed the estrus cycle at doses of
0.13mg/kg of body weight per day and above and had significant
effects on fertility and pregnancy at doses of 0.06mg/kg of body
weight per day and above: at 0.06mg/kg of body weight per day, only
one of seven dams became pregnant, whereas at 0.03mg/kg of body
weight per day all dams became pregnant. While the incidence of
resorption in animals treated at 0.03mg/kg of body weight per day and
above was twice that in controls, there was no difference in litter size.
The body weights of pups of treated dams during lactation were not
statistically significantly different from those in the control group,
although their birth weights were higher. Dams dosed at 0.06mg/kg of
body weight per day and above showed a significant increase in the
serum prolactin concentration and significant decreases in the serum
progesterone concentration and in the weights of the adrenals, ovaries
and uterus. The histological appearance of the ovaries and uterus was
consistent with progestational activity. The NOEL for reproductive
toxicity was 0.03mg/kg of body weight per day.
The effect of melengestrol acetate on reproductive performance in
beagle dogs was evaluated in the 2-year study described above in
which melengestrol acetate was administered orally at a dose of 0,1 or
2ug/kg of body weight per day, or at 8mg/kg of body weight per day
for 1 year followed by 4 mg/kg of body weight per day for another year.
Animals treated at the same dose were bred, and females in the
lowest-dose group were bred with males in the highest-dose group.
Treatment of females was begun 120 days after estrus. Melengestrol
acetate at 4 or 8 mg/kg of body weight per day suppressed estrus, but

73
 estrus resumed within 12-201 days of cessation of treatment. Fewer
females treated at the highest dose became pregnant, and parturition
was impaired in animals at this dose during the second year, resulting
in significantly greater pup loss. The percentage of male pups and
the mean birth weight were slightly decreased at the highest dose.
Melengestrol acetate did not appear to affect the fertility of male
dogs. The NOEL for reproductive performance was 2mg/kg of body
weight per day.
Cows and heifers that were Fl or F2 progeny of melengestrol acetate-
treated heifers received the drug in their diet at a concentration
equal to 2 mg/kg of body weight per day for up to about 2 years,
except during the breeding period. Melengestrol acetate completely
suppressed estrus. The conception and pregnancy rates were not dif-
ferent from those of controls, except for a temporarily reduced con-
ception rate at estrus after the last feeding of melengestrol acetate.
The calves weighed less than those of controls at birth but not at
weaning. At necropsy, the only treatment-related change was reduced
adrenal weight. When bull calves received the same treatment for
about 2 years, no adverse effect was seen on fertility, and the only
effect was a reduction in adrenal weight.
In a study of developmental toxicity, pregnant rats received a single
subcutaneous dose of 0, 15, 25, 50 or l00mg/kg of body weight of a
sustained-release formulation of melengestrol acetate on day 6 of
gestation and were killed on day 20. Reduced litter weights and
average pup weights, increased numbers of sites of fetal resorption, a
larger percentage of pups with retarded development, and skeletal
and visceral abnormalities were observed at doses of 25 mg/kg of body
weight and above. The Committee concluded that melengestrol ac-
etate was embryotoxic and teratogenic at these doses. A NOEL could
not be identified because no information was available on the
toxicokinetics of the sustained-release formulation that would allow
estimation of the systemic exposure of the dams.
In another study of developmental toxicity, melengestrol acetate was
administered orally to pregnant rabbits at concentrations equivalent
to 0,0.016,0.064,0.16,0.4,0.8,1.6,3.2 or 6.4mg/kg of body weight per
day on days 6-18 of gestation. The fetuses were removed surgically on
day 28. The body weights of the does treated with doses of up to
0.16 mg/kg of body weight per day were increased, and those of animals
given higher doses were slightly decreased. Melengestrol acetate was
embryotoxic and fetotoxic at doses of 0.8 mg/kg of body weight per day
and above, as indicated by a large increase in the numbers of sites of
fetal resorption and dead fetuses. The percentage of embryonic deaths

