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    INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY



    ENVIRONMENTAL HEALTH CRITERIA 104





    PRINCIPLES FOR THE TOXICOLOGICAL
    ASSESSMENT OF PESTICIDE RESIDUES IN FOOD









    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 United Nations Environment Programme, the International
    Labour Organisation, or the World Health Organization.

    Published under the joint sponsorship of
    the United Nations Environment Programme,
    the International Labour Organisation,
    and the World Health Organization

    World Health Organization
    Geneva, 1990


         The International Programme on Chemical Safety (IPCS) is a
    joint venture of the United Nations Environment Programme, the
    International Labour Organisation, and the World Health
    Organization. The main objective of the IPCS is to carry out and
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    risk-assessment methods that could produce internationally
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    toxicology. Other activities carried out by the IPCS include the
    development of know-how for coping with chemical accidents,
    coordination of laboratory testing and epidemiological studies, and
    promotion of research on the mechanisms of the biological action of
    chemicals.

    WHO Library Cataloguing in Publication Data

    Prinicples for the toxicological assessment of pesticide residues
    in food.

        (Environmental health criteria ; 104)

        1.Pesticide residues - analysis - toxicity  2. Food contamination 
        I.Series

        ISBN 92 4 157104 7        (NLM Classification: WA 240)
        ISSN 0250-863X

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CONTENTS

PRINCIPLES FOR THE TOXICOLOGICAL ASSESSMENT OF PESTICIDE RESIDUES IN FOOD

FOREWORD                

PREFACE                 

1. INTRODUCTION        

2. GENERAL HISTORICAL BACKGROUND   

3. JMPR ASSESSMENT PROCESS 

4. CONSIDERATIONS OF IDENTITY, PURITY, AND STABILITY   
    4.1. Background        
    4.2. Principles        
          4.2.1. Identity  
          4.2.2. Purity        
          4.2.3. Stability 

5. AVAILABILITY AND QUALITY OF DATA    
    5.1. Background        
    5.2. Principles        

6. HUMAN DATA          
    6.1. Background        
    6.2. Current position  
    6.3. Principles        

7. STRUCTURE-ACTIVITY RELATIONSHIPS    
    7.1. Principle     

8. TEST METHODOLOGIES      
    8.1. Background        
    8.2. General considerations    
          8.2.1. Choice of species and strain  
          8.2.2. Group size    
          8.2.3. Selection of dose levels  
          8.2.4. Test duration 
          8.2.5. Pathological procedures   
          8.2.6. Historical control data   
    8.3. Conduct and evaluation of studies 
          8.3.1. Short- and long-term toxicity studies 
          8.3.2. Carcinogenicity studies   
                  8.3.2.1   Background  
                  8.3.2.2   Routes of exposure  
                  8.3.2.3   Commonly occurring tumours  
                  8.3.2.4   Pathological classification of neoplasms    
                  8.3.2.5   Evaluation of carcinogenicity studies   
                  8.3.2.6   Extrapolation from animals to man   
                  8.3.2.7   Principles  
          8.3.3. Reproduction studies  
                  8.3.3.1   Multigeneration reproduction studies    
                  8.3.3.2   Teratology studies  
                  8.3.3.3   Screening studies in teratology 
                  8.3.3.4   Principles  

          8.3.4. Neurotoxicity studies 
                  8.3.4.1   Delayed neurotoxicity   
                  8.3.4.2   Acute neurotoxicity 
                  8.3.4.3   Chronic neurotoxicity   
                  8.3.4.4   Pyrethroid-induced neurotoxicity    
                  8.3.4.5   Neurobehavioural toxicity   
                  8.3.4.6   Principles  
          8.3.5. Genotoxicity studies  
                  8.3.5.1   Principles  
          8.3.6. Immunotoxicity studies    
                  8.3.6.1   Background  
                  8.3.6.2   Current position    
                  8.3.6.3   Principles  
          8.3.7. Absorption, distribution, metabolism, and excretion   
                  8.3.7.1   Background  
                  8.3.7.2   Current position    
                  8.3.7.3   Principles  

9. EVALUATION OF DATA      
    9.1. Extrapolation of animal data to humans    
    9.2. Safety factors    
          9.2.1. Background    
          9.2.2. Principles    
    9.3. Allocating the ADI    
          9.3.1. Background    
          9.3.2. Temporary ADIs    
          9.3.3. Present position  

10. EVALUATION OF MIXTURES  
    10.1. Introduction      
    10.2. Background        
    10.3. Principle     

11. RE-EVALUATION OF PESTICIDES 

12. BIOTECHNOLOGY   

13. SPECIAL CONSIDERATIONS FOR INDIVIDUAL CLASSES OF PESTICIDES     
    13.1. Organophosphates - ophthalmological effects   
    13.2. Organophosphates - aliesterase (carboxylesterase) inhibition
    13.3. The need for carcinogenicity testing of organophosphates
    13.4. Ocular toxicity of bipyridilium compounds 
    13.5. Goitrogenic carcinogens   

REFERENCES              

ANNEX I: GLOSSARY  

ANNEX II: APPROXIMATE RELATION OF PARTS PER MILLION IN THE DIET TO MG/KG 
BODY WEIGHT PER DAY 

INDEX                   

WHO TASK GROUP ON PRINCIPLES FOR THE TOXICOLOGICAL ASSESSMENT OF PESTICIDE
RESIDUES IN FOOD


Dr N. Aldridge, The Robens Institute of Industrial & Environmental
   Health & Safety, University of Surrey, Guildford, Surrey, United
   Kingdoma

Dr G. Becking, International Programme on Chemical Safety, World Health
   Organization, Research Triangle Park, North Carolina, USAa,e

Professor C.L. Berry, Department of Morbid Anatomy, The London Hospital
   Medical College, London, United Kingdoma,e

Dr A.L. Black, Department of Community Services and Health, Woden,
   Australiab,d,e

Professor J.F. Borzelleca, Department of Pharmacology and Toxicology,
   Medical College of Virginia, Virginia Commonwealth University,
   Richmond, USAd,e

Dr G. Burin, Health Effects Division, Office of Pesticide Programs, US
   Environmental Protection Agency, Washington, DC, USAb,c,e

Dr J.R.P. Cabral, Unit of Mechanisms of Carcinogenesis, International
   Agency for Research on Cancer, Lyon, Francea

Dr D.B. Clayson, Toxicology Research Division, Bureau of Chemical
   Safety, Food Directorate, Health and Welfare Canada, Ottawa,
   Ontario, Canadae

Mr D.J. Clegg, Agricultural Chemicals Section, Toxicological Evaluation
   Division, Food Directorate, Health Protection Branch, Ottawa,
   Canadaa,b,c,d,e

Professor B. Goldstein, Rutgers Medical College, Busch Campus,
   Pescataway, New Jersey, USAa

Dr J.L. Herrman, International Programme on Chemical Safety, World
   Health Organization, Geneva, Switzerlanda,b,c

Professor M. Ikeda, Department of Environmental Health, Tohoku 
   University School of Medicine, Sendai, Japana

Dr S.E. Jaggers, ICI Central Toxicology Laboratory, Cheshire, United
      Kingdome

Dr F.-W. Kopisch-Obuch, Plant Protection Service, Food and Agriculture
   Organization, Rome, Italye

Dr R. Kroes, National Institute of Public Health and Environmental
   Hygiene, Bilthoven, The Netherlandsa

Professor M. Lotti, Istituto di Medicina del Lavoro, UniversitÓ di
   Padova, Padova, Italya,b,d,e

Dr L. Magos, Toxicology Unit, Medical Research Council Laboratories,
   Woodmansterne Road, Carshalton, Surrey, United Kingdoma

Dr K. Miller, Immunotoxicology Department, British Industrial Biologi-
   cal Research Association, Surrey, United Kingdome

Professor R. Nilsson, The National Swedish Chemicals Inspectorate, De-
   partment for Scientific Documentation and Research, Solna, Swedene

Dr A.K. Palmer, Reproductive Studies, Huntingdon Research Centre Ltd.,
   Huntingdon, Cambridgeshire, United Kingdomb,e

Professor D.V. Parke, Department of Biochemistry, University of Surrey,
   Guildford, United Kingdoma,e

Dr O.E. Paynter, Health Effects Division, US Environmental Protection
   Agency, Washington, DC, USAa,b,c,e

Dr R. Plestina, Division of Vector Biology and Control, World Health
   Organization, Geneva, Switzerlandb

Dr F.R. Puga, Section of Toxicology, Instituto Biol˛gico, Sao Paulo,
   Brazild

Professor A. Rico, Ecole Nationale VÚtÚrinaire, Toulouse, Francee

Dr L. Shuker, Unit of Carcinogen Identification and Evaluation, Inter-
   national Agency for Research on Cancer, Lyon, Franceb,e

Dr J. Steadman, Department of Health & Social Security, Hannibal House,
   Elephant and Castle, London, United Kingdoma

Dr E.M. den Tonkelaar, Toxicology Advisory Center, National Institute
   of Public Health and Environmental Protection, Bilthoven, The
   Netherlandse

Dr G. Ungvary, Section of Toxicology, National Institute of Occu-
   pational Health, Budapest, Hungaryd,e

Dr G. Vettorazzi, International Toxicology Information Centre, San
   Sebastian, Spaina,b,c,e


-------------------------------
a   Present at strategy meeting, Carshalton, United Kingdom, 2-6 March
    1987
b   Present at consultation, Geneva, Switzerland, 14-16 September 1988
c   Member of editorial committee
d   Member of WHO Expert Group on Pesticide Residues (1989 JMPR)
e   Submitter of written comments

NOTE TO READERS OF THE CRITERIA DOCUMENTS

    Every effort has been made to present information in the criteria
documents as accurately as possible without unduly delaying their pub-
lication. In the interest of all users of the environmental health cri-
teria documents, readers are kindly requested to communicate any errors
that may have occurred to the Manager of the International Programme on
Chemical Safety, World Health Organization, Geneva, Switzerland, in
order that they may be included in corrigenda, which will appear in
subsequent volumes.


                            *     *     *

FOREWORD

    The  WHO activities concerned with  the safety assessment of  food
chemicals  were incorporated into the International Programme on Chemi-
cal  Safety (IPCS) in 1980.  These activities include administering the
WHO  Expert Group on Pesticide Residues, which meets regularly with the
FAO  Panel of Experts on Pesticide Residues in Food and the Environment
in the well-known Joint FAO/WHO Meeting on Pesticide Residues, or JMPR.
The  objectives of the  WHO Expert Group  are consistent with  those of
IPCS,  which  include  the  formulation  of  "guiding  principles  for
exposure  limits, such as acceptable  daily intakes for food  additives
and  pesticide residues, and tolerances  for toxic substances in  food,
air, water, soil, and the working environment." The inclusion  of  the
present  publication  as a  methodology  document in  the Environmental
Health Criteria series will make it readily available to all  of  those
who  have an  interest in  the toxicological  assessment  of  pesticide
residues in food.

    The IPCS gratefully acknowledges the financial and  other  support
of  the Canadian Health Protection Branch, the US Environmental Protec-
tion Agency, and the United Kingdom Department of Health.  This support
was indispensable for the completion of the project.


                                            Dr M. Mercier
                                            Manager
                                            International Programme on
                                              Chemical Safety

PREFACE

    Since  the early  1960s the  Joint FAO/WHO  Meeting  on  Pesticide
Residues,  usually known as the  JMPR, has evaluated a  large number of
pesticides.   The WHO component of these Joint Meetings, the WHO Expert
Group  on Pesticide Residues, has,  during that time, relied  upon pro-
cedures  developed by other  expert groups, such  as the Joint  FAO/WHO
Expert  Committee  on Food  Additives  (JECFA), and  developed specific
principles  for evaluating the various  classes of pesticides that  are
used  on food crops and may leave residues on them.  The publication of
WHO Environmental Health Criteria 70: Principles for the safety assess-
ment of food additives and contaminants in food, which  summarizes  the
assessment  procedures used by JECFA,  has been used by  the WHO Expert
Group  on Pesticide Residues  since its publication.   Other principles
specific  to pesticides, however, have  until now been scattered  among
the  various JMPR  reports, which  has made  it difficult  for the  WHO
Expert  Groups to use them  in a consistent manner  during their evalu-
ations. In addition, many of the reports date back many years, and some
of the advice given in earlier reports is no longer valid.

    Recognizing  the importance of maintaining  consistency, an inter-
country  meeting that was  held in 1985  in Ottawa, Ontario,  Canada to
consider  ways of strengthening the  role of JMPR in  its evaluation of
pesticide  residues in food recommended  that the principles that  have
been elaborated by JMPR through the years be codified and updated where
appropriate  and consolidated in a  single publication.  The 1985  JMPR
supported  such  an effort  and  recommended "that  this international
meeting  be requested  to consider  the toxicological  basis  and  data
requirements  for  the estimation  of an ADI  or temporary ADI,  and to
provide general guidance on relevant toxicological methodology."

    An  IPCS planning meeting  was held in  March 1987 in  Carshalton,
Surrey,  UK in response to  these recommendations, at which  time areas
were  identified for consideration,  which were incorporated  into  the
first draft.  This draft was reviewed at a task group meeting in Geneva
in  September 1988, after which extensive revisions were made.  An edi-
torial group meeting in Geneva in June 1989 produced the  final  draft,
which  was considered by the WHO Expert Group at the 1989 JMPR.  Drafts
were  widely distributed at several stages, and the comments which were
received  from  a  wide  range  of  international  experts  have   been
incorporated into the final publication.

    The  present publication therefore reflects  the views of a  large
number of international experts who are involved with the toxicological
assessment  of pesticides.  In addition,  by concentrating on the  pro-
cedures used by the WHO Expert Group on Pesticide Residues,  it  faith-
fully reflects the principles used in the evaluation of pesticide resi-
dues by JMPR.  It is expected, therefore, that the future use  of  this
publication  by the WHO Expert  Group will ensure consistent  decision-
making using up-to-date principles.  Those involved in  the  production
of this publication also hope that it will be of significant  value  to
government officials responsible for establishing safe levels of pesti-
cide  residues on food  commodities and by  companies producing  safety
data on pesticides.

    The  WHO Expert Group on Pesticide Residues has been the responsi-
bility of the International Programme on Chemical Safety  (IPCS)  since
the  inception of the Programme in 1980. The preparation of this publi-
cation provides an indication of the importance that IPCS places on the
work  of the WHO  Expert Group in  particular and on  the toxicological
assessment  of pesticides in general.  I am confident that  those of us
responsible  for the toxicological  assessment of pesticides  will find
the  resources that have  been put into  the production of  this publi-
cation  to have been  well-spent, and that  the publication will  be of
enormous value in our work.