74
approached 100% in the does treated at 3.2mg/kg of body weight per
day. The litter size was reduced at 1.6mg/kg of body weight per day.
The number of live fetuses and the mean litter and fetal weights were
significantly lower at doses of 0.8mg/kg of body weight per day and
above. Other effects observed at 0.8 and 1.6mg/kg of body weight
per day included cleft palate, talipes, umbilical hernia and incomplete
skeletal ossification. At 1.6mg/kg of body weight per day, the
male: female ratio was reduced to 0.36. The Committee concluded that
the fetotoxic and teratogenic effects of melengestrol acetate in rabbits
are due to its corticosteroid activity. The NOEL for embryotoxicity
and teratogenicity was 0.4mg/kg of body weight per day.
In a second study of developmental toxicity in rabbits, females re-
ceived a subcutaneous injection of a sustained-release formulation of
melengestrol acetate at 0, 5 or 15mg/kg of body weight on day 6 after
artificial insemination. The fetuses were delivered surgically on day 28
of gestation. At the highest dose, only one live but undersized fetus
was found in the 12 litters examined. At 5mg/kg of body weight, five
live but undersized fetuses were found in the 14 litters examined.
Nearly all live and dead fetuses of treated does had cleft palate. The
other abnormalities observed were exencephaly, agenesis of the lens,
irregular brain conformation, umbilical hernia, ablepharia, and en-
larged liver. The Committee concluded that melengestrol acetate was
teratogenic and embryotoxic at 15mg/kg of body weight and fetotoxic
at doses of 5mg/kg of body weight and above. The study was not
appropriate for identifying a NOEL because no information was
available on the toxicokinetics of the sustained-release formulation
that would allow estimation of the systemic exposure of the dams.
Observations in regularly ovulating women (number not stated) indi-
cated that melengestrol acetate delayed the onset of menses at daily
doses of 7.5 and 10 mg but not 5mg. In three volunteers, daily doses of
2.5 mg of melengestrol acetate and 0.05 mg of ethinylestradiol sup-
pressed endometrial proliferation. Single doses of 5, 7.5 or l0mg of
melengestrol acetate or repeated daily doses of 2.5mg induced with-
drawal bleeding in 11 estrogen-primed amenorrhoeic women.
The Committee concluded that the most appropriate end-point for
evaluating the safety of residues of melengestrol acetate is the pro-
gestational effect in non-human primates. An ADI of 0-0.03mug/kg
of body weight was established by applying a safety factor of 200 to
the minimally effective dose of 5 jig/kg of body weight per day of
melengestrol acetate for effects on the menstrual cycle of cynomolgus
monkeys in a study over three menstrual cycles. This safety factor was
used because the ADI is not based on a clear NOEL.

75
 Pharmacokinetic and residue data
Studies with [3H]melengestrol acetate labelled in the C6 position on
the methyl group and with [14C]melengestrol acetate labelled in the
C6 position on the methyl group and in the C16 position on the
methylene group were conducted in young heifers. In one study, four
animals received ground feed supplemented with 0.5 mg of unlabelled
melengestrol acetate daily for 4 months, followed by either daily oral
doses (in capsules) of approximately 0.5 mg of [3H]melengestrol
acetate for 21 days (three animals) or daily oral doses (in capsules)
of approximately 0.5 mg of [14C]melengestrol acetate for 7 days (one
animal). The animals were killed 6h after administration of the last
capsule, and samples of their body fluids and tissues were collected
and analysed for total residues. The radiolabel was eliminated in the
faeces and urine in a 6:1 ratio. Independent studies of animals with
bile cannulae showed that biliary excretion closely parallelled total
faecal output. In the study with four animals, the highest concen-
tration of total residues was found in liver; however, the highest
percentage of parent drug was found in fat, with similar concentra-
tions in visceral, omental and perirenal fat. The results obtained for
fat and liver were similar in all four treated animals and the concen-
trations of melengestrol acetate residues found in muscle were all
close to or below the limit of quantification of the analytical method
used (Table 4). Individual metabolites were not identified because
they occurred at such low concentrations.
In another study, in which 4.0mg of [3H]melengestrol acetate was
administered orally daily for 15 days to three Holstein heifers to
achieve a steady-state concentration of the drug, 83% (standard de-
viation, 13%) of the daily dose was recovered in urine and faeces on
the same day. The animals were killed 1, 4 and 10 days after the last
dose, and the total residues in selected edible tissues were deter-
mined. The results confirmed that the concentrations of total residues
in perirenal, visceral and omental fat were similar and decreased at
similar rates. Although the dose was eight times the recommended
dose, no residues were found in muscle.
In order to estimate the concentrations of residues of melengestrol
acetate in edible tissues, one heifer was treated with l000mg orally
(corresponding to 2000 times the highest recommended dose) for 5
days, and then received 500mg by subcutaneous injection and another
500 mg intramuscularly on the fifth day. The animal was killed on
the sixth day, and samples of the edible tissues were collected and
analysed for residues of the parent drug. The residue concentrations
were highest in fat (3300mg/kg), followed by liver (880mg/kg), muscle
(220mg/kg) and kidney (120mg/kg). These results are consistent with

76
Table 4
Residue concentrations of melengestrol acetate in tissues of four heifers given
unlabelled melengestrol acetate in their feed for 4 months, followed by either
[3H]melengestrol acetate orally for 21 days or [14C]melengestrol acetate orally for
7 days3
Tissue Concentration of total Percentage of total residues
residues (jig/kg) accounted for by parent drug
Muscle 0.6 31b
1.0 72b
0.5 40b
<LOQ 45C
Liver 12 30b
15 30b
9.0 28b
8.2 37C
Kidney 1.7 24b
1.8 34b
1.2 130b
1.6 30C
Fat (perirenal) 7.5 78b
7.7 86b
8.0 94b
3.6 75°

LOQ: limit of quantification (0.5mg/kg).

a
Animals received unlabelled melengestrol acetate in their feed at 0.5mg daily for 4 months,
followed by either [3H]melengestrol acetate orally at 0.5mg/day for 21 days or
[14C]melengestrol acetate orally at 0.5mg/day for 7 days.
b 3
[ H]Melengestrol acetate.
c
[ C]Melengestrol acetate.

the lack of detectable residues in muscle reported in the studies in

which the animals were treated at the recommended doses.
The Committee reviewed a large number of residue-depletion studies
of melengestrol acetate. In one pivotal study, 174 samples of perirenal
fat were obtained from heifers in 25 feedlots which had been given a
conventional solid supplement containing melengestrol acetate at the
highest recommended dose of 0.5 mg/animal per day. Another 84 fat
samples were obtained from heifers in 12 other feedlots which had
received a dose of 0.5 mg/animal per day of a liquid formulation
delivered into the complete feed. Of the 37 groups of cattle, 27 were
slaughtered less than 10 h after the last treatment, eight were slaugh-
tered between 11 and 16 h after the last treatment, and two were
slaughtered 18 and 27.5 h after the last treatment. The concentrations
of residues of melengestrol acetate in samples of fat from animals
treated with the liquid formulation were significantly lower than
those in samples from animals given the solid formulation. The 99th

77
 percentile concentration of residues of melengestrol acetate in fat
samples from all animals was about 18mg/kg, and the median concen-
tration was about 6 mg/kg.
A relationship was established between the administered dose and
the concentrations of residues of melengestrol acetate found during
treatment on the basis of the results of several similar studies con-
ducted with doses ranging from 0.3 to l0mg/animal per day. This
relationship was used to estimate that the 99th percentile concen-
tration of residues of parent melengestrol acetate in fat would be
about 10mg/kg when the approved dose of 0.25 mg/animal per day was
administered.

Analytical methods
The available analytical methods were developed before the intro-
duction of GLP. Validation of the method used in the studies summa-
rized by the Committee was based mainly on the analytical recovery
of melengestrol acetate. The procedure involves several solvent
partition and chromatographic separation and purification steps,
followed by gas-liquid chromatography (GLC) and detection by
electron capture. In a collaborative study in seven laboratories in
1975, the method was tested on samples of muscle, liver, kidney and
fat of cattle. Its accuracy and recovery were considered to be satisfac-
tory. The authors concluded that the method could be used to distin-
guish samples containing residues at a concentration of l0mg/kg
from samples that did not contain residues of melengestrol acetate.
However, it was not suitable for differentiating samples containing
residues at concentrations of 10mg/kg and 20mg/kg.
This collaborative study resulted in Official Method 976.36 of the
Association of Official Analytical Chemists (AOAC International),
which is applicable for the analysis of residues of melengestrol acetate
in animal tissues (muscle, liver, kidney and fat) at concentrations
down to l0mg/kg when a calibration curve that includes concentra-
tions of 10 and 30mg/kg is used. However, the method requires large
volumes of organic solvents, including benzene, use of which is pro-
hibited in many parts of the world because of its carcinogenicity.
A GLC-MS procedure can be used to confirm the results for fat
samples containing melengestrol acetate at about l0mg/kg. The char-
acteristic mass ions of melengestrol acetate are m/z 311, 321,336, 337
and 354 atomic mass units.

Choice of marker residue and target tissue

If the usual food factors are applied, liver would be the main dietary
source of residues of melengestrol acetate. However, liver and fat are

78
equally important sources of residues. The contributions of residues
in muscle and kidney are minor. Of the four standard edible tissues,
fat contains the highest concentrations of melengestrol acetate. In
studies in which radiolabelled melengestrol acetate was administered
to cattle, the parent drug accounted for 33% (range, 28-37%) of the
total residues in liver and 85% (range, 75-94%) of those in fat. There-
fore, melengestrol acetate and fat are appropriate as the marker residue
and target tissue, respectively.

Maximum Residue Limits

In reaching its decision on MRLs for melengestrol acetate, the Com-
mittee took the following factors into account:
• An ADI of 0-0.03 mg/kg of body weight was established, which is
equivalent to a maximum ADI of 1.8mgfor a 60-kg person.
• The available analytical methods date from 1968-76 and do
not meet current standards for regulatory use. The Committee
noted that analytical instrumentation and data processing for
use in residue analysis have improved dramatically since that
time.
• The sponsors did not submit an analytical method suitable for
monitoring purposes. A variety of potentially suitable, modern
methods for regulatory purposes, which have been validated in a
single laboratory and are cost-effective and efficient, are described
in the literature.
• Only fat and liver contain concentrations of the marker residue
that are quantifiable on a routine basis; methods with limits of
quantification greater than 0.3 mg/kg are unlikely to permit quan-
tification of residues in muscle from animals treated with recom-
mended doses of melengestrol acetate.
• The steady-state concentrations of melengestrol acetate residues in
muscle and kidney are about 0.7 and 1.6mg/kg respectively, with the
parent drug accounting for approximately 45% of the total residues
in both tissues.
• As inadequate information was available on the structure and ac-
tivity of metabolites of melengestrol acetate, they were assumed to
be as progestogenic as the parent drug and were taken into account
by the Committee in recommending MRLs.
The Committee recommended temporary MRLs for melengestrol
acetate of 2mg/kg for liver and 5mg/kg for fat in cattle, expressed as
parent drug. MRLs were not recommended for muscle and kidney as
the concentrations of residues in these tissues are generally low and,
in the case of muscle, often at or below the limit of quantification of
the analytical method. The MRLs were made temporary, pending the

79
Table 5
Theoretical maximum daily intake of melengestrol acetate residues
Tissue Recommended Estimate of total Theoretical maximum daily intakeb
MRL (mg/kg)a residues (mg/kg) (mg melengestrol acetate equivalents)
Muscle —° —
Liver 2 6d 0.6
Kidney —° —
Fat 5 6e 0.3
Total 0.9
a
Expressed as parent drug.
b
Based on a daily intake of 300g of muscle, 100g of liver, and 50g each of kidney and fat.
0
MRLs were not recommended for muscle and kidney.
d
Melengestrol acetate accounted for 33% of the total residues in liver.
8
Melengestrol acetate accounted for 85% of the total residues in fat.

receipt of information on an analytical method suitable for quanti-

fying residues of melengestrol acetate in liver and fat tissue. This
information is required for evaluation in 2002.