                                                Dr J.L. Herrman
                                                WHO Joint Secretary
                                                Joint FAO/WHO Meeting on
                                                  Pesticide Residues

1.  INTRODUCTION

    The  World Health Assembly noted in 1953 that the increasing use of
various  chemicals in the food industry had in recent decades created a
new  public health  problem.  In  response to  this, the  World  Health
Organization,  in conjunction with  the Food and  Agriculture  Organiz-
ation, initiated two series of annual meetings on food additives (Joint
FAO/WHO  Expert Committee on  Food Additives, JECFA)  and on  pesticide
residues. The first meeting on food additives was held in 1956 and that
on pesticide residues in 1963.

    Joint Meetings of the FAO Panel of Experts on Pesticide Residues in
Food and the Environment and the WHO Expert Group on Pesticide Residues
(usually  referred to as the  Joint FAO/WHO Meeting on  Pesticide Resi-
dues,  or JMPR) have provided  an authoritative voice on  the levels of
pesticides  that can be ingested daily by man without appreciable risk;
this  has  been accomplished  through  the establishment  of acceptable
daily intakes (ADIs).  Since 1966, JMPR has been  establishing  maximum
residue limits (MRLs) of pesticides in food commodities.

    This monograph has been prepared on behalf of the Central  Unit  of
the International Programme on Chemical Safety (IPCS) and its aim is to
provide an update of the principles utilized by the WHO Expert Group on
Pesticide Residues.  It does not address the work of the FAO Panel.

    Certain  toxicological principles pertinent to JMPR have previously
been discussed in the JMPR reports.  These principles usually relate to
advances  in scientific knowledge, which  have modified both test  pro-
cedures and the evaluation of test results.  The contents of  the  JMPR
reports were collated in 1977 [164].  Many of the basic principles were
initially adopted from the deliberations of JECFA and are  detailed  in
"Principles for the Safety Assessment of Food Additives  and  Contami-
nants  in Food" [176].  In some of the areas where the principles used
by  JECFA and JMPR are  identical, direct quotes from  that publication
have  been included in  this monograph.  In  this context, however,  it
should be recognized that the two committees, while utilizing data from
similar types of studies, differ in their approach to the evaluation of
the available data.  This difference in approach arises  because  JECFA
usually  evaluates compounds intended for  addition to food, which  are
usually  of low toxic  potential, whereas JMPR  deals with residues  of
compounds that are toxic to at least some groups of living organisms.

    The types of data that are evaluated when assessing the safety of a
pesticide include those from biochemical and toxicological studies and,
when available, observations in humans.  The recent JMPR  viewpoint  is
that  an  understanding  of  the  pharmacokinetic  and  pharmacodynamic
characteristics  of a pesticide is extremely important and that such an
understanding  will even compensate  for inadequacies in  the available
data base.  On the other hand, this approach will sometimes lead to the
requirement for either additional parameters to be investigated in rou-
tine  studies or for additional specific studies not routinely required
for the particular class of pesticide.

    It  has been  recognized that  data from  studies using  routes  of
exposure  other than oral are of value in the overall evaluation of the
safety of pesticides.  However, these studies are not directly relevant
for  the calculation of  the ADI, so  this monograph will  not consider
these other routes of exposure in detail.

    The more recent additions to the battery of toxicity  tests  avail-
able for use in safety assessment are discussed in this monograph. Some
of these tests, especially in the fields of immunotoxicity  and  behav-
ioural  toxicity, are not yet at the stage of development where results
are  consistently  reproducible  and therefore  readily  utilizable  in
safety  assessment.  In addition,  criteria for interpretation  of such
studies have not yet been sufficiently developed to be of value in rou-
tine safety assessment.  Therefore, only the potential of these studies
is discussed in this document.

    The development of knowledge in the field of toxicology  in  recent
years has been quite remarkable.  The history of JMPR and  the  changes
in  its principles of safety assessment reflect this development.  Thus
decisions  taken by JMPR are always provisional and ADIs are subject to
re-evaluation as new significant data become available.

    Each  chapter in this monograph provides a rational background to a
specific  area, describes the history of relevant changes in principles
according  to the  development of  scientific knowledge,  and offers  a
short summary of the current position of JMPR.  It also  indicates  the
principles being followed at present by the WHO Expert Group  in  their
evaluations  of pesticide residues in  food and recommendations on  how
the studies may be performed to provide meaningful results.

2.  GENERAL HISTORICAL BACKGROUND

    The concept of JMPR was first proposed in 1959, when an  FAO  Panel
of  Experts on the Use  of Pesticides in Agriculture  [29], recommended
that FAO and WHO should jointly study:

 *  the hazard to consumers arising from pesticide residues in  and  on
    food and feedstuffs;
 *  the  establishment of principles governing the setting up of pesti-
    cide tolerances; and
 *  the  feasibility of preparing an International Code for toxicologi-
    cal and residue data required in achieving the safe use of a pesti-
    cide.

    Consequently,  in 1961, a Joint Meeting of the FAO Panel of Experts
on the Use of Pesticides in Agriculture and the WHO Expert Committee on
Pesticide Residues was convened.  The report of the 1961  Meeting  [32]
recommended to the Directors-General of FAO and of WHO  the  evaluation
of  "toxicological and other pertinent  data . . . on those pesticides
known  to leave residues in  food when used according  to good agricul-
tural  practice".  The evaluations  would include the  estimate of  an
acceptable daily intake and an explanation of its derivation.

    To implement this recommendation the first Joint Meeting of the FAO
Committee  on Pesticide Residues in Agriculture and the WHO Expert Com-
mittee on Pesticide Residues was convened in September, 1963 [35]. This
Meeting adopted the concept of the acceptable daily intake,  which  was
based on:

 *  the chemical nature of the residue,
 *  the  toxicity of the chemical based on data from acute, short-term,
    and  long-term studies, and  knowledge of metabolism,  mechanism of
    action, and possible carcinogenicity of residue chemicals when con-
    sumed (usually determined in animals);
 *  knowledge of the effects of these chemicals on humans.

    The  1963 JMPR [35] adopted  the use of "safety  factors" for ex-
trapolating animal data to humans and to allow for  variability  within
the human population.  It also noted other points to be considered when
establishing ADIs, including additive effects of multiple pesticides in
the  diet, potentiation between pesticide residues, and genetic differ-
ences (especially in enzyme composition) within the exposed human popu-
lation.

    The  1963 and 1965  Joint Meetings [35; 36]  were concerned  solely
with the acceptable daily intake and did not consider tolerances.  Sep-
arate meetings of an FAO Working Party on Pesticide  Residues  examined
the  issue of tolerances approximately two months after the Joint Meet-
ings  and issued separate  reports. The first  report considered  prin-
ciples  [34] and the second  proposed tolerances for pesticides  on raw
cereals [37].

    In 1966, the JMPR report [38] considered both ADIs  and  tolerances
for the first time.  Joint Meetings have since been held  yearly,  and,
after  each one, reports and evaluations have been published.  The JMPR

has  evolved principles consistent with the changing state of knowledge
in toxicology and chemistry, and the evaluation of new data  has  often
prompted  adjustments  in  previous conclusions  on  various chemicals.
However, the products of the Joint Meetings (which include  ADIs,  tem-
porary  ADIs, MRLs (MRL  replacing the term  "tolerance"),  temporary
MRLs,  guideline levels, and  extraneous residue limits)  have remained
essentially unchanged.

3.  JMPR ASSESSMENT PROCESS

    JMPR comprises two separate groups of scientists. The FAO Panel has
responsibility  for reviewing pesticide use patterns, data on the chem-
istry  and composition of pesticides, and methods of analysis of pesti-
cide  residues, and for recommending MRLs that might occur in food com-
modities following the use of pesticides according to good agricultural
practices. The WHO Group has responsibility for reviewing toxicological
and related data and for estimating (where possible) an ADI for humans.
During  the Joint Meetings,  the two groups  coordinate activities  and
issue a joint report.  However, the present section  on  interpretation
of data is limited to the procedures used by the WHO Expert Group.

    The  data used in the assessment of the toxicity of pesticide resi-
dues  generally  comprise  acute studies,  short-term  feeding studies,
long-term  feeding studies, and biochemical  studies (including absorp-
tion, tissue distribution, excretion, metabolism, biological half-life,
and  effects on enzymes).   In addition, studies  on specific  effects,
e.g., carcinogenicity, reproduction, teratogenicity, and, for some com-
pounds,  neurotoxicity, are usually  necessary.  Human data  and  other
information,  e.g.,  SAR  (structure-activity relationships),  are also
considered when available.

    The  overall objective  of the  evaluation is  to determine  a  no-
observed-adverse-effect  level (NOAEL), based upon consideration of the
total toxicology data base,  which will be utilized in conjunction with
an appropriate safety factor to determine the ADI.  The  initial  stage
of  the evaluation has to  be a critical examination  of the individual
studies.  In some cases, a study initially considered to be of marginal
value  may, in fact, be  useful when considered in  the context of  the
entire data base.  Integration of the results from all studies can then
permit an appraisal of the toxicity of the compound.

    Data from acute oral studies are rarely considered to  be  relevant
to  the establishment of the ADI. However, such data may provide infor-
mation  that permits a ranking of the sensitivity of different species,
may assist in the selection of dose levels in subsequent  studies,  and
may indicate types of pharmacological activity, degree  of  absorption,
or potential target organs.  JMPR has, on occasion, required additional
acute data to determine the relative toxicity of salts of  a  pesticide
(e.g., imazalil [57]) or required further metabolic studies  to  deter-
mine species differences in acute toxicity (e.g., triazophos [67]).

    Historically,  short-term feeding studies  have provided the  basis
for  the determination of ADIs  for a number of  compounds evaluated by
JMPR.   Prior to  1971 [48],  before long-term  toxicity  studies  were
indicated  to be an essential  part of the toxicological  data base for
evaluating  the safety of pesticides, ADIs were established for several
pesticides  based on short-term  toxicity studies (e.g.,  demeton [45],
parathion-methyl [182], dimethoate [182; 41], diazinon [182], azinphos-
methyl  [182], methyl bromide  [39], and dichlorvos  [39]).   Temporary
ADIs (TADIs)  based on such studies have also been  established,  e.g.,
omethoate  [49], fenthion [49].   Since long-term feeding  studies have
become  an essential part of the toxicological data base, the major use
of  the  data from  short-term toxicity studies  has been to  determine

suitable  dose levels  to be  utilized in  long-term  and  reproduction
studies. However, studies lasting more than two years are rarely avail-
able in dogs.  Thus, in situations where this species is more sensitive
to  a toxic  effect or  more appropriate  for use  in extrapolation  to
humans  than  are  the rodent species, the ADI is usually based on data
generated  in studies covering  less than 50%  of the lifespan  of this
species, e.g., methamidophos [182], diflubenzuron [182], and phenthoate
[63; 73].  Occasionally, an ADI may also be based on short-term studies
in  rodent species, e.g., diphenylamine [56; 68; 73], even though long-
term studies may exist which indicate higher NOAELs. This situation may
arise  from adaptation,  resulting in  the disappearance  of an  effect
after long-term exposure.

    Multigeneration  reproduction and teratogenicity studies  have also
been  used for establishing  ADIs for certain  compounds, e.g.,  chlor-
mequat [51] and dinocap [183].

    Delayed  neurotoxicity has been  identified as a  potential problem
for a number of compounds evaluated by JMPR.  To date,  only  leptophos
has had an ADI withdrawn because of this effect. However, withdrawal of
the ADI was not because of possible hazards from exposure to food resi-
dues  but  because of  withdrawal of the  product from the  market as a
result of effects on heavily exposed individuals during its manufacture
[59].  ADIs have been allocated for other delayed neurotoxicants, e.g.,
isofenphos  [76]. JMPR has  indicated that the  exaggerated doses  (ex-
ceeding the LD50)   used in the standard hen studies for delayed neuro-
toxicity  are not  necessarily applicable  to the  assessment of  human
hazard arising from the intake of residues in food.

    For  the effects discussed above,  the basic interpretation of  the
available data depends upon the identification of a toxic  effect,  the
establishment of incremental increases  in the incidence of this effect
with  increasing exposure (i.e.  a dose-effect relationship),  and  the
establishment of a threshold.

    The  primary consideration in  determining whether a  compound  can
induce  a  toxic  effect is the dose of test material to which the test
organism  is exposed.  A basic  concept of toxicology is  the statement
made by Paracelsus in 1538, which translated indicates: "Only the dose
decides that a thing is not poisonous" [1]. Thus, if a series of doses
used  in  a  study fails  to elicit  a toxic  response, then  an insuf-
ficiently high dose level has been used.  This philosophy is applicable
to all toxicity studies. However, provided an adequate margin of safety
exists  between the highest dose tested and the possible human exposure
from pesticide residues, then a study in which a toxic effect  was  not
observed  may  be considered  to be acceptable  for the purpose  of as-
sessing  the safety  of such  residues.  The  major difficulty  in  the
absence  of toxicity is  the determination of  the safety factor  to be
applied  to the highest dose tested, since there would be no indication
of  the  type,  importance, or severity of the effect that might be in-
duced with increased dose levels.

    A  second factor is  the determination of  which effects should  be
considered to be toxic.  A judgment must be made, based on  the  nature
of the effect, on whether it should be considered adverse. As indicated

in  section 8.3.6.2,  plasma  cholinesterase depression  should  not be
considered to be a toxic phenomenon since, although it is an effect, it
is not apparently a toxic effect. A reversible increase in liver weight
may be an adaptive response rather than a toxic effect. However, ancil-
lary studies may be required.

    The determination of dose-response relationships in an experimental
population is based on the concept that the incidence or severity of an
adverse effect is related to dose.   A time-response  relationship  may
also  occur, i.e. where  the incidence of  an induced effect  increases
with  the duration of  dosing at a  constant level.  Comparison  of the
results  of experiments with differing durations (performed on the same
species  and strain, preferably in the same laboratory, and under simi-
lar  environmental conditions) may  be necessary to  demonstrate  time-
effect  relationships if interim kills have not been performed.  In the
absence  of interim kills  or shorter-term experiments  for comparative
examination, it is sometimes possible to calculate the approximate time
of appearance of a major lesion based on findings in dying  animals  or
those  sacrificed in moribund condition during the course of the study,
particularly if the lesion is associated with the cause of death.

    The  integrated  nature  of mammalian  reproductive  processes  may
complicate  the  establishment of  dose/time/response relationships for
reproductive effects. Events that are initiated during  early  develop-
ment  may be moderated considerably in subsequent developmental stages.
The  defense mechanisms which have evolved to minimize the consequences
of  insult may repair minimal  damage or discard that  which is damaged
beyond effective repair (e.g., resorptions or abortions). Consequently,
in  reproduction studies,  the demonstrated  dose response  is often  a
reflection  of a progressive  involvement of multiple  variables rather
than a temporal change in a single variable.

    When  evaluating toxicological data, relevant parameters are evalu-
ated  statistically so that  their significance is  established on  the
basis of predetermined criteria. A statistically significant difference
between  experimental and control  groups should be  considered in  the
light of its biological relevance.  Thus, an increased incidence  of  a
rare  tumour  type in  treated animals may  be of concern,  even if the
incidence is not significantly different statistically from  its  inci-
dence in the concurrent control animals.  Conversely,  a  statistically
significant change in an isolated parameter, e.g.,  erythrocyte  count,
would  usually not  be considered  to be  biologically relevant  unless
supported by changes in other parameters, e.g., bone marrow  or  spleen
histopathology or methaemoglobin formation.

    A high background incidence of a specific lesion frequently compli-
cates the interpretation of data generated in toxicity studies. To some
extent,  especially in the case of a specific tumour type, this problem
can  be avoided by judicious  choice of species or  strains of animals.
For  example, if  the target  organ is  known to  be  the  kidney,  the
interpretation  of results would be difficult in long-term studies in a
rat strain with a high incidence of geriatric nephropathy.

    The  use  of more  than one species  for the same  type of toxicity
study  may complicate  interpretation in  those cases  where an  effect
occurs in one species but not in a second species, or where one species
is  much  more  sensitive to the agent than the other species.  In such
cases it is often difficult to determine the most  appropriate  species
for extrapolation to man. Generally, unless adequate data are available
to indicate the most appropriate species (usually comparative pharmaco-
kinetic or pharmacodynamic data), the most sensitive species (i.e., the
species in which the adverse effect occurred at the lower dose) is used
in determining the NOAEL and allocating the ADI.

    In interpreting carcinogenicity data, JMPR bases its evaluations on
the  threshold concept, which  is the basis  for evaluating most  other
toxicological  effects [170]. In assessing tumour incidence, benign and
malignant  tumours have  been considered  as separate  entities in  the
majority of cases [176].  For further discussion of  the  determination
of NOAELs in carcinogenicity studies, see section 8.3.4.5.

    The 1986 Joint Meeting stressed the importance of understanding the
mechanism  of action that  results in the  expression of toxicity.   It
noted that: "Current knowledge of mechanisms of toxicity  is  limited,
but there is already a sufficient understanding in some cases to permit
better   design,  performance,  and  interpretation   of  toxicological
studies.   Mechanistic studies are therefore encouraged, since a knowl-
edge  of  mechanism of  action is likely  to result in  a more rational
assessment of the risk to man." [76, p. 2].

4.  CONSIDERATIONS OF IDENTITY, PURITY, AND STABILITY

4.1.  Background

    The report of the WHO Scientific Group on Procedures  for  Investi-
gating Intentional and Unintentional Food Additives indicated  in  1967
that:  "adequate  specifications for  identity  and purity  should  be
available  before toxicological work  is initiated.  Toxicologists  and
regulatory  bodies need assurance that the material to be tested corre-
sponds to that to be used in practice."  It also stated that: "levels
of impurities that, according to current knowledge, are  considered  to
be   toxicologically  significant . . . must  appear  in  the  specifi-
cations." [169, p. 8].

    The need for accurate specifications for pesticides was stressed by
the 1968 JMPR [42] during its deliberations on toxaphene and on techni-
cal  grades of benzene hexachloride  (BHC).  Because of the  unknown or
variable  composition of these compounds, the JMPR was unable to relate
the  existing toxicological data to the products in actual agricultural
use.   Consequently, ADIs could not  be allocated.  Attention was  also
drawn to the likelihood of variability between similar  chemicals  pro-
duced by different manufacturers.

    The possible influence of known or unknown impurities on  the  tox-
icity of technical grade chemicals and of residues resulting from their
use was discussed by the 1974 JMPR [53].  This JMPR noted that toxicity
studies are generally performed on technical grade  materials  produced
by commercial-scale processes and that the resulting toxicological data
normally, therefore, take into account the presence of impurities (pro-
vided  that the manufacturing process  remains the same).  However,  it
noted the problems encountered with trace amounts of  biologically  ac-
tive  materials, e.g., 2,3,7,8-tetrachloro-dibenzo- p  -dioxin  in 2,4,5-
trichloroacetic acid. It further noted that: "specifications  such  as
those  issued by FAO and WHO are seldom designed to take note of trace-
level  impurities, unless the importance of such impurities has already
been revealed by biological studies." [53, p. 15].

    The  1977 JMPR [57]  noted that data  on the nature  and  level  of
impurities, intermediates, and by-products in technical pesticides were
often available, but, because such data could provide  valuable  infor-
mation  to competitors they  were normally considered  to be a  "trade
secret". The Joint Meeting, therefore, agreed that such data would not
normally be published in the JMPR reports or monographs.

    In  considering the applicability of  recommendations to pesticides
from different feedstocks produced by different manufacturers, the 1978
JMPR [59] indicated that evaluations and recommendations are valid only
for the specific technical grade of pesticide  examined.   Considerable
care  and knowledge  of the  detailed specifications  are  required  to
extrapolate  evaluations and recommendations  to products of  differing
quality or composition.

    Subsequent Joint Meetings [60; 62; 72] have stressed the importance
of  information on the  presence of impurities  in technical  pesticide
products  (e.g., the presence  of hexachlorobenzene in  various  pesti-
cides,  impurities  in phenthoate,  and  dimethyl hydrazine  in  maleic
hydrazide).  The need for technical grade pesticides to meet FAO speci-
fications  has  also  been stressed. It was noted by the 1984 JMPR that
occasionally data have been rejected because the test  material  failed
to comply with these specifications [72].

    The 1987 JMPR [78] noted that ADIs based on studies using compounds
of specific purity can be relevant to products of different  origin  or
purity  but that there are examples of changes in the amount or type of
impurity in the technical material that markedly influence the toxicity
of a compound.

    Toxicity  tests should normally be performed on the technical grade
of  the  pesticide (except  for acute toxicity,  for which both  formu-
lations  and technical materials must  be tested to assess  the risk to
the  applicator).   However, the  percentage  of active  ingredient and
impurities  in the technical grade  material may vary among  production
batches  and  may  differ at  various  stages  of product  development.
Furthermore,   since some toxicity  testing is likely  to be  performed
with  the product in the  early stages of development,  the preliminary
studies (usually designed to assess potential acute hazards to individ-
uals  who  will  be involved in the development of the material) may be
performed  on batches of material produced within the laboratory.  Sub-
sequent studies may be performed on material produced in a pilot plant,
while  other toxicity studies may be performed on the marketed product,
which  will be produced in  a full-scale manufacturing plant.   At each
step  in this sequence, there is a potential for change in the percent-
age  of the active ingredient in the "technical-grade" material and a
potential  for change in  the quantity and  identity of the  impurities
that constitute the remainder of the "technical-grade"  product.   It
is,  therefore, essential that  detailed specifications should  be pro-
vided for the test material utilized in each study.

    In  certain cases the pesticidally  active ingredient may exist  in
two or more forms, e.g., as a diastereoisomeric mixture.  In  the  case
of  the synthetic pyrethroids, this  is normally the case.   Under such
conditions, the ratio of isomers in the test material must  be  clearly
specified  since it  has been  documented that  different isomers  fre-
quently have different toxicological activities [60; 87].  For example,
an ADI for permethrin (40% cis : 60% trans) was allocated in 1982 [67],
whereas  the ADI for permethrin (25% cis : 75% trans) was not allocated
until 1987 [78].

    Data on the stability of the test material is also  of  importance.
The  percentage of the active material will decrease and that of break-
down  products will increase with  time if a test  compound is unstable
under the conditions of storage.  This will be of major  importance  in
the  evaluation of the results of studies where a single batch of tech-
nical material is utilized for a long-term study or  a  multigeneration
study.  Variation in the amount of degradation occurring  in  different
batches (i.e., batches of different post-manufacturing age) may compli-
cate  the  interpretation of  a study.  Finally,  reaction of the  test

compound  with components of the test diet may result in the production
of  toxic reaction products in the diet, which may affect the nutritive
value  of the diet and will result in a decreased concentration of test
compound.   The NOAEL may  well be overestimated  if the percentage  of
active  ingredient  decreases with  time.   Conversely, if  a breakdown
product is more toxic than the parent active material, then  the  NOAEL
of the parent compound may be underestimated.  Either  situation  would
result in the establishment of an inaccurate ADI.

    Up to now, JMPR has evaluated only the active ingredients of pesti-
cide formulations.  The toxicity of "inert ingredients"  (e.g.,  sol-
vents, emulsifiers, preservatives) that may occur as residues  in  food
has not been considered.

4.2.  Principles

4.2.1.  Identity

(a) A detailed specification of the test material used in each individ-
    ual study must be provided.

(b) Where  isomeric mixtures exist,  the ratio of  isomers in the  test
    material must be clearly specified, since it has been  amply  docu-
    mented that different isomers frequently have different toxicologi-
    cal activities.

(c) JMPR  recommendations relate  to a  specific technical  grade of  a
    pesticide.   They  will not  necessarily  be applicable  to similar
    materials  produced  by  different manufacturers  or where specifi-
    cations of new material used in the manufacturing process  are  not
    consistent.

4.2.2.  Purity

(a) The percentage of the active ingredient in any  technical  material
    used  in a toxicity test  or proposed for marketing  must be speci-
    fied.

(b) Levels of all identifiable impurities should be specified.

(c) Data  on manufacturing processes may be required to permit determi-
    nation of potential impurities. However, because of confidentiality
    and  industrial secrecy, such  data will not  be published in  JMPR
    monographs.

4.2.3.  Stability

(a) Stability  of the test material during storage and in the diet must
    be adequately investigated and reported.

(b) Where  instability in diets is observed, possible reaction products
    and the nutritional quality of the diet should be investigated.

5.  AVAILABILITY AND QUALITY OF DATA

5.1.  Background

    Most  of  the data  utilized by JMPR  consists of unpublished  pro-
prietary  data, as  well as  information submitted  by governments  and
other  interested parties.  When  available, relevant reports  from the
open  literature  are considered.   However,  published data  on  major
studies must provide sufficient information to permit  evaluation  [38;
57]. This precludes reliance on summary or abstract publications.  Such
information  must include complete  descriptions of experimental  tech-
niques  and data adequate to  permit assessment of the  validity of the
results [38].  It is preferable that published reports be from refereed
journals.   Although all available  relevant data are  considered  [46;
54],  unpublished data must meet certain criteria, i.e. reports must be
complete,  study supervisors should be  qualified to perform the  study
and should be identified, and the time at which the study was performed
must be identified [38; 46].

    Only  those  data which  are available to  all members can  be con-
sidered  during a Joint Meeting  [55]. This requirement applies  to all
supporting data and cited material.  This has been a subject of ongoing
concern  and has been addressed frequently in JMPR reports [53; 54; 55;
57; 59; 60; 62; 65; 67; 70; 78; 172].

    On some occasions, important information is omitted from the report
of  a study.   Examples  of such information  include the identity  and
specifications of test material (section 4), information on the quality
of  experimental  animal diets,  and  information on  their nutritional
composition.

    Present-day standards generally require that data should be subject
to quality control and that the study should conform to  the  standards
specified  under codes of  good laboratory practice  (GLP)  [160; 161].
Studies  performed in compliance  with GLP codes  help assure that  the
quality of unpublished data is acceptable. However, compliance with GLP
codes does not provide a substitute for scientific quality. An inappro-
priate  study is still unacceptable  even though it may  have been con-
ducted according to GLP standards.

    The  validity of data submitted  for evaluation has always  been of
concern.  Recent scandals reported  in the scientific  literature,  in-
volving  inaccurate or falsified data  [12], highlight this problem  of
data validity. However, the application of "good laboratory practice"
and "quality assurance" techniques should reduce, but  probably  will
not eliminate, the problems of data validation.

    JMPR does not have the resources to validate studies  [59].  There-
fore, it accepts submitted data as being valid unless there is evidence
to the contrary [57].  The 1977 JMPR [57] was informed of the suspicion
that  serious deficiencies  existed in  several studies  that had  been
utilized  by JMPR  in allocating  some ADIs  and TADIs.   In 1982,  the
Meeting  re-evaluated a number of pesticides that had been supported by

data  from Industrial Bio-Test  Laboratories.  As a  general principle,
where  studies  supporting the  ADI could not  be validated, and  where
alternative studies were unavailable, the ADI was converted to  a  tem-
porary ADI. Furthermore, if the studies were not validated or replaced,
the ADI was withdrawn.

    The  format for data  presentation requires that  a summary of  all
pertinent studies be prepared [57], together with reports of each study
with  complete supporting data.   Complete supporting data  are usually
considered  to be individual animal data, although occasionally, if GLP
codes  have been followed and  quality control assurance is  available,
this requirement has been waived.  The submission of an  evaluation  of
the  compound by the sponsor is encouraged.  Since the working language
of  JMPR is  English [65],  translations of  reports into  English  are
appreciated.

5.2.  Principles

1.  To  evaluate the safety  of pesticide residues,  JMPR is  dependent
    upon the receipt of acceptable data.  Data for major studies should
    not be in abstract or summary format and should be of  good  scien-
    tific  quality  from  laboratories utilizing  acceptable laboratory
    practices [46; 54; 57; 59; 60; 62].

2.  Compliance  with recognized  GLP codes  (e.g., those  of  OECD)  is
    encouraged.

3.  Submitted data should be in such a form that the integrity  of  the
    study can be ascertained.

6.  HUMAN DATA

6.1.  Background

    Human  data on pesticides are  collected from a variety  of sources
including  accidental, occupational, and experimental  exposures.  Data
from  experimental exposures of human volunteers can provide quantitat-
ive  information on dose-effect  and dose-response relationships  which
may be applied directly in establishing an ADI.  Data on accidental and
occupational exposure can serve as supporting information.

    A  Joint FAO/WHO Meeting in 1961 highlighted the relevance of human
data for toxicological evaluations and the need to  study  occupational
exposures  during production, handling,  and uses of  pesticides, since
exposure is generally higher than that of the general population [32].

    In  1967, a WHO Scientific  Group was convened to  provide guidance
for the review of intentional and unintentional food  additives.   This
group addressed the general problem of investigations in human subjects
and  recommended the conduct of human metabolic studies.  It was recog-
nized that adequate preliminary tests in animals are  necessary  before
 in vivo  human studies can be performed [169].  In addition, studies in
volunteers  might be required to  confirm the predicted safety  margin.
However,  several  conditions were  listed  which should  be  fulfilled
before such studies can be undertaken, including the  demonstration  of
need and full information on toxicity in experimental animals  and  the
reversibility  of toxic effects.   The Scientific Group  indicated that
experimental studies on the toxic effects of pesticides in  humans  are
not acceptable.

    At  the 1968 and 1969  Joint Meetings [42; 44], it  was stated that
the  availability of adequate human data might justify the use of lower
safety factors in setting the ADI [46].

    The use of modern quantitative analytical toxicology  concepts  was
introduced at the 1973 JMPR [52], with suggestions of the  analysis  of
tissue  and body fluids for  a given pesticide.  This  is of particular
importance in accidental poisonings.  This Joint Meeting also suggested
the follow-up of workers exposed to pesticides.  Observations  in  such
studies  may reveal effects specific to humans.  The 1975 Joint Meeting
recommended  to  WHO  that cooperation  should  be  sought with  Poison
Control  Centres and other  organizations to develop  appropriate  data
banks [54].