From these MRLs, the theoretical maximum daily intake of residues

would be 0.9 mg (see Table 5).

4 Future work
The Committee noted several issues that might be addressed in rela-
tion to its risk assessment procedures on the basis of a preliminary
review of the document on Procedures for recommending Maximum
Residue Limits — residues of veterinary drugs in food (1987-1999) (7).
These issues should be addressed during the process of updating
the principles of assessment, as recommended at the Conference on
International Food Trade Beyond the Year 2000 (!}.

5 Recommendations
1. Recommendations relating to specific veterinary drugs, including
ADIs and MRLs, are given in section 3 and Annex 2.
2. In view of the continuing need for evaluations of veterinary drugs,
meetings of the Joint FAO/WHO Expert Committee on Food
Additives should be held regularly for this purpose.

80
Acknowledgement
The Committee wishes to thank Mrs E. Heseltine, Communication in Science,
Lajarthe, Saint-Leon-sur-Vezere, France, for her assistance in the preparation of
the report.

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Working Party of Experts on Pesticide Residues and the WHO Expert
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of the United Nations, 1972 (FAO Agricultural Studies, No. 88); Geneva,
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Working Party of Experts on Pesticide Residues and the WHO Expert
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82
 Annex 1
Reports and other documents resulting from
previous meetings of the Joint FAO/WHO Expert
Committee on Food Additives
1. General principles governing the use of food additives (First report of the Joint
FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings
Report Series, No. 15,1957; WHO Technical Report Series, No. 129,1957 (out
of print).
2. Procedures for the testing of intentional food additives to establish their safety for
use (Second report of the Joint FAO/WHO Expert Committee on Food Addi-
tives). FAO Nutrition Meetings Report Series, No. 17, 1958; WHO Technical
Report Series, No. 144,1958 (out of print).
3. Specifications for identity and purity of food additives (antimicrobial preserva-
tives and antioxidants) (Third report of the Joint FAO/WHO Expert Committee
on Food Additives). These specifications were subsequently revised and pub-
lished as Specifications for identity and purity of food additives, vol. I. Antimicro-
bial preservatives and antioxidants. Rome, Food and Agriculture Organization
of the United Nations, 1962 (out of print).
4. Specifications for identity and purity of food additives (food colours) (Fourth
report of the Joint FAO/WHO Expert Committee on Food Additives). These
specifications were subsequently revised and published as Specifications for
identity and purity of food additives, vol. II. Food colours. Rome, Food and
Agriculture Organization of the United Nations, 1963 (out of print).
5. Evaluation of the carcinogenic hazards of food additives (Fifth report of the
Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meet-
ings Report Series, No. 29,1961; WHO Technical Report Series, No. 220,1961
(out of print).
6. Evaluation of the toxicity of a number of antimicrobials and antioxidants (Sixth
report of the Joint FAO/WHO Expert Committee on Food Additives). FAO
Nutrition Meetings Report Series, No. 31,1962; WHO Technical Report Series,
No. 228,1962 (out of print).
7. Specifications for the identity and purity of food additives and their toxicological
evaluation: emulsifiers, stabilizers, bleaching and maturing agents (Seventh
report of the Joint FAO/WHO Expert Committee on Food Additives). FAO
Nutrition Meetings Report Series, No. 35,1964; WHO Technical Report Series,
No. 281,1964 (out of print).
8. Specifications for the identity and purity of food additives and their toxicological
evaluation: food colours and some antimicrobials and antioxidants (Eighth
report of the Joint FAO/WHO Expert Committee on Food Additives). FAO
Nutrition Meetings Report Series, No. 38,1965; WHO Technical Report Series,
No. 309, 1965 (out of print).
9. Specifications for identity and purity and toxicological evaluation of some antimi-
crobials and antioxidants. FAO Nutrition Meetings Report Series, No. 38A,
1965; WHO/Food Add/24.65 (out of print).
10. Specifications for identity and purity and toxicological evaluation of food colours.
FAO Nutrition Meetings Report Series, No. 38B, 1966; WHO/Food Add/66.25
(out of print).
11. Specifications for the identity and purity of food additives and their toxicological
evaluation: some antimicrobials, antioxidants, emulsifiers, stabilizers, flour-
treatment agents, acids, and bases (Ninth report of the Joint FAO/WHO Expert
Committee on Food Additives). FAO Nutrition Meetings Report Series, No. 40,
1966; WHO Technical Report Series, No. 339,1966 (out of print).
12. Toxicological evaluation of some antimicrobials, antioxidants, emulsifiers,