    Data from humans continued to be required in relation to  a  number
of pesticides until the time of the 1976 JMPR [55].  After  that  time,
because  of ethical problems  and the increasing  difficulties of  per-
forming studies in humans, JMPR reports indicated that data  on  humans
were  "desirable".  Since 1982 [67], JMPR has generally, when toxico-
logical  assessments have been performed, indicated the desirability of
data  on observations in humans.   When considering again the  need for
comparative  biotransformation  data, the  1987  JMPR [62]  stated that
these might also be obtained with  in vitro  experiments.  It  should  be

noted that there are limitations in the use of  in vitro  data,  in  that
absorption and subsequent distribution as well as  possible  activation
mechanisms must be considered before extrapolating such data to the   in
 vivo  situation and the subsequent establishment of an ADI.

    The  1989 JMPR re-emphasized the necessity of obtaining human data.
It indicated that human data may confirm a common mechanism of toxicity
between humans and the test species or may be used to compare doses and
effects between species [183].

6.2.  Current Position

    All  human  data  (accidental, occupational,  and  experimental ex-
posures)  are fundamental for  the overall toxicological  evaluation of
pesticides and their residues in food.  Data on  accidental  poisonings
may  identify  target  organs, dose-effect  and dose-response relation-
ships,  and the reversibility  of toxic effects,  provided that  modern
standards  of analytical toxicology (e.g.,  identity and purity of  the
pesticide, blood levels of the parent compound and/or  breakdown  prod-
ucts,  gastric  lavage  content,  and  urinary  metabolites)  have been
applied  to the study.  A careful assessment of the dose and perhaps of
the  effects (e.g., plasma  and erythrocyte cholinesterase  inhibition)
may  enable comparison with animal data.  Unfortunately, available data
rarely permit such comparison.  Follow-up studies in workers may enable
the  validity of extrapolations from  animal data to humans  to be con-
firmed  and  unexpected  adverse  effects  specific  to  humans  to  be
detected.

    The JMPR mandate is to consider the safety of pesticide residues in
food. Dietary exposure on a daily basis is almost always relatively low
compared to occupational exposure, and therefore it might  be  expected
that  an effect on  the exposed worker  would be more  easily detected.
Unfortunately,  there  are  limitations in  attempting  to  extrapolate
observations in the occupational setting to dietary exposure. The major
route of exposure to pesticides for workers is generally  dermal.   The
extent  and  rate of  absorption via the  dermal route usually  differs
markedly from that observed after oral exposure. Ingested compounds may
be  metabolized by intestinal microflora  and may be subject  to metab-
olism  within the liver directly after absorption from the gastrointes-
tinal  tract and transport by  the hepatic portal system.   Thus target
tissues  may be exposed to  a different pattern of  parent compound and
metabolites after dermal or inhalation administration than  after  oral
administration.  Data on the identity and levels of parent compound and
metabolite(s),  following administration by  the different routes,  are
desirable  to  assist  in the  interpretation  of  the  observed  toxic
effects.

    When  large groups of individuals  are exposed to pesticides,  epi-
demiological  studies can be  of considerable value.   Often,  however,
workers  in manufacturing plants and pesticide mixer/loaders and appli-
cators are also exposed to several other compounds and it may be diffi-
cult  to determine a cause-effect  relationship for a given  pesticide.
Results are also often confounded by the difficulty in finding suitable
control  populations, the large number of other variables involved, the
long latency period for certain effects such as cancer, and small study

populations,  especially in manufacturing facilities.   Exposure levels
may also be difficult to quantify.  Further guidance in the conduct and
interpretation  of  epidemiological  studies is  given in Environmental
Health Criteria 27 [173].

    Studies  on human volunteers are sometimes of considerable value in
allocating ADIs. However, before human  in vivo  studies are considered,
ethical  considerations  must  be  taken  into  account.   The Proposed
Guidelines on Biomedical Research Involving Human Subjects, issued as a
joint project by WHO and the Council for International Organizations of
Medical Sciences [21], have been endorsed by JMPR.

    The value of human data was expressed cogently by Paget [127], when
he wrote:

     "The question is not whether or not human subjects should be used in  
toxicity experiments but rather whether such chemicals, deemed from animal 
toxicity studies to be relatively safe, should be released first to 
controlled, carefully monitored groups of human subjects, instead of being 
released indiscriminately to large populations with no monitoring and with 
little or no opportunity to observe adverse effects." 

6.3.  Principles

1.  The  submission of human data,  with the aim of  establishing dose-
    effect  and  dose-response  relationships in  humans,  is  strongly
    encouraged.

2.  Studies on volunteers are of key relevance for extrapolating animal
    data to humans. However, attention to ethical issues is necessary.

3.  The  use of  comparative metabolic  data between  humans and  other
    animal species for the purpose of extrapolation is recommended.

7.  STRUCTURE-ACTIVITY RELATIONSHIPS

    The Joint FAO/WHO Meeting on Principles Governing  Consumer  Safety
in  Relation to Pesticide Residues recognized that toxicological "pro-
cedures must be determined by the chemical and physical  properties  of
the  pesticide . . . " [1, p. 8].  A  subsequent WHO Scientific  Group
stated that: "If a series of chemical analogues can be shown  to  give
rise to the same main metabolic product and other compounds  which  are
already present in the organism in greater quantities, or that  can  be
readily and safely metabolized, it may be sufficient to carry out toxi-
cological studies on a suitable representative of the  series."  [169,
p. 7].   The same Meeting, in considering the duration of studies, also
indicated  that: "Where adequate biochemical and toxicological data on
closely  related  compounds are  available,  the objective  becomes the
detection of any deviation from the established pattern" [169, p. 13].
This  latter principle  has been  exemplified by  some  evaluations  of
dithiocarbamate  pesticides, where related compounds were considered as
a group.

    Structure-activity  considerations can influence the  testing needs
of  a pesticide.  Thus the organophosphorus compounds, especially those
with  the P-S configuration, are routinely tested for delayed neurotox-
icity,  while the  majority of  other pesticides  are not.   Similarly,
neurotoxicity  is carefully considered in  assessing the safety of  the
synthetic pyrethroid compounds.

    The  limitations of the use of structure-activity relationships has
been  discussed in the recent document on Principles for the Assessment
of Food Additives and Contaminants in Food:

    "Structure-activity  relationships appear to provide  a reasonably
good  basis for predicting toxicity  for some categories of  compounds,
primarily  carcinogens, which are characterized  by specific functional
groups  (e.g., nitrosamines, carbamates, epoxides, and aromatic amines)
or by structural features and specific atomic arrangements (e.g., poly-
cyclic aromatic hydrocarbons and aflatoxins). However, all these chemi-
cal groups have some members that do not seem to be carcinogenic or are
only weakly so." [176, p. 27-28].

    For detailed information on the various chemical classes associated
with  carcinogenesis,  the  reader  is  referred  to  published  review
articles [146; 178].

7.1.  Principle

    For the determination of ADIs, JMPR relies primarily on data gener-
ated  on  individual chemicals.   Structure-activity considerations are
used only as ancillary information.

8.  TEST METHODOLOGIES

    The design and conduct of toxicological investigations  has  always
been, and still remains, the responsibility of competent experts in the
field.   Therefore, the following  sections and subsections  should  be
considered only as guidelines unless stated otherwise.

8.1.  Background

    The  second and fifth  reports of JECFA  addressed the conduct  and
uses  of acute, short-term, long-term, biochemical, and carcinogenicity
studies in the safety evaluation of food additives [31; 33]. While many
of  the proposals included in these documents have changed with advanc-
ing  knowledge in toxicology, some are still deemed to be valid.  These
include:

 *  the need for short-term studies in rodents and non-rodents (defined
    as  studies comprising repeated doses over a period of up to 10% of
    the  expected lifespan of the animal, i.e., usually 90 days in rats
    and 1 year in dogs);

 *  the  non-requirement for determining LD50 values  when no mortality
    occurs at doses of 5 g/kg body weight or more;

 *  the need to initiate short- and long-term studies in  young  (post-
    weaning) animals;

 *  the  need for uniform distribution of the test compound in the diet
    when feeding studies are utilized;

 *  the  requirement to use both sexes in  acute, short-term, long-term
    (chronic), and carcinogenicity studies;

 *  the need for initiating studies with sufficient animals  to  ensure
    adequate  numbers of survivors to provide data for proper statisti-
    cal analysis;

 *  the need to restrict the amount of test compound to less  than  10%
    of the diet when performing feeding studies (although today  it  is
    generally  recommended not to  exceed a dietary  level of 1%  for a
    pesticide);

 *  the need, on a routine basis, for data on absorption, distribution,
    and  excretion, and, where  possible, identification of  the  major
    metabolites;

 *  investigation  of  the effects  of dose level  and duration on  the
    metabolism of the test material;

 *  the need to test contaminants in food for carcinogenicity  by  oral
    administration;

 *  the requirement to maintain an adequate nutritional status  of  the
    test  animal  in  feeding studies,  especially  in  carcinogenicity
    studies.  Information on the quality and composition of  the  diets
    used in toxicology studies should be provided.

    The first JMPR [35] indicated that the biological data required for
allocation  of  an ADI  should  include biochemical,  acute, short-term
(defined as repeated administration for less than half  the  lifespan),
and long-term studies.  The 1976 JMPR outlined the data  necessary  for
the  evaluation  of  pesticides.  These  included  short- and long-term
studies,  special  studies  on  carcinogenicity,  mutagenicity,  repro-
duction,  and teratology, observations  in humans, and  information  on
metabolism,  pharmacokinetics,  and  biochemical effects  [55, p. 8-9].
These are the studies that are now generally available  for  pesticides
used  on food items.  Salient  aspects of the toxicological  tests most
often  used in determining the safety of pesticide residues in food are
discussed in the following sections.

8.2.  General Considerations

8.2.1.  Choice of species and strain

    Limitations  are  inherent in  the  selection of  laboratory animal
species.  The most readily available test species are the  rat,  mouse,
hamster,  guinea-pig, rabbit, cat, dog,  pig, and monkey.  More  exotic
animals  (e.g., Tupia) are  also utilized but  only rarely.  The  major
reasons for the use of such a limited number of species  include  econ-
omics  (cost of obtaining and maintaining animals), lifespan, behaviour
and  survival in captivity,  handling, and, perhaps  most  importantly,
knowledge  of the "normal"  physiology and pathology  of the  species
(see section 8.2.6).

    In 1967, a WHO Scientific Group indicated the need to  utilize  the
most appropriate species in extrapolating to man, i.e.,  "the  species
most  similar  to man  with regard to  its metabolic, biochemical,  and
toxicological characteristics in relation to the subject  under  test"
[169, p. 9].  The choice of an ideal test species requires considerable
knowledge of the absorption and biotransformation of the test material,
not only in the experimental animal species, but also in humans. Unfor-
tunately,  other  considerations (e.g.,  cost  or availability  of test
species,  duration of the study) must also be considered, and it is not
always practical to use the optimum test species.

    It  is  necessary to  consider  both quantitative  and  qualitative
responses in laboratory animals when establishing the ADI. For example,
it is recognized that compound-induced peroxisome proliferation is con-
siderably greater in mice, rats, and hamsters than in  humans  [9; 176;
180].   Thus, these species may be inappropriate for investigating this
effect  in man.  Since specific knowledge of comparative metabolism and
the basis for differences in species sensitivity are often unavailable,
the  effects noted in the  most sensitive species usually  provides the
basis for the ADI assessment.

    JMPR, recognizing the difficulties of obtaining  in vivo human data,
has  proposed as  a compromise  the generation  of  in vitro  data  using
human tissues or cultured human cell lines [78].  Comparison  can  then
be  made (a) between  in vitro  data generated in a number of species and
(b)  between the  in vitro  and  in vivo  data in the test species. Such a
procedure  would markedly assist in the selection of the most appropri-
ate species for studies involving multiple daily administrations and in
the extrapolation of data.  A comparison of this nature  for  methylene
chloride has recently generated a great deal of interest and  has  been
proposed for use in safety assessment [3].

    The choice of species should also depend upon the susceptibility of
the  species (or strain) to the toxic effect being investigated.  Thus,
in teratogenic studies, the test species or strain should be  known  to
be susceptible to teratogenic agents.  As new strains of  rabbits  have
been  introduced for teratology studies,  JMPR has had to  request evi-
dence  (from exposure  to known  teratogens) of  their sensitivity  and
hence their appropriateness for such studies, e.g.,  methacrifos  [67].
In  addition, the time  of specific embryological  events in  different
mouse strains may result in the absence of insult at crucial  times  in
teratology studies [120].

    The  normal incidence of a  pathological lesion may also  influence
the choice of test species or strain. For instance, the use of a strain
in  which the incidence of tumours in a particular organ is excessively
high  in untreated animals (e.g., the incidence of pituitary tumours in
most strains of rat) would be contra-indicated if there was information
indicating  that elements of  the endocrine system  could be among  the
target organs (i.e., if hormonal imbalance were suspected).  Similarly,
the  high incidence of liver  tumours in control male  B6C3F1 mice  may
also mask a neoplastic response in treated animals.  Thus,  a  thorough
knowledge  of the strain being considered for the study is essential to
determine its suitability for a specific type of experiment.

8.2.2.  Group size

    Group size in toxicity studies is dependent upon a number  of  fac-
tors, including the purpose of the experiment, the required sensitivity
of the study, the lifespan of the species under test, the design of the
study,  the reproductive  capacity and  the fertility  of the  species,
economic aspects, and the availability of animals.  This  section  con-
tains a brief discussion of group sizes acceptable for various toxicity
tests followed by a more detailed discussion of numbers of  animals  to
be utilized in long term/oncogenicity studies.

    In  acute oral toxicity tests in rodent species, the number of ani-
mals  utilized depends upon the degree of accuracy required. LD50   de-
terminations (as indicated by 95% confidence limits)  are  approximate,
rather  than accurate. To obtain these approximations, five animals per
sex  per dose level are usually used. Because of the problems of avail-
ability  and because  of economic  factors involved  in utilizing  non-
rodent  species, smaller numbers  of non-rodents (resulting  in reduced
accuracy) are frequently utilized in acute toxicity studies, especially
when  the objective of the study is to examine the comparative toxicity
between species.

    In  teratology studies, because the objective is to obtain adequate
numbers of litters from treated females, the actual number  of  animals
required  is dependent on fertility and the difficulties encountered in
breeding.  Most protocols for studies with rodent species specify 20-25
pregnant  females per dose level.  When other species are used, such as
the rabbit, smaller group sizes (usually producing a minimum of 12 lit-
ters)  are utilized.  However,  when equivocal data  are obtained  from
such  studies (e.g., an  incidence of congenital  malformations, which,
although not statistically significant, shows positive trend analysis),
increased  group size or the  provision of adequate historical  control
data may be necessary.