S3
 stabilizer's, flour-treatment agents, acids, and bases. FAO Nutrition Meetings
Report Series, No. 40A, B, C, 1967; WHO/Food Add/67.29 (out of print).
13. Specifications for the identity and purity of food additives and their toxicological
evaluation: some emulsifiers and stabilizers and certain other substances (Tenth
report of the Joint FAO/WHO Expert Committee on Food Additives). FAO
Nutrition Meetings Report Series, No. 43,1967; WHO Technical Report Series,
No. 373, 1967 (out of print).
14. Specifications for the identity and purity of food additives and their toxicological
evaluation: some flavouring substances and non-nutritive sweetening agents
(Eleventh report of the Joint FAO/WHO Expert Committee on Food Addi-
tives). FAO Nutrition Meetings Report Series, No. 44, 1968; WHO Technical
Report Series, No. 383,1968 (out of print).
15. Toxicological evaluation of some flavouring substances and non-nutritive sweet-
ening agents. FAO Nutrition Meetings Report Series, No. 44A, 1968; WHO/
Food Add/68.33 (out of print).
16. Specifications and criteria for identity and purity of some flavouring substances
and non-nutritive sweetening agents. FAO Nutrition Meetings Report Series,
No. 44B, 1969; WHO/Food Add/69.31 (out of print).
17. Specifications for the identity and purity of food additives and their toxicological
evaluation: some antibiotics (Twelfth report of the Joint FAO/WHO Expert
Committee on Food Additives). FAO Nutrition Meetings Report Series, No. 45,
1969; WHO Technical Report Series, No. 430, 1969 (out of print).
18. Specifications for the identity and purity of some antibiotics. FAO Nutrition
Meetings Report Series, No. 45A, 1969; WHO/Food Add/69.34 (out of print).
19. Specifications for the identity and purity of food additives and their toxicological
evaluation: some food colours, emulsifiers, stabilizers, anticaking agents, and
certain other substances (Thirteenth report of the Joint FAO/WHO Expert
Committee on Food Additives). FAO Nutrition Meetings Report Series, No. 46,
1970; WHO Technical Report Series, No. 445, 1970 (out of print).
20. Toxicological evaluation of some food colours, emulsifiers, stabilizers, anticaking
agents, and certain other substances. FAO Nutrition Meetings Report Series, No.
46A, 1970; WHO/Food Add/70.36 (out of print).
21. Specifications for the identity and purity of some food colours, emulsifiers, stabi-
lizers, anticaking agents, and certain other food additives. FAO Nutrition Meet-
ings Report Series, No. 46B, 1970; WHO/Food Add/70.37 (out of print).
22. Evaluation of food additives: specifications for the identity and purity of food
additives and their toxicological evaluation: some extraction solvents and certain
other substances; and a review of the technological efficacy of some antimicrobial
agents (Fourteenth report of the Joint FAO/WHO Expert Committee on Food
Additives). FAO Nutrition Meetings Report Series, No. 48,1971; WHO Tech-
nical Report Series, No. 462, 1971 (out of print).
23. Toxicological evaluation of some extraction solvents and certain other substances.
FAO Nutrition Meetings Report Series, No. 48A, 1971; WHO/Food Add/70.39
(out of print).
24. Specifications for the identity and purity of some extraction solvents and certain
other substances. FAO Nutrition Meetings Report Series, No. 48B, 1971; WHO/
Food Add/70.40 (out of print).
25. A review of the technological efficacy of some antimicrobial agents. FAO Nutri-
tion Meetings Report Series, No. 48C, 1971; WHO/Food Add/70.41 (out of print).
26. Evaluation of food additives: some enzymes, modified starches, and certain other
substances: toxicological evaluations and specifications and a review of the tech-
nological efficacy of some antioxidants (Fifteenth report of the Joint FAO/WHO
Expert Committee on Food Additives). FAO Nutrition Meetings Report Series,
No. 50, 1972; WHO Technical Report Series, No. 488, 1972.
27. Toxicological evaluation of some enzymes, modified starches, and certain other
substances. FAO Nutrition Meetings Report Series, No. 50A, 1972; WHO Food
Additives Series, No. 1, 1972.

84
28. Specifications for the identity and purity of some enzymes and certain other
substances. FAO Nutrition Meetings Report Series, No. 50B, 1972; WHO Food
Additives Series, No. 2,1972 (out of print).
29. A review of the technological efficacy of some antioxidants and synergists. FAO
Nutrition Meetings Report Series, No. 50C, 1972; WHO Food Additives Series,
No. 3, 1972 (out of print).
30. Evaluation of certain food additives and the contaminants mercury, lead, and
cadmium (Sixteenth report of the Joint FAO/WHO Expert Committee on
Food Additives). FAO Nutrition Meetings Report Series, No. 51, 1972; WHO
Technical Report Series, No. 505, 1972, and corrigendum (out of print).
31. Evaluation of mercury, lead, cadmium, and the food additives amaranth,
diethylpyrocarbonate, and octyl gallate. FAO Nutrition Meetings Report Series,
No. 51 A, 1972; WHO Food Additives Series, No. 4, 1972.
32. Toxicological evaluation of certain food additives with a review of general prin-
ciples and of specifications (Seventeenth report of the Joint FAO/WHO Expert
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1974; WHO Technical Report Series, No. 539, 1974, and corrigendum (out of
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33. Toxicological evaluation of certain food additives including anticaking agents,
antimicrobials, antioxidants, emulsifiers, and thickening agents. FAO Nutrition
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34. Specifications for identity and purity of thickening agents, anticaking agents,
antimicrobials, antioxidants and emulsifiers. FAO Food and Nutrition Paper,
No. 4, 1978.
35. Evaluation of certain food additives (Eighteenth report of the Joint FAO/WHO
Expert Committee on Food Additives). FAO Nutrition Meetings Report Series,
No. 54, 1974; WHO Technical Report Series, No. 557, 1974, and corrigendum
(out of print).
36. Toxicological evaluation of some food colours, enzymes, flavour enhancers,
thickening agents, and certain other food additives. FAO Nutrition Meetings
Report Series, No. 54A, 1975; WHO Food Additives Series, No. 6, 1975.
37. Specifications for the identity and purity of some food colours, flavour enhancers,
thickening agents, and certain food additives. FAO Nutrition Meetings Report
Series, No. 54B, 1975; WHO Food Additives Series, No. 7, 1975.
38. Evaluation of certain food additives: some food colours, thickening agents,
smoke condensates, and certain other substances (Nineteenth report of the Joint
FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings
Report Series, No. 55,1975; WHO Technical Report Series, No. 576,1975 (out
of print).
39. Toxicological evaluation of some food colours, thickening agents, and certain
other substances. FAO Nutrition Meetings Report Series, No. 55A, 1975; WHO
Food Additives Series, No. 8, 1975.
40. Specifications for the identity and purity of certain food additives. FAO Nutrition
Meetings Report Series, No. 55B, 1976; WHO Food Additives Series, No. 9,
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41. Evaluation of certain food additives (Twentieth report of the Joint FAO/WHO
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42. Toxicological evaluation of certain food additives. WHO Food Additives Series,
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43. Specifications for the identity and purity of some food additives. FAO Food
and Nutrition Series, No. IB, 1977; WHO Food Additives Series, No. 11,
1977.
44. Evaluation of certain food additives (Twenty-first report of the Joint FAO/
WHO Expert Committee on Food Additives). WHO Technical Report Series,
No. 617, 1978.

85
 45. Summary of toxicological data of certain food additives. WHO Food Additives
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46. Specifications for identity and purity of some food additives, including antioxi-
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47. Evaluation of certain food additives and contaminants (Twenty-second report of
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50. Evaluation of certain food additives (Twenty-third report of the Joint FAO/
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125. Evaluation of certain veterinary drug residues in food (Forty-seventh report of
the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical
Report Series, No. 876, 1998.
126. Toxicological evaluation of certain veterinary drug residues in food. WHO Food
Additives Series, No. 38, 1996.
127. Residues of some veterinary drugs in animals and foods. FAO Food and Nutri-
tion Paper, No. 41/9,1997.
128. Evaluation of certain veterinary drug residues in food (Forty-eighth report of the
Joint FAO/WHO Expert Committee on Food Additives). WHO Technical
Report Series, No. 879, 1998.
129. Toxicological evaluation of certain veterinary drug residues in food. WHO Food
Additives Series, No. 39, 1997.
130. Residues of some veterinary drugs in animals and foods. FAO Food and Nutri-
tion Paper, No. 41/10,1998.
131. Evaluation of certain food additives and contaminants (Forty-ninth report of the
Joint FAO/WHO Expert Committee on Food Additives). WHO Technical
Report Series, No. 884,1999.
132. Safety evaluation of certain food additives and contaminants. WHO Food Addi-
tives Series, No. 40, 1998.
133. Compendium of food additive specifications, addendum 5. FAO Food and Nutri-
tion Paper, No. 52, Add. 5, 1997.
134. Evaluation of certain veterinary drug residues in food (Fiftieth report of the Joint
FAO/WHO Expert Committee on Food Additives). WHO Technical Report
Series, No. 888, 1999, and corrigendum.
135. Toxicological evaluation of certain veterinary drug residues in food. WHO Food
Additives Series, No. 41, 1998.
136. Residues of some veterinary drugs in animals and foods. FAO Food and Nutri-
tion Paper, No. 41/11, 1999.

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 137. Evaluation of certain food additives (Fifty-first report of the Joint FAO/WHO
Expert Committee on Food Additives). WHO Technical Report Series, No.
891, 2000.
138. Safety evaluation of certain food additives. WHO Food Additives Series, No. 42,
1999.
139. Compendium of food additive specifications, addendum 6. FAO Food and Nutri-
tion Paper, No. 52, Add. 6,1998.
140. Evaluation of certain veterinary drug residues in food (Fifty-second report of the
Joint FAO/WHO Expert Committee on Food Additives). WHO Technical
Report Series, No. 893, 2000.
141. Toxicological evaluation of certain veterinary drug residues in food. WHO Food
Additives Series, No. 43, 2000.
142. Residues of some veterinary drugs in animals and foods. FAO Food and Nutri-
tion Paper, No. 41/12, 2000.
143. Evaluation of certain food additives and contaminants (Fifty-third report of the
Joint FAO/WHO Expert Committee on Food Additives). WHO Technical
Report Series, No. 896, 2000.
144. Safety evaluation of certain food additives and contaminants. WHO Food Addi-
tives Series, No. 44, 2000.
145. Compendium of food additive specifications, addendum 7. FAO Food and Nutri-
tion Paper, No. 52, Add. 7,1999.