    In  multigeneration studies in rats,  a minimum of 20 pregnant  fe-
males per dose level per mating are usually used.  As  with  teratology
studies, fertility and breeding ability in captivity must be considered
when determining group size at each dose level. In addition, sufficient
litters  are required from the mating of the generation that is used as
the  source of parent animals  for the next generation.   Ideally, suf-
ficient litters should be available at each dose level to permit selec-
tion of future parental animals for the next generation on the basis of
1 male  and 1 female per litter.  Again, this factor must be considered
in  initial  determinations of  group size.  This  ideal is not  always
achievable,  since, if  some females  do not  produce offspring,  or  a
litter  contains animals of only  one sex, then group  size will dimin-
ish as the study proceeds.  Under these circumstances, the selection of
parental  animals for the next generation should be based on the widest
distribution permissible from the available litters.  It should also be
noted  that, if closely inbred strains are being used, the distribution
of  future parents becomes less critical. The limiting factor in multi-
generation studies is usually the logistics of the study  which,  since
animals  do not mate or  deliver to order, become  increasingly complex
with each mating and with each generation.

    Appropriate  group sizes in short-term studies depend upon the pur-
pose of the study. These studies are often designed to  provide  infor-
mation useful for the selection of dose levels to be used in subsequent
long-term  studies.  They are, however, sometimes used as the basis for
the  ADI. In these cases, increased group size is desirable. The short-
term study utilized for selection of doses in future studies requires a
minimum  of 10 animals of  each sex per  dose level in  rodent species.
Smaller group sizes (e.g., 4-6 of each sex per dose level)  are  gener-
ally accepted for non-rodent species such as the dog.

    In  considering  long-term/oncogenicity studies,  the protocol fre-
quently  separates the two  components of the  study.  The basic  group
size  is  based on  the oncogenicity study,  with ancillary groups  for
intermediate sacrifices and for investigation of haematological, clini-
cal  chemistry, and urinalysis  parameters.  Group sizes  must be  suf-
ficient  to ensure that adequate numbers of animals survive to the ter-
mination of the study.  Furthermore, the study design must be such that
the  sensitivity of the study,  i.e., its ability to  detect an adverse
effect, is acceptable. A recent publication by the International Agency
for  Research on Cancer (IARC)  [100] has addressed the  sensitivity of
carcinogenic studies. Tables 1 and 2, reproduced from this publication,

indicate  the numbers of animals of each sex per dose level required to
attain specified sensitivities in a two-dose-level study. (It should be
noted  that three dose levels are generally required for safety assess-
ments; see section 8.2.3).

Table 1.  Minimum group sizes required to ensure a false-negative
rate of 10% or lessa
--------------------------------------------------------
 Excess tumour       Tumour incidence in control group
incidence in test   ------------------------------------
  group (%)b         0%     1%     5%     10%     20%
--------------------------------------------------------
        1           819   2611   9084   16 287  28 110
        5           162    243    503      783    1232
       10            80    100    166      233     339
       15            53     61     90      119     163
       20            39     44     59       75      98
       25            31     34     43       53      67
--------------------------------------------------------
a  Based  on Fischer exact test (p < 0.05) with n animals in each of a con-
   trol  and a test group,  and assuming that all  animals respond indepen-
   dently.
b  Difference  between  the  response rates  in  the  test and  the control
   groups, respectively.

    As  can be seen from these data, test sensitivity is a major factor
in  determining  group size.   Furthermore,  these data  emphasize  the
importance of the background incidence of tumours in untreated animals,
which  in turn underlines the importance of species or strain selection
for oncogenicity studies (see section 8.2.1 and 8.2.6).

    Group  sizes utilized in  oncogenicity studies are  usually in  the
range of 50 to 100 animals of each sex per dose level.  For  additional
information on group sizes in oncogenicity studies, Annex 2  of  refer-
ence 176 should be consulted.

Table 2.  Number of animals per group required to obtain false-positive
rates of 5% and false-negative rates of 10% based on tests for
linear trend with three equally spaced doses
-----------------------------------------------------
             Tumour response rates
            ----------------------    Number of
Control     Low Dose    High Dose   animals/group
-----------------------------------------------------
0.02          0.04         0.06          420
0.02          0.07         0.12          112
0.02          0.12         0.22           44
0.10          0.12         0.14         1150
0.10          0.15         0.20          224
0.10          0.20         0.30           70
0.20          0.22         0.24         1860
0.20          0.25         0.30          328
0.20          0.30         0.40           93
-----------------------------------------------------

    The size of ancillary groups depends upon the basic study protocol.
The  utilization of a procedure  for interim kills requires  sufficient
animals to be sacrificed at each kill to provide adequate  numbers  for
histological  analysis.  Groups designated for haematological, clinical
chemistry,  and urine  analyses must  be of  adequate size  for  proper
statistical  analysis of the data  that are generated and  to allow for
anticipated  mortality as the study proceeds.  In general, a minimum of
10 animals of each sex per dose level should be available for each sub-
group required.

8.2.3.  Selection of dose levels

    Data obtained from acute toxicity studies can sometimes  assist  in
the  selection of appropriate dose levels for use in short-term feeding
studies.   Thus, when  acute toxicity  data are  available, it  is  not
unusual  for some fraction of the LD50 or  of the LD01 determined  from
acute toxicity studies to be employed.  When available, data on pharma-
cokinetics  or metabolism can be helpful in determining dose levels for
short-term  toxicity studies, particularly if there is evidence of bio-
accumulation of the test compound or of its metabolites, or if there is
evidence  of dose-dependent changes in detoxification. Since the deter-
mination  of a dose-response curve  is one of the  objectives of short-
term studies, at least three dose levels are normally required, as well
as a control.

    The selection of dose levels in long-term or  oncogenicity  studies
should  be based on the information derived from pharmacokinetic, phar-
macodynamic,  and short-term toxicity studies.  Frequently, the highest
dose level selected is the maximum tolerated dose (MTD), estimated from
short-term  feeding studies.  However, there are problems in attempting
to extrapolate data obtained at high dose levels in  experimental  ani-
mals  to probable human exposure levels.  This concept was discussed by
the 1987 JMPR, which made the following statement:

    "The Meeting was concerned at  (sic)   the difficulties  of  inter-
pretation of the results of long-term studies in which high  doses  had
been used. In reproduction and teratology studies the use of maternally
toxic doses has also caused concern.  The Meeting discussed the maximum
tolerated dose (MTD), which has been defined `as a dose that  does  not
shorten  life expectancy nor produce signs of toxicity other than those
due to cancer' and `operationally, as the maximum dose level at which a
substance  induces a decrement in weight gain of no greater than 10% in
a subchronic toxicity test' [176]. To identify agents with particularly
low orders of toxicity, exposure conditions are often maximized.  These
may  include the use of  very high doses and  gavage administration.  A
number of assumptions are implicit in the use of the MTD: (i)  the  ab-
sorption,  distribution, biotransformation, and excretion of a chemical
are not dose-dependent (that is, their kinetics are the same at low and
high doses); (ii) both the rate and extent of reparative processes (for
example,  DNA repair)  are independent  of dose  and of  the extent  of
damage; (iii) the response to a chemical is not age-dependent; (iv) the
dose-dependent response is linear; (v) doses tested in animals need not
bear any relationship to human exposure levels." [78, p. 3].

    At the 1987 JMPR meeting, these assumptions were questioned. Thus:

(i)     absorption,  distribution, biotransformation, and excretion  of a
        compound  are dependent on several factors, e.g., physicochemical
        properties, degree of protein binding, bioavailability, and satu-
        ration  of routes of metabolism  (resulting in variations in  the
        proportions of different metabolites or complete changes in meta-
        bolic pathways with dose (e.g., 2-phenylphenol));

(ii)    DNA  repair  is dependent on dose and/or degree of damage both  in
         vivo  and  in vitro  [7; 139];

(iii)   the response to many chemicals is age-dependent (e.g., acute tox-
        icity of DDT or malathion [116]);

(iv)    the  US NCTR study on 2-acetylaminofluorene (the megamouse study)
        did not demonstrate a linear response for bladder tumours [99];

(v)     results  of studies at dose levels many orders of magnitude above
        the  level of human exposure  to pesticide residues in  food have
        little relevance to the safety assessment of  pesticide  residues
        in the diet (e.g., 2-phenylphenol [75]).

    The JMPR has indicated that, instead of using the MTD to select the
top-dose  level, the use of properly designed biotransformation studies
over a range of doses (including human exposure levels) may  provide  a
more rational basis for dose selection in long-term animal studies.

8.2.4.  Test duration

    In  certain studies, e.g., teratology  and multigeneration studies,
the  duration of the study  is determined by the  biological character-
istics of the test species or strain.  The duration of these  types  of
studies is considered in sections 8.3.5.1 and 8.3.5.2.

    The  duration of other  studies is determined,  to some extent,  by
definition.  Thus, an acute study was originally defined as  a  single-
dose  study, observation of  the treated animal  continuing for 2  to 4
weeks following dosing [37].  The concept of an acute study has changed
slightly through the years; it is now considered to be a study  of  the
effects  of a dose administered  either singly or on  several occasions
over a period of 24 hours.  The observation period is  usually  14 days
[124].

    A short-term study has been defined as having a duration lasting up
to  10%  of  the animal's lifespan [31], 90 days in rats and mice, or 1
year in dogs.  It has also been defined as a study covering  less  than
half the animal's lifespan [37].

    Long-term/oncogenicity studies are usually defined as studies last-
ing for the greater part of the lifespan of the  animal  [176, p. 113].
Studies  of this type usually  fall into one of  two categories: (a)  a
specific  duration; (b) until mortality  in the most susceptible  group
attains a fixed level, usually 80%. Fixed-term studies vary in duration

with species and strain, depending on lifespan.  The  late  development
of  many  types  of tumours  requires  that  the study  be permitted to
continue  as  long as  possible. In addition,  reduced liver or  kidney
function  with increasing age and  a consequent increase in  the plasma
levels  of toxins  in older  animals may  result in  manifestations  of
toxicity  not otherwise seen.  However, low survival  rates and  normal
geriatric  changes may complicate  study interpretation and  limit  the
sensitivity of comparison between groups. Thus, the goal of a long-term
oncogenicity  study is to determine  the optimum balance between  these
factors.

    The report of the 1967 WHO Scientific Group [169] concluded that it
is  better to terminate  toxicity studies before  the complications  of
senescence  arise in the test  animals.  Although many effects  of sen-
escence  are now  recognized, further  data are  still required  before
scientifically supportable generalizations on the duration of long-term
studies are possible.  If a finite mortality is the definitive endpoint
of the study, then care must be taken:

 *  to ensure that mortality does not exceed the predetermined limit in
    any group (including the control);

 *  to consider whether the mortality arises because of tumour develop-
    ment;

 *  that autopsies are performed as soon as possible on  animals  dying
    during  the study,  thereby avoiding  loss of  information  due  to
    autolysis or cannibalism.

8.2.5.  Pathological procedures

    Three  steps are involved in the pathological examination of exper-
imental animals:

 *  gross pathological examination at the time of post-mortem;

 *  histopathological examination of the tissues;

 *  a review of these data by an independent pathologist.

For the last of these steps, JMPR has recommended to WHO that  a  mech-
anism  should be established to permit independent pathological assess-
ment  of questionable or disputed findings that are brought forward for
review [65].

    Pathological  examinations and the way  in which they are  reported
can give rise to a number of problems.

    In acute toxicity studies, gross pathological examination  of  ani-
mals both dying during the study and killed at the termination  of  the
observation  period is desirable, because  one of the objectives  of an
acute  oral study is to  obtain information on potential  target organs
and  on possible dose levels to be used in subsequent repeated adminis-
tration studies. Such information should, therefore, be  as  comprehen-
sive as possible and should include gross pathology examination. Unfor-
tunately, such examinations are not always performed or reported.

    In  short- and long-term studies,  pathology is a  major  endpoint.
However,  the  presentation of  pathological  data is  often confusing.
Gross pathological data (frequently reported separately from  the  data
on  histopathological  examinations)  are difficult  to  correlate with
histopathological  findings. It is not unusual to find gross pathologi-
cal  notations of "lumps and bumps", petechial haemorrhages, etc., in
an  organ, for which the  histopathological notation is "normal".   A
high  frequency of such  apparent discrepancies in  the absence of  any
comment is unsatisfactory.  The explanation may be either a  mix-up  in
specimens, or that the sections cut for  histopathological  examination
failed to intersect a "lump or bump".  Either way, the study probably
has  not achieved its objectives.  Partial resolution of these problems
can sometimes be achieved by cutting multiple sections  throughout  the
area of the gross lesion.

    Pathological  terminology is also confusing since several different
names  may be used for the same lesion. Therefore, an adequate descrip-
tion  of the lesion  and an indication  of its size  and  frequency  is
essential  in pathological reports.  Furthermore, a standard  classifi-
cation  of lesions should always  be used in reports,  e.g., the Inter-
national Agency for Research on Cancer (IARC) Tumour Register [183].

    A high incidence of tissue autolysis is occasionally noted  in  the
histopathological  reports.   Even  fairly advanced  autolysis does not
necessarily preclude the identification of a tumour, despite  the  fact
that  the specific cellular  characteristics are obscured  by autolytic
activity.   Although such tumours cannot always be reported in adequate
detail, their presence can be recorded.

    The  percentage incidence of tumours is of importance in the evalu-
ation, but data are often such that it is extremely difficult to deter-
mine how many animals were actually examined with respect to a specific
tissue.   In the absence  of such information,  although the number  of
diagnosed tumours is known, percentage incidence cannot be determined.

    The precise site of tumours may be of major importance. In a recent
evaluation of folpet, tumours in the duodenum and jejunum of the exper-
imental animals were noted and a probable mechanism for  the  induction
of these tumours was proposed [77].  The data were inadequate to deter-
mine  whether these tumours were a "spill-over" (related to the irri-
tant  properties of the compound) or whether they were induced indepen-
dently of the postulated mechanism.  Additional data were  required  to
resolve this problem and, hence, to arrive at a valid evaluation of the
safety of the compound [76].

    The increasing emphasis on mechanism of action in  evaluating  tox-
icity  studies  may  be  supported  by  histopathological  examinations
utilizing  special stains for  identification of cell  elements  (e.g.,
Sudan III  for  fat  droplets) or  involving  histochemical techniques.
Electron  microscopic examination should  also be considered  when bio-
chemical  or other data indicate the need to examine cell organelles or
membrane structures.

    Many  protocols for multigeneration studies require histopathologi-
cal  examination of a representative  selection of pups at  one or more
points  in the study.   The need for  such examination is  questionable
(see section 8.3.5.1).

8.2.6.  Historical control data

    In almost all toxicity studies, quantitative and  qualitative  data
from several treated animal groups are compared with data from  one  or
more concurrent untreated or vehicle-treated control groups. The appli-
cation of appropriate statistical procedures will indicate,  with  some
predetermined  probability, which of  the observed differences  are not
likely to be attributable to chance.  In such procedures, the data from
untreated animals become the standard reference.  Yet it is known that,
even  with random assignment  of individual animals  to each group  and
strict  adherence to GLP, the  incidence of spontaneous neoplastic  and
other morphologic lesions is often highly variable among control groups
of  the same species and strain in different studies conducted within a
single laboratory, as well as in different laboratories [119; 152; 157;
167; 168].

    To  be indicative of  a treatment-induced change,  the  differences
between  control  and  treatment  groups  should  show  a dose-response
relationship  and delineate a trend away from the expected norm for the
particular  species and strain of experimental animal used.  Since data
from  the concurrent control group  are used as the  standard reference
for treatment group responses, and since control data in any particular
study  may be unpredictably variable, qualitative and quantitative cri-
teria must be used to evaluate whether the concurrent control data con-
stitute  the typical species/strain  pattern, i.e. whether  they corre-
spond  to an expected  norm.  Historical control  data relating to  the
specific  species/strain  used in  the  study provides  such evaluation
criteria [23; 126; 154; 155]. This type of information must  be  viewed
as an auxiliary aid to interpretation of data from the study. It should
not be used as a complete substitution for concurrent control data.

    The  following have been proposed for use in the evaluation of car-
cinogenicity  data by a Task Force of Past Presidents of the Society Of
Toxicology [155] and may have utility for the evaluation of other forms
of toxicity as well:

 *  If  the incidence rate or  other observed effect in  the concurrent
    control  group is lower than  in the historical control  groups but
    these  same effects in the treated groups are within the historical
    control range, the differences between treated and  control  groups
    are not biologically relevant.

 *  If  the incidence rates  or other observed  effects in the  treated
    groups are higher than the historical control range but  not  stat-
    istically  significantly greater than the  concurrent control inci-
    dence,  the conclusion would be that there is no relation to treat-
    ment (but with the reservation that this result could  have  arisen
    by chance or because of flaws in the assay and may therefore  be  a
    false negative).

 *  If  the incidence rates  or other observed  effects in the  treated
    groups  are significantly greater  than in the  concurrent controls
    and greater than the historical control range, a  treatment  effect
    is  probably  present  which is  unlikely  to  be a  false positive
    result.

    The  best  historical  control data  are  obtained  using the  same
species and strain, from the same supplier, maintained under  the  same
routine conditions in the same laboratory that generated the study data
being  evaluated.  The data should be from control animals from contem-
poraneous  studies.  Statistical procedures can  be used to relate  the
overall historical incidence to that in a specific study. However, this
leaves  much to be desired  since the incidence of  spontaneous lesions
and  the averages of  quantitative data can  vary considerably  between
groups  of animals. This type of variation is not apparent if the inci-
dence in combined historical control animals is used [157].

    To  assess variability, historical control data should be presented
as  discrete group incidences,  segregated by sex  and age and  updated
with each new study that is performed [135]. It is also  highly  desir-
able that additional information on each discrete control group be made
available.  This information should include the following:

 *  identification  of  species,  strain,  name  of  the  supplier, and
    specific  colony identification if the  supplier has more than  one
    geographical location;

 *  name  of the laboratory  and time during  which the study  was per-
    formed;

 *  description  of  general conditions  under  which the  animals were
    maintained,  including the type or  brand of diet and,  where poss-
    ible, the amount consumed;

 *  the  approximate age, in days, of the control animals at the begin-
    ning of the study and at the time of killing or death;

 *  description  of the control group mortality pattern observed during
    or  at the end of the study and of any other pertinent observations
    (e.g., diseases, infections);

 *  name  of the pathology laboratory and the examining pathologist who
    was  responsible  for  gathering and  interpreting the pathological
    data from the study;

 *  what tumours may have been combined to produce any of the incidence
    data.

8.3.  Conduct and Evaluation of Different Types of Studies

8.3.1.  Short-term and long-term toxicity studies

    Both  short- and long-term feeding studies utilize the same method-
ologies  and differ only in  the duration of the  test.  The parameters
investigated usually include body and organ weights,  clinical  chemis-
try and haematological effects, and gross and  histopathological  exam-
inations.

    Short- and long-term toxicity studies are designed to determine the
NOAEL for the test substance and to provide information relevant to the
determination  of the safety factor  to be applied in  extrapolating to
humans (see section 2.2).

    The  majority  of  protocols  available  for  toxicity  testing are
intended  as  guidelines, thus  leaving the final  study design to  the
individual investigator.  It is usually the case that by the  time  the
long-term  toxicity studies are  initiated, the investigator  will have
access  to the information from earlier studies (acute, short-term, and
metabolic  studies) and hence will  be able to judge  the most suitable
design for long-term studies.

    The  selection of species and of dose levels have been discussed in
sections 8.2.1 and 8.2.3.

    In  long-term oral toxicity studies, the test substance is normally
incorporated in the diet and administered for the majority of the life-
time  (see section 8.2.4), on a daily basis (7 days per week). Lifetime
exposure  is required due to  the fact that, during  the aging process,
factors  such  as altered  tissue  sensitivity, changing  metabolic and
physiological  capability, and spontaneous disease may alter the nature
of  the toxic response [171].  Spontaneous diseases include age-related
increases in the incidence of heart disease, chronic renal failure, and
neoplasia, which are observed in most mammalian species.

    To ensure that the objectives of the long-term toxicity  study  are
achieved,  statistical principles must  be used to  determine  adequate
group  sizes for reducing  the incidence of  false positives and  false
negatives to a minimum (section 8.2.2).  Similarly, the use  of  random
numbers  or comparable statistical techniques, both for allocating ani-
mals to experimental groups and for ensuring that the  distribution  of
cages  of animals within housing racks is random, is essential to mini-
mize  bias  in selecting  animals  and minimize  possible environmental
effects  (e.g., temperature, humidity,  light) within the  animal house
[176, Annex II].

    In  conducting  these studies,  the  principles of  GLP  [161; 160]
should  be followed to ensure both acceptable conditions of animal hus-
bandry  and adequate conduct  of the experiment.   Full records on  all
animals must be kept, detailing all observations, results of  any  lab-
oratory  techniques  (e.g.,  bleeding and  subsequent haematological or
clinical  chemistry studies), and  information on pathological  examin-
ations at the end of the study.

    Since  one  of the  objectives of a  feeding study is  to determine
changes  in  toxic  signs and  manifestations,  it  is  axiomatic  that
periodic detailed examinations be performed on at least a proportion of
the experimental animals.  Non-invasive procedures such as the measure-
ment  of  body  weight  and  food  consumption,  palpation, behavioural
observations, and assessment of general condition of  the  experimental
animals  (both control and  exposed) can be  performed regularly.   The
frequency  of  handling may  be limited by  the potential for  creating
stress  in the experimental  animal, particularly if  the frequency  is
increased  towards  termination  of  the  study  (i.e.  in oncogenicity
studies).   Urinalysis,  the remaining  routine non-invasive technique,
should  also  be  performed regularly,  but  at  longer time  intervals
(usually at 3, 6, 12, 18, and 24 months in rat studies). The process of
collecting  urine may cause stress,  depending upon the type  of caging
used.   Thus, if animals are housed one per cage, the use of metabolism
cages  for single animals will  induce minimal stress.  However,  where

multiple  caging is  the norm,  sudden isolation  can induce  a  stress
condition, with consequent physiological changes in the  animal.   This
should be considered when the results of urinalysis are interpreted.

    In  general, urinalysis utilizes insufficient animals (often as few
as  five of each sex per dose level) or an insufficient acclimatization
period  in the metabolism cage(s) to be very useful, since variability,
even in the same individual, can be high [150].  Even if the numbers of
animals  or acclimatization time is  adequate, further problems may  be
encountered.  Dissolved carbon dioxide may dissipate and thus alter pH,
the  appearance  of  the urine may vary according to the time of day at
which sampling takes place, and bacterial concentration and composition
may change even if preservatives are used.  However, useful data can be
obtained in clinical chemistry studies on urine, such as concentrations
of proteins, ketones (elevated in starvation or with  low  carbohydrate
diets),  glucose  (diabetes,  hypoglycaemia), and  porphyrins (elevated
with liver disorders), osmolality (reflecting kidney function, but data
on  water consumption is needed  to interpret kidney concentration  ef-
fects), urinary haemoglobin (often elevated in toxic  situations),  and
high crystal content (possibly predictive of kidney or bladder stones).
In  addition, periodic urine collection and analysis for metabolites of
the  test  substance may  yield data on  age-related changes in  metab-
olism.

    Invasive  techniques  (usually  involving blood  sampling) normally
utilize a pre-designated ancillary group of animals identified for that
purpose  prior to the onset of the study.  Thus the effects of repeated
bleeding  at specific intervals (the intervals usually being similar to
those  delineated  for  urinalysis) on  terminal  pathological manifes-
tations  are recognizable in animals in the ancillary group. The ancil-
lary groups (which must allow for mortality with increasing duration of
the  study) should comprise  at least 12 animals  of each sex  per dose
level for each group to provide groups of at least 10 animals  of  each
sex  per dose  level for  haematological and  other clinical  chemistry
examinations.

    End-points normally measured in haematological examinations include
erythrocyte  counts,  leucocyte counts,  differential leucocyte counts,
haemoglobin,  haematocrit,  and  platelet and  reticulocyte counts.  In
addition,  erythrocyte  fragility, sedimentation  rate, and coagulation
factors are frequently measured and bone marrow is examined.

    End-points  traditionally  examined by  clinical chemistry measure-
ments include:

 *  serum bilirubin (liver and haematological effects);

 *  serum glucose;

 *  lactate  dehydrogenase (a non-specific  indicator of tissue  damage
    seen  in myocardial infarction, renal toxicity, pulmonary embolism,
    and pernicious anaemia);

 *  serum  alkaline phosphatase (which,  it should be  noted, decreases
    with  age and with  nutritional status, and  cannot be regarded  as
    specifically  indicative of a disease  process because of its  wide
    distribution in many organs);

 *  alanine  aminotransferase (previously serum glutamic-pyruvic trans-
    aminase) and aspartate aminotransferase (previously serum glutamic-
    oxalic transaminase) (both indicators of liver toxicity);

 *  amylase  (increased  in  renal insufficiency  and pancreatitis, de-
    creased with hepatobiliary toxicity);

 *  creatinine (renal failure);

 *  creatinine  phosphorylase (elevated with myocardial  infarction and
    lung disorders);

 *  cholinesterase (decreased by organophosphates and carbamates);

 *  serum protein;

 *  blood urea nitrogen (elevated with renal toxicity,  depressed  with
    liver toxicity);

 *  serum  electrolytes (see reference  [93] for a  comprehensive  dis-
    cussion of the interpretation of clinical chemistry measurements).

    It  has been  proposed that  clinical chemistry  studies  be  aimed
mainly  at known target organs  that are identified in  short-term tox-
icity studies [150].  However, long-term toxicity studies may result in
changes  in the degree of toxicity to specific organs (e.g., adaptation
of  initial target organs, secondary  effects arising from the  initial
effects  noted in short-term studies, and changes in circulating enzyme
or hormone levels due to tumour development).   Consequently,  limiting
clinical  chemistry  studies  to  parameters  suggested  by  short-term
studies is not encouraged.

    In  certain cases, clinical chemistry  studies may be necessary  to
investigate endocrine organ function.  For example, delayed  growth  or
metabolic dysfunction may be the result of thyroid dysfunction, induced
either by direct toxic action of the test material on the thyroid or by
decreased  thyrotropin  release  by the  pituitary.  Similarly, altered
liver  carbohydrate metabolism may  be due to  pancreatic  dysfunction,
adrenal dysfunction may result in disturbed kidney function, changes in
fertility or reproductive performance may be mediated by  gonadal  hor-
monal  changes, and tumour formation may arise due to enhanced hormonal
stimulation,  either  in endocrine  organs  or in  non-endocrine organs
(e.g.,  the mammary gland).  A  recent publication [163] discusses  the
practical  problems  and  describes methods  of investigating endocrine
toxicity.

    While  clinical  chemistry data  are  often non-specific,  they  do
permit the progress of an effect to be followed  in vivo.   When  histo-
pathological data are available (usually only at the times  of  interim
and  terminal  sacrifices),  they  may  supersede  clinical   chemistry
findings.

    The pathological data derived from feeding studies are of paramount
importance.   Such data in long-term feeding studies should be obtained
from at least two specified sacrifice periods, one (usually  a  minimum
of  10 rats of each sex per dose) at a point in time prior to the onset
of  senescence and the second at termination of the study.  All animals

(including all non-scheduled deaths, or animals sacrificed in  a  mori-
bund condition) should be examined at least grossly, and tissues should
be  preserved where possible  for histological examination.   To  avoid
undue  loss of tissues due  to autolysis, animals should  be checked at
least 2 or 3 times daily. A high incidence of autolyzed animals results
in  loss of data and  raises concerns about the  quality of the  animal
husbandry and standard of laboratory expertise.

    Histopathological examination should cover a wide range  of  organs
and  tissues.  However, recognizing the  economics of histopathological
examinations,  examination of tissues from mid- and low-dose groups may
be  limited to those tissues  where differences occurred between  those
from control and high-dose groups.

    The  NOAEL is frequently based  on the results of  the pathological
examination of the test animals.  The initial (gross) examination notes
any   abnormalities  in  the  tissues   (e.g.,  masses,  discoloration,
necrosis). This is followed by removal and weighing of specific organs.
Because  of  the  high rate of autolysis of some organs (e.g., the kid-
ney), removal, weighing, and preservation should be performed  as  rap-
idly  as  is consistent  with accurate work.   Paired organs should  be
weighed  separately  to  avoid  inaccuracies  arising  from  unilateral
lesions  (e.g., tumours) that are not grossly visible.  Organs normally
weighed  include the liver,  kidneys, heart, adrenals,  gonads, spleen,
and  brain.  Results of such  weighings should be reported  as absolute
weights, and also as a ratio to body weight and to brain weight.

    In assessing data from short- and long-term toxicity  studies,  the
following factors should be considered:

 *  Comparison  of mean values of  body weights for specific  groups of
    animals  may not  necessarily be  the most  appropriate  method  of
    detecting potentially toxic effects.  The use of body  weight  gain
    differences  should also be considered,  as should changes in  food
    intake.

 *  Clinical chemistry data can provide a useful indicator  of  toxico-
    logical effects. However, they are limited in sensitivity and frank
    pathological changes are often observed at dose levels less than or
    equal to those resulting in significant clinical chemistry effects.
    When  studies  include  a post-treatment  recovery period, clinical
    chemistry data are frequently of value in assessing the progress of
    recovery.  In many cases, the specificity of the test system, e.g.,
    serum  alkaline  phosphatase,  is insufficient  to  permit  precise
    identification  of target tissues or organs.  In other cases, e.g.,
    acetylcholinesterase  measurements, clinical chemistry data  may be
    the major toxicological effect measured.