90
 Annex 2
Recommendations on compounds on the agenda
and further information required

Anthelminthic agent
Ivermectin
ADI: 0-1 mg/kg of body weight (established at the for-
tieth meeting of the Committee (WHO Techni-
cal Report Series, No. 832,1993)).

Residue definition: 22,23-Dihydroavermectin Bla.

Species Recommended MRLs (mg/kg)

Muscle Liver Kidney Fat Milk
a a
Cattle — 100 — 40 10b
Other species — 15C — 20C —
a
These MRLs were recommended at the fortieth meeting of the Committee (WHO
Technical Report Series, No. 832, 1993).
b
Temporary MRL, pending the receipt of the full set of data for validation of the analytical
method and information on other routes of application of ivermectin to cattle. This
information is required for evaluation in 2002.
c
These MRLs were recommended at the thirty-sixth meeting of the Committee (WHO
Technical Report Series, No. 799, 1990).

Antimicrobial agents
Flumequine
ADI: 0-30mg/kg of body weight (established at the
forty-eighth meeting of the Committee (WHO
Technical Report Series, No. 879,1998)).

Residue definition: Flumequine.

Species Recommended MRLs ((mg/kg)

Muscle Liver Kidney Fat

Cattle 500 500 3000 1000

Pigs 500 500 3000 1000
Sheep 500 500 3000 1000
Chickens 500 500 3000 1000
Trout 500a — — —
a
Muscle/skin in normal proportions.

91
 Lincomycin
ADI: 0-30mg/kg of body weight.
Residue definition: Lincomycin.

Species Recommended MRLs (mg/kg)

Muscle Liver Kidney Fat Milk Eggs
a a a a
Cattle 100 500 1500 100 150 —
Pigs 100 500 1500 100 — —
Sheep 100a 500a 1500a 100a — —
Chickens 100a 500a 1500a 100a — —b
a
Temporary MRLs, pending the receipt of data from residue-depletion studies in cattle,
sheep and chickens, which show that lincomycin is the major microbiologically active
residue in the edible tissues. These data are required for evaluation in 2002.
b
Before recommending an MRL for chickens' eggs, the Committee would wish to see the
following:
— data from residue-depletion studies showing that lincomycin is the major
microbiologically active residue in eggs;
— the results of a residue-depletion study in which the GC-MS method is used to
analyse residues in eggs.
This information is required for evaluation in 2002.

Oxytetracycline
ADI: 0-30mg/kg of body weight (group ADI for tetra-
cycline, oxytetracycline and chlortetracycline;
established at the fiftieth meeting of the Com-
mittee (WHO Technical Report Series, No. 888,
1999)).
Residue definition: Oxytetracycline, alone or in combination with
chlortetracycline and tetracycline.

Species Recommended MRL (mg/kg)

Muscle Liver Kidney Fat Milk Eggs
a a a b a
Cattle 200 600 1200 — 100 —
Pigs 200a 600a 1200a —b — —
Sheep 200a 600a 1200a —b 100a —
Poultry 200a 600a 1200a —b — 400a
Fish 200C — — — — —
Giant tiger prawn 200ad — — — — —
(Penaeus monodon)
a
These MRLs were recommended at the fiftieth meeting of the Committee (WHO
Technical Report Series, No. 888, 1999).
b
At its forty-seventh meeting (WHO Technical Report Series, No, 876, 1998), the
Committee recommended that the MRL for oxytetracycline in fat be withdrawn, and
decided that the MRLs for chlortetracycline and tetracycline in fat were not required.
0
Temporary MRL, pending the results of a residue-depletion study and information on the
validated analytical method for fish. These data are required for evaluation in 2002.
d
Applies only to oxytetracycline.

92
 Tilmicosin
ADI: 0-40mg/kg of body weight (established at the
forty-seventh meeting of the Committee (WHO
Technical Report Series, No. 876, 1998)).
Residue definition: Tilmicosin.

Species Recomm ended MRLs (mc3/kg)

Muscle Liver Kidney Fat Milk
a a a
Cattle 100 1000 a
300 100 —
Pigs 100a 1500a 1000a 100a
Sheep 100a 1000a 300a 100a b

a
These MRLs were recommended at the forty-seventh meeting of the Committee (WHO
Technical Report Series, No. 876, 1998).
b
The temporary MRL recommended at the forty-seventh meeting of the Committee (WHO
Technical Report Series, No. 876, 1998) was not extended, as the results of the
requested study with radiolabelled tilmicosin in lactating sheep, to determine the
relationship between total residues and the parent drug in milk, were not available.

Insecticides
Cyhalothrin
ADI: 0-2mg/kg of body weight (designated as tempo-
rary, pending the results of studies appropriate
for identifying a NOEL for neurobehavioural
effects in laboratory animals; these results are
required for evaluation in 2002).
Residue definition: Sum of the concentrations of the isomers of
cyhalothrin.

Species Recommiended MRLs (mC)/kg)a

Muscle Liver Kidney Fat Milk

Cattle 20 20 20 400 30
Pigs 20 20 20 400
Sheep 20 20b 20 400
Temporary MRLs.
Data on the validation of the analytical method for sheep liver, to confirm the limit of
quantification of 10mg/kg, are required for evaluation in 2002.

Cypermethrin
ADI: 0-50mg/kg of body weight (established at the
forty-seventh meeting of the Committee (WHO
Technical Report Series, No. 876, 1998)).
Residue definition: Sum of the concentrations of the isomers of
cypermethrin.

93
 Species Recommended MRLs (mg/kg)
Muscle Liver Kidney Fat Milk Eggs
a a a a a
Cattle — — — — — —
Sheep —a —a —a —a — —
Chickens —a —a —a —a — —a
a
The temporary MRLs recommended at the forty-seventh meeting of the Committee
(WHO Technical Report Series, No. 876, 1998) were not extended, as the requested
information was not provided and there was no indication that it would be provided in
the future.

a-Cypermethrin
ADI: 0-20mg/kg of body weight (established at the
forty-seventh meeting of the Committee (WHO
Technical Report Series, No. 876,1998)).
Residue definition: Sum of the concentrations of the isomers of a-
cypermethrin.

Species Recommended MRLs (mg/kg)

Muscle Liver Kidney Fat Milk Eggs
Cattle ~* ~* ~* ~* ~* ^~
Sheep —a —a —a —a — —
Chickens —a —a —a —a — —a
a
The temporary MRLs recommended at the forty-seventh meeting of the Committee
(WHO Technical Report Series, No. 876, 1998) were not extended, as the requested
information was not provided and there was no indication that it would be provided in
the future.

Dicyclanil
ADI: 0-7mg/kg of body weight.
Residue definition: Dicyclanil.

Species Recommended MRLs (mg/kg)

Muscle Liver Kidney Fat
Sheep 200 400 400 150

Permethrin
ADI: 0-50mg/kg of body weight (for technical-grade
permethrin containing the cis- and Jra/w-isomers
in ratios of between 25:75 and 40:60; estab-
lished by the 1999 Joint FAO/WHO Meeting on
Pesticide Residues (FAO Plant Production and
Protection Paper, No. 153, 2000; available on
the Internet at http://www.fao.org/ag/agp/agpp/

94
 pesticid/)). The Committee was unable to estab-
lish an ADI for the 80:20 cis: trans isomeric
mixture proposed for use as a veterinary drug
because of the lack of information on toxicity.
Residue definition: Sum of the concentrations of the isomers of
permethrin. The Committee was unable to rec-
ommend MRLs for the 80:20 cis: trans isomeric
mixture of permethrin in the absence of an
ADI.

Metrifonate (trichlorfon)
ADI: 0-20mg/kg of body weight.
Residue definition: Metrifonate.
Species Recommended MRLs (mg/kg)
Muscle Liver Kidney Fat Milk
a a a a
Cattle 50 50 50 50 50
a
The Committee noted that the concentrations of residues were very low in muscle, liver,
kidney and fat. These MRLs are for guidance only and are based on the limit of
quantification of the analytical method.

Production aid
Melengestrol acetate
ADI: 0-0.03 mg/kg of body weight.
Residue definition: Melengestrol acetate.
Species Recommended MRLs (mg/kg)
Muscle Liver Kidney Fat Milk
a b
Cattle ~ ~ 2 ~ * 5 * ~
a
MRLs were not recommended for muscle or kidney as the concentrations of residues
were below the limit of quantification of the analytical method.
b
Temporary MRL, pending the receipt of information on an analytical method suitable for
quantifying residues of melengestrol acetate in liver and fat tissue. This information is
required for evaluation in 2002.
0
An MRL was not recommended for milk as the drug is not used in dairy cows.

95
 Annex 3
Proposed draft definitions of commodities for
Volume 3 of Residues of veterinary drugs in foods1
Meat
Proposed draft definition
The skeletal muscular tissue of an animal carcass or cuts of such tissue
from an animal carcass. It includes interstitial and intramuscular fat. It
may also include bone, connective tissue and tendons, as well as
nerves and lymph nodes in natural proportions. It does not include
edible offal or trimmable fat.

Portion of the commodity to which the MRL applies

The whole commodity without bones.

Current definition
The edible part of any mammal.

Muscle and fat

Proposed draft definitions
Muscle
Muscle tissue only (definition established and adopted by the Joint
FAO/WHO Expert Committee on Food Additives).

Fat
The lipid-based tissue that is trimmable from an animal carcass or cuts
from an animal carcass. It may include subcutaneous, omental or
perirenal fat. It does not include interstitial or intramuscular carcass
fat or milk fat.

Portion of the commodity to which the MRLs apply

The whole commodity. For fat-soluble compounds, the MRL applies
to the trimmable fat. For those compounds where the trimmable fat is
insufficient to provide a suitable test sample, the whole commodity
(muscle and fat but without bone) is analysed and the MRL applies to
the whole commodity (e.g. rabbit meat).

Current definitions
None.
1
Codex Alimentarius Commission. Residues of veterinary drugs in foods, 2nd ed. Volume
3. Rome, Food and Agriculture Organization of the United Nations, 1996 (rev. 1995)
(available from FAO or WHO).

96
 SELECTED WHO PUBLICATIONS OF RELATEDINTEREST

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