 *  Changes in a single haematological parameter unsupported by further
    changes  in  other  haematological parameters  or  by  pathological
    changes in bone marrow or spleen are rarely of  toxicological  sig-
    nificance.

 *  Organ weight changes should always be examined on an  absolute  and
    organ/body  weight ratio basis.   Organ/body weight ratios  can  be

    misleading when a change in body weight occurs.  Mathematical  pro-
    cedures  for correcting for  this situation exist.   When the  body
    weight  per se  is affected, there  is a tendency  to place  greater
    reliance on organ/brain weight ratios.

 *  Gross   and  histopathological  examinations  should  be  carefully
    checked for correspondence. A detailed description of the lesion(s)
    or photomicrographs may be necessary since the terminology used for
    certain  lesions is variable  and there is  some degree of  subjec-
    tivity in the interpretation of lesions (see also section 8.2.5).

    No  discussion of toxicity studies  would be complete without  some
consideration of the dose actually ingested.  Dose-level  selection  is
discussed  in section 8.2.3 and stability  of the test material  in the
diet  in section 4.  Assuming that the stability is acceptable and that
the homogeneity of the test material in the diet has been measured on a
number  of occasions during the study, one major variable remains, i.e.
the  food consumption per unit  of body weight.  This  varies with age,
being  highest in the  young animal and  decreasing as the  animal ages
(the  special  case  of  lactating  females  is  discussed  in  section
8.3.3.1). When data are available, the actual dose ingested  is  calcu-
lated from the concentration of test substance in the diet and the food
consumption.   Under these circumstances, the JMPR evaluation indicates
the NOAEL as X ppm  equal to Y mg/kg body weight per day, and is usually
based on the mean intake of the test substance over the  lifespan.   In
other  cases, when the calculation  of intake in mg/kg  body weight per
day  is not feasible because  of inadequate food intake  data, the JMPR
evaluation uses the standard conversion factors for ppm to  mg/kg  body
weight  per day ([114], reproduced as Annex II in this monograph), this
being reported as X ppm  equivalent to Y mg/kg body weight per day.  The
former method is preferable.

8.3.2.  Carcinogenicity studies

    From  its inception, JMPR has  recognized the need to  evaluate the
carcinogenicity of pesticide residues in food [32].  It has adopted the
principle  that carcinogenicity testing should utilize adequate numbers
of animals, generally of two or more species (e.g., rat and mouse), and
a suitably high dose level of the substance should be fed for the life-
time of the animals [33].

8.3.2.1     Background

    The  1977 Joint Meeting noted that an evaluation of carcinogenicity
should be undertaken routinely for:

 *  pesticides whose use results in substantial residues in  crops  di-
    rectly or indirectly used for human food;

 *  pesticides with structural similarity to known carcinogens;

 *  pesticides  that are metabolized  to, or leave  residues that  are,
    known carcinogens or closely related to such compounds;

 *  pesticides that give rise to early pathology suggestive  of  poten-
    tial tumorigenicity;

 *  pesticides with pharmacokinetic properties "suggestive of covalent
    binding to tissues" or bioaccumulation [57].

    JMPR has sometimes recommended that certain compounds should not be
used  where residues may occur in food, due to their potential carcino-
genicity  (e.g., hexachlorobenzene, captafol).  At  other times, either
TADIs  or ADIs have been set, even though there was limited evidence of
carcinogenicity  in animals (e.g., several chlorinated organic insecti-
cides).  Overall, JMPR has maintained the philosophy that  a  pesticide
for  which there  is limited  evidence of  carcinogenicity  should  not
necessarily be prohibited (see section 8.3.2.7).

8.3.2.2     Routes of exposure

    The  oral route of administration  is the most appropriate  one for
determining  in  experimental  animals the  carcinogenic  potential  of
pesticides leaving residues in food.

    The 1966 JMPR [38] noted the comments of the report of a WHO Scien-
tific Group [169] concerning experimentally induced local sarcomas that
apparently  result  from  the  physical  characteristics  of  the  test
material.  This Joint Meeting concluded that for the routine testing of
pesticide residues, the subcutaneous route is not  generally  appropri-
ate.  The occurrence of local sarcomas following subcutaneous injection
should  not alone be considered  sufficient evidence of a  carcinogenic
hazard  following  ingestion [169].   It  does, however,  indicate that
further studies would be desirable.

    The  1989 JMPR noted that  severe local effects may  interfere with
the  interpretation of data, e.g.,  the production of forestomach  epi-
thelial  hyperplasia  and  papilloma formation  following  the adminis-
tration  of gastric  irritants.  It  was recommended  that  methods  of
administration other than feeding be justified.

8.3.2.3     Commonly  occurring tumours and factors influencing tumour
            incidence in different species

    Some rodents commonly used for  in vivo bioassays exhibit high inci-
dences of some tumours. In evaluating toxicological data, it is import-
ant  to determine whether  an increased incidence  of tumours and/or  a
decreased time to tumour in exposed animals are related  to  treatment.
The  incidence of such tumours in control animals may vary considerably
with  time.  As an example, ten years ago the occurrence of Leydig cell
tumours  in rats was rarely  reported. By 1987, some  laboratories were
reporting  that the occurrence of such tumours sometimes reached 50% in
control rats. It is not known whether this change in incidence  is  due
to a genetic shift in certain rat strains or to more careful pathologi-
cal examinations of the rat testes. The importance of such  factors  is
discussed in section 8.2.6.

    As noted in Environmental Health Criteria 70 [176]:

    "The evaluation of studies in which commonly-occurring tumours are
a complicating factor needs careful individual assessment.  The tumours
that  have given rise to the most controversy in recent years are hepa-
tomas  (particularly in the mouse),  pheochromocytomas in the rat  (see
below), lymphomas and lung adenomas in the mouse,  pancreatic  adenomas

and  gastric papillomas in  the rat, and  certain  endocrine-associated
tumours, including pituitary, mammary, and thyroid tumours in both rats
and  mice.  Some of these tumours, such as hepatomas and lung adenomas,
may occur in the majority of untreated animals.

    "With  the  exception  of lymphomas,  some  of  which are  virally
associated, the endocrine-associated tumours, and possibly hepatomas in
high-incidence strains of mice, which may involve oncogenes [82], there
is  no  clue  as to the origin of tumours that occur commonly in exper-
imentally-used  rodents. Indeed, there  is not even  any cogent  specu-
lation  as  to  the mechanisms  by  which  these tumour  incidences are
increased." [176, p. 44].

    Since  the publication of Environmental Health Criteria 70, a great
deal  of additional research  has been carried  out on the  etiology of
cancer, particularly with respect to the important role of oncogenes in
neoplasia.  Nevertheless, additional investigation into the initiation,
promotion,  and progression of cancer is necessary to assist the incor-
poration of such mechanistic considerations into human  hazard  assess-
ment for carcinogens.

    JMPR has generally considered it unwise to classify a compound as a
carcinogen  solely on the basis of an increased incidence of tumours of
a  kind  that commonly  occur spontaneously in  the species and  strain
under  study and at a frequency that may seriously reduce the statisti-
cal power of the study.  Data are usually required in one or preferably
two alternate species, and the overall evidence is then considered.

    The significance of mouse liver tumours was first considered by the
1970  JMPR [46].   These tumours  were then  becoming  more  frequently
observed  in carcinogenicity studies, especially  following exposure to
the  chlorinated organic pesticides. Subsequent Joint Meetings [46; 48;
52; 53; 57; 72; 74] have also considered the problem of pesticides that
induce  mouse hepatic tumours.  A  number of hypotheses concerning  the
etiology of mouse liver tumours have been considered by  JMPR  [2; 132;
133; 162; 170].   Biochemical  differences  between the  mouse and many
other  species, including humans, are  highly pertinent to mouse  hepa-
tomas  [72].  In addition,  degranulation of endoplasmic  reticulum  is
known  to be  associated with  carcinogenesis in  the mouse  [130; 131;
134].  Both dieldrin and phenobarbitone degranulate the  hepatic  endo-
plasmic reticulum of CF1 mice, a strain susceptible to dieldrin-induced
tumorigenesis, but do not degranulate the endoplasmic reticulum of LACG
mice, a non-susceptible strain, nor that of rats or humans. The current
position of JMPR is that mouse liver tumours are of little relevance in
predicting human cancer risk. It is inadvisable to classify a substance
as  likely to  be a  carcinogen to  humans solely  on the  basis of  an
increased incidence of mouse liver tumours [72].

    Other  tumours occurring with a high relative frequency are adrenal
medullary lesions in rats. As noted in Environmental Health Criteria 70
[176]:

    "An  overview of the literature  indicates that untreated rats  of
various  strains  may exhibit  widely  differing incidences  of lesions
described  as `pheochromocytomas' [69; 141; 142].   There are no  clear
criteria for distinguishing between prominent foci of  hyperplasia  and

benign neoplasms, and pathologists differ in the criteria that they use
for  distinguishing  between  benign and  malignant  adrenal  medullary
tumours.

    "Rats fed  ad libitum  on highly nutritious diets tend to develop a
wide  variety of neoplasms,  particularly of the  endocrine glands,  in
much higher incidences than animals provided with enough food  to  meet
their  nutritional needs  but not  enough to  render them  obese.   The
adrenal  medulla is just one of the sites affected by overfeeding. Con-
trolled  feeding . . . reduces the life-time expectation  of developing
either hyperplasia or neoplasia of the adrenal medulla in rats." [176,
p. 44].

    Thus,  food intake can be  a major factor in  experimental carcino-
genesis.  Restricted food intake in rodents is known to  increase  life
expectancy  and to reduce the incidence of naturally occurring and some
induced tumours.  However, restricted dietary intake may  also  require
other  considerations (e.g., study duration)  be taken into account  in
designing protocols for carcinogenicity studies.

8.3.2.4     Pathological classification of neoplasms

    The  need  for guidelines  leading  to consistency  in pathological
diagnosis  is apparent.  As noted  in Environmental Health Criteria  70
[176],  tumours should be classified and analyzed on the basis of their
histogenic  origin  in order  to  prevent different  malignant tumours,
occurring  in the same  organ, from being  grouped inappropriately  for
statistical analysis.  This is particularly important when brain tumour
incidences  are being considered, since different tumour types are fre-
quently, but incorrectly, grouped together for analysis.

    Accurate determination of histogenic origin is clearly important in
determining the significance of benign tumours, since this is  often  a
complicating  factor in assessing carcinogenicity studies.  As noted in
Environmental Health Criteria 70:

    "If  benign and malignant tumours are observed in an animal tissue
and there is evidence of progression from the benign to  the  malignant
state, then it is appropriate to combine the tumour types  before  per-
forming  statistical analysis. It is, however, still advisable to exam-
ine  incidences of benign and malignant tumours separately.  Assessment
of  the relative numbers of benign and malignant tumours at the various
dose  levels  in  the study can help determine the dose response of the
animal to the compound under test.  On the other hand, if  only  benign
tumours are observed and there is no indication that they  progress  to
malignancy,  then, in most cases, it is not appropriate to consider the
compound  to be a  frank carcinogen, under  the conditions of  the test
(this finding may suggest further study)." [176, pp. 44-45].

    The  1983 JMPR [70]  indicated possible approaches  (e.g.,  interim
sacrifice  of satellite groups, morphometric measurement of tumours) to
the problem of latency, which is an important component of  the  evalu-
ation of carcinogenic potential.

8.3.2.5     Evaluation of carcinogenicity studies

    Various  classification  schemes  have been  proposed for potential
chemical carcinogens.  For example, IARC Working Groups  evaluate  evi-
dence on the carcinogenicity of agents in humans and describe  them  in
standard  terms of "sufficient", "limited",  or "inadequate" evi-
dence  of  carcinogenicity or  "evidence  suggesting lack  of carcino-
genicity". These categories refer only to the strength of the evidence
that an agent is carcinogenic and not to the extent of its carcinogenic
activity (potency) nor to the mechanisms involved.  Finally  the  total
body  of evidence (including,  where relevant, supporting  evidence  of
carcinogenicity  from other data such  as genetic and related  effects)
from humans and experimental systems is taken into account and an agent
is categorized into one of four groups [101]:

    Group 1: carcinogenic to humans,

    Group 2A: probably carcinogenic to humans,

    Group 2B: possibly carcinogenic to humans,

    Group 3: not classifiable as to carcinogenicity to humans, and

    Group 4: probably not carcinogenic to humans.

    JMPR  considers, where possible, both carcinogenic potency and bio-
logical  relevance in its evaluations.  It does not utilize a classifi-
cation  system for carcinogenic pesticides, preferring to evaluate com-
pounds  on a case-by-case basis,  rather than allocating a  compound to
"the best fit" position in existing classification systems.

8.3.2.6     Extrapolation from animals to man

    Different approaches to the extrapolation of animal carcinogenicity
data  to humans have been utilized. One of these approaches relies on a
knowledge  of  the comparative  metabolism in the  test species and  in
humans. If data are available indicating that a crucial metabolic path-
way  is overloaded, an increase  in tumour incidence occurring  only at
dose  levels exceeding those resulting in the overload, then confidence
in  the NOAEL is increased.   If comparative metabolic data  indicate a
similar situation in humans, the task of extrapolation is simplified.

    Another approach is based on pathological considerations. When data
are  available to  demonstrate a  fixed pattern  of tumour  development
(e.g.,  progression from hyperplasia,  through nodular hyperplasia  and
benign  tumour, to  malignant tumour),  then a  dose level  below  that
resulting in the initial pathological change is unlikely to be carcino-
genic (see also section 13.5).

    In  1969,  JMPR  [44]  urged  the  consideration  of  dose-response
relationships  and possible NOAELs for carcinogens.  The 1974 JMPR [53]
adopted several of the principles put forth by a WHO  Scientific  Group
[170] concerning preliminary changes such as hyperplasia,  the  effects
of hormonal compounds, and tumours apparently induced by  the  physical

character of the carcinogen. The 1974 Joint Meeting noted that prelimi-
nary changes such as hyperplasia are associated with a number  of  car-
cinogenic  compounds.  Furthermore, some chemicals apparently give rise
to  neoplasms only  after the  induction of  a particular  pathological
effect [19].

    The  1983 JMPR recognized that  most of the mechanisms  of chemical
carcinogenesis were not fully understood.  In view of  the  uncertainty
surrounding  the use of various mathematical models for carcinogenicity
assessment, the Meeting decided that the use of safety factors remained
a  reasonable approach.  It  also recognized the  importance of  taking
into  account all biological activities of such agents in arriving at a
safety  assessment.   This pragmatic  approach is used  by JMPR in  the
absence of satisfactory alternatives (see section 9.2).

    In  determining  the acceptable  level  of pesticide  residues  for
humans,  the safety factor utilized reflects the confidence in the data
base and the degree of concern for the toxic effect. This is especially
true for carcinogenic effects.  Where there is the need for a very high
safety  factor due to concern about the safety of the pesticide, it may
be  prudent to recommend  that the pesticide  should not be  used where
residues in food may occur.

8.3.2.7     Principles

1.  An  evaluation of carcinogenicity  should be undertaken  for  those
    pesticides that:

     *  may give rise to substantial residues in crops  used  directly
        or indirectly for human food;

     *  have a chemical structure similar to known carcinogens or give
        rise  to metabolites or residues that are known carcinogens or
        closely related compounds;

     *  give rise to histological changes that are suggestive  of  po-
        tential neoplasia.

2.  The  oral route of  administration to experimental  animals is  the
    most  appropriate  route  for determining  the  carcinogenicity  of
    pesticide residues in food.

3.  All available data should be considered in the evaluation  and  as-
    sessment of carcinogenic activity.

4.  A  pesticide for which there is limited evidence of carcinogenicity
    in animals should not necessarily be prohibited for use.

5.  Mechanistic considerations are of major importance in the extrapol-
    ation of animal carcinogenicity data to humans.

8.3.3.  Reproduction studies

    Multigeneration  reproduction  studies  and teratology  studies are
routinely required for pesticides.  Although experimental designs exist
that  combine teratology studies  with reproduction studies,  these two
types of study will be considered separately in this monograph.

8.3.3.1     Multigeneration reproduction studies

    The 1961 FAO/WHO Meeting on Consumer Safety in Relation  to  Pesti-
cide  Residues stated that  one of the  aims of toxicological  investi-
gations  of a pesticide  is to ascertain  "the amount of  pesticide to
which man and farm animals can be exposed daily for a  lifetime"  [32,
p. 10].   With  respect  to the effect of age on toxicity, a WHO Scien-
tific  Group stated: "In general, but not invariably, the young animal
is more sensitive to the toxic effects of exposure to chemicals" [169,
p. 10].   It also pointed  out the effects  of different gut  flora and
changes  in  enzymes with  age  (e.g., poorly  developed mixed-function
oxidase  enzymes in newborn rodents). The Group indicated that "perti-
nent  information observed from reproductive  (multigeneration) studies
provides  some assurance  on the  safety of  compounds which  might  be
present  in the diet of babies" [169, p. 12] and concluded that "use-
ful information may be obtained from studies in newborn or  young  ani-
mals, from reproduction studies and biochemical studies" [169, p. 23].
It  also indicated the need for further studies on "the development of
enzyme  systems in the human  young, with particular emphasis  on those
enzymes responsible for dealing with foreign chemicals" [169, p. 25].

    JMPR addressed the problem of toxicity to juveniles  indirectly  in
1963  when it stated in  its report that "the  Meeting considered that
foods,  such  as  milk, which figure largely in the diets of babies and
invalids,  should be essentially  free from pesticide  residues"  [35,
p. 6].   However, it was not until 1976 that JMPR indicated that repro-
duction  studies should be  available as part  of the basic  toxicology
data  package required for allocating  an ADI [55].  The  need for such
studies  was  repeated in  subsequent Meetings [59; 60; 62; 65; 67; 70;
72; 74]. It should be noted, however, that multigeneration studies on a
number  of compounds had been submitted and evaluated before that time,
some as early as 1963 (e.g., aldrin, dieldrin, heptachlor epoxide).

    In evaluating a multigeneration study, there is a tendency to focus
on  the conceptus, the neonate, and the immature animal, because of the
known variations in toxicity in these stages of development compared to
those observed in adult animals. It must also be recognized  that  pro-
found  physical, physiological, and psychological  changes occur during
pregnancy, which may affect the susceptibility of the dam to  the  tox-
icity  of a specific  chemical.  Attention must  therefore be given  to
maternal toxicity during pregnancy and lactation.

    A  number of  basic protocols  for the  conduct of  multigeneration
studies  have been developed [92; 125; 159; 162; 174].   None, however,
have  gained unanimous approval and proposals for alternatives continue
to be suggested [16; 112; 128; 129].

    The  multigeneration study may best  be viewed as a  screening test
for toxicity in reproducing animals because, although the  emphasis  is
on  detecting effects specific to  reproduction, it is also  useful for
detecting  the enhancement of general toxic effects that may occur as a
consequence  of physiological changes associated  with reproduction and
development.

    The major asset of a multigeneration study that is  well  designed,
conducted,  and interpreted is that it has the ability to detect a wide
range  of indirect  or direct  effects on  reproduction.  This  ability
arises  from the complex integration of reproductive processes, so that
minimal effects that may be difficult to demonstrate in  isolation  may
combine  and  cascade to  generate a more  notable deviation in  a more
distal  end-point (e.g., litter weight).  Observations in the premating
period provide a setting for assessing subsequent observations; initial
observations during mating can identify lack of libido or a disturbance
of hormone (oestrous) cycles. Subsequent data are generated to indicate
effects   on  fertility,  fecundity,  prenatal  toxicity,  parturition,
lactation, weaning, and postnatal growth and development  of  offspring
through  puberty to maturity.  However, those features that enhance the
ability  of the  study to  detect an  effect have  the disadvantage  of
making  it difficult to ascertain  the primary cause when  an effect is
obtained.   Where multigeneration studies  provide an indication  of an
effect  on reproduction, it is usually advisable, or even mandatory, to
perform  follow-up studies for  further elucidation.  Recent  proposals
[112; 128; 129]  seek to alleviate  this limitation of  protocols  cur-
rently in use by allowing flexibility of operation once an  effect  has
been detected or is suspected. A wide range of options is available for
follow-up studies, including separate male and female studies,  use  of
the  three segment  designs applied  in drug  testing, and  use of  the
dominant lethal assay as a male fertility study.

    A number of factors in the experimental design  of  multigeneration
studies  have  been,  or are,  points  of  controversy.  The  following
examples may be cited:

(a) The  duration of the pre-treatment  period of the first  generation
    (F0)    has been the subject of much discussion.  A period equal to
    one  spermatic cycle plus epididymal transit time is generally used
    for  males  and  a period  of  five  estrous cycles  is advised for
    females.  A period of 100 days prior to pairing was originally pro-
    posed.   However, in some rat  strains, such a prolonged  treatment
    period  results in the test animals having passed peak reproductive
    capacity by the time mating is initiated. At present, a 70-day pre-
    mating treatment period is generally used. If two  breeding  gener-
    ations  are employed, the  problem becomes largely  academic, since
    the  second  (F1)   generation  cannot  reproduce until  it reaches
    maturity  and  it will  have  been exposed  continuously throughout
    development to sexual maturity.

(b) The need for second litters in each generation has also been a sub-
    ject  of controversy.  Two  recent studies [20; 113]  indicate that
    second  litters are more  sensitive, with respect  to certain  par-
    ameters, than are first litters.  However, although the sensitivity
    of  the  second litters  is increased in  some areas, there  are no
    recorded cases where effects been observed that were not present in
    the first litters. Thus, provided adequate dose levels are utilized
    and no adverse effects are recorded in the first litter,  a  second
    litter  should not be necessary.  Exceptions to this generalization
    apply  to studies in which findings in the first litter are equivo-
    cal. They also apply when compounds with long biological half-lives
    are being tested and plateau levels have not been attained  at  the
    time of first mating.

(c) Litter  size in multigeneration  studies is often  "standardized"
    (i.e.  culled, usually to eight pups on day 4 post partum). Culling
    may  introduce  bias and  reduce  sensitivity.  Surveys  of routine
    studies show that some supposed advantages of culling  (e.g.,  pre-
    vention  of high mortality in large litters and reduced variability
    of pup weight) are more imagined than real.  If first  litters  are
    culled  and it is suspected that this may have masked the detection
    of  an effect, then production  of a second unculled  litter in the
    same generation is recommended.

(d) The  requirement  for  histopathological  examination  of  pups  at
    weaning has also been a point of discussion. The majority of histo-
    pathological  changes will  normally be  similar to  those seen  in
    routine  short- or  long-term  studies.  The  need for histological
    examination should be on a case-by-case basis, depending  upon  the
    results of the other available studies and gross observations.

(e) It  has been proposed that, since the food intake in the female rat
    during lactation may be as much as 2.5-fold higher than in the non-
    lactating female rat, it would be reasonable to reduce the level of
    test substance in the diet during this period, in order to  give  a
    more constant exposure in terms of mg/kg body weight. However, this
    is  not considered  advisable as  a routine  procedure because  the
    study  would no longer model the human situation, in which maternal
    exposure  to pesticide residues increases during lactation.  Such a
    procedure  adds complexity to  the study and  complicates extrapol-
    ation of the results to humans.

    The  evaluation of the data  from the multigeneration study  starts
with  a  scan  of the entire study for effects and then focuses in more
detail on specific areas, bearing in mind the following points:

(a) Data  from the premating period  (and from other toxicity  studies)
    provide  the baseline information  against which effects  on repro-
    duction  per se  are compared. These data provide a check on whether
    the  exposures  were too  high or too  low or whether  the interval
    between dosages were too wide to establish  dose-related  responses
    of  any effect.   The better  the baseline  information,  the  more
    reliable the judgements on subsequent reproductive effects.

(b) Data  from the initiation  of mating to  parturition provide,  with
    respect  to  adults, information  on  libido, precoital  time, fer-
    tility,  fecundity, duration of gestation, parturition and toxicity
    to  the pregnant  female.  With  respect to  the offspring,  litter
    values (size, number of live pups, and pup weight) at birth provide
    information that would indicate prenatal toxicity.

(c) Data  from birth to  weaning provide information  on the  potential
    susceptibility  of the lactating female  to the test substance  and
    its effect on her nursing ability.  Pup weight and  survival  allow
    the assessment of effects on postnatal growth,  toxins  transmitted
    via the milk, and development of the offspring. Effects seen during
    this period could be delayed responses to earlier (prenatal) insult
    or a combination of post- and pre-natal effects.

(d) Data  on the offspring  during the period  from weaning to  puberty
    provide  information on the  persistence or permanence  of  earlier
    effects  and on direct effects  on the still-immature animal.   The
    profound changes associated with puberty provide a stress point for
    the detection of delayed manifestation of earlier,  latent  effects
    or enhancement of direct effects.

    Comparison  of results obtained  from the two  parental generations
may  be informative. The F0    parental animals (first parental  gener-
ation)  have  not been  exposed to the  test material  in utero   during
lactation  or  during early  post-weaning  development, whereas  the F1
parents  (second  parental  generation), have  been  exposed throughout
development.   Consequently, if an  effect is observed  (on  fertility,
libido, parental body weight, or general condition), comparison between
the  F0 and  F1 parental  animal data  may yield useful information  on
the  time at which the effect is initiated. Thus, because oocyte devel-
opment  is completed in the  female prior to birth,  adverse effects on
fertility  observed only in  female F1 parents  would  suggest that  an
area for further investigation would be the  in utero  oogenesis.  Simi-
larly, disturbance in the development of male hormonal systems  in utero
may  be produced (e.g., fenarimol [99]), resulting in reduced libido of
the F1 males.

    In  considering fertility, the protocol  chosen often has a  marked
influence on the ability to determine which sex is involved.  Protocols
vary with regard to methods of pairing (e.g., one male to  one  female,
one male to two females), duration of pairing (1-3 oestrus cycles), use
of  replacement males in  non-successful pairings, follow-up  of appar-
ently  infertile males,  and use  of proven  males.   Cross-matings  of
untreated  males with treated females and  vice versa may be required to
ascertain the sex of the infertile partner.  Once this  is  determined,
histopathological  examination  of  the reproductive  organs  may yield
information  indicating the type  of effect. Studies  may also be  per-
formed on circulating hormone levels.  Further details on  these  types
of studies are given in reference [166].

    The  initial data on newborn pups are usually limited to the number
of  pups born alive.  Data  on stillbirths and the  number of malformed
pups  may be inaccurate because of cannibalism. Thus, although a multi-
generation  study may  give indications  of high  prenatal  losses  and
developmental  toxicity, it  cannot be  considered to  be a  definitive
teratology study.  Often the only indicator of  prenatal  developmental
toxicity in the reproduction study is reduced litter size at  the  time
of  the  first observation,  usually  several hours  after parturition.
However, if dose levels administered in the multigeneration  study  are
sufficiently high, then the lack of any effect provides  some  reassur-
ance  regarding potential teratogenicity.  Furthermore,  the continuous
exposure to the test substance over a long period of time in the multi-
generation feeding study may lead to changes in metabolism. It may also
lead to changes in the dose of parent compound or  metabolite  reaching
the placenta or fetus or to higher blood plasma levels in the  case  of
chemicals with long half-lives.  This can lead to divergence, in either
direction,  of  the results  of  multigeneration and  prenatal toxicity
(teratogenicity) studies.

    The rate of growth and survival of post-partum pups may be affected
by  a  number  of factors  including  general  maternal  care,  effects
initiated  in utero, reduced lactation by the mother, or the presence of
toxicants in the milk. When the need to determine the cause of pup mor-
tality or reduced pup weight gain arises, the initial step  is  usually
histopathological examination of pups failing to survive.  If lactation
has been affected (either in terms of quantity or quality of the milk),
the  normal routine for  investigation is cross-fostering  of pups,  in
which pups from treated mothers are weaned by untreated  maternal  ani-
mals and  vice versa.

    It  needs to be borne  in mind that the  sensitivity of the  repro-
duction study is low for specific end-points. This is particularly true
for  discrete end-points  such as  infertility and  total litter  loss.
Where  such end-points are concerned, more sensitive indicators must be
found  or the dimensions of  the study must be  greatly increased.  For
example, if male infertility is suspected, studies of  sperm  motility,
mobility,  and morphology  may be  undertaken.  In  the case  of  male-
mediated reproductive toxicity, the commonly used multigeneration study
design  is particularly insensitive and specialized studies are necess-
ary if male fertility is believed to be effected.   Sperm  measurements
are now being conducted in conjunction with some  short- and  long-term
studies and this may partially alleviate this problem.

    In  addition, a commonly available, and sometimes neglected, source
of  supporting information may be provided by histopathological examin-
ation of the reproductive organs (after proper fixation) in the chronic
toxicity studies.

8.3.3.2     Teratology studies

    In 1967, the WHO Scientific Group on Procedures  for  Investigating
Intentional  and Unintentional Food  Additives [169] stated  that  "at
present,  no  specific tests  can be recommended  for the detection  of
teratogens,  but  some safeguard  can  be provided  by  multigeneration
studies" [169, p. 24]. In 1976 [57] and more recently [59; 60; 62; 64;
65; 67; 70; 72; 74],  JMPR has stated that teratology studies should be
an  integral part of the  toxicology data base required  for evaluation
and for allocation of the ADI.

    The basic teratology study (also defined by IPCS as an embryo/feto-
toxicity  study) involves the treatment of the pregnant animal through-
out the period of organogenesis. Since this begins at or around implan-
tation  of the blastocyst into the endometrium, pre-implantation losses
are  not usually of concern.  However, an "apparent" pre-implantation
loss could be a failure to detect blastocyst implantation losses.

    The route of exposure, in teratology as in other studies, can mark-
edly influence results.  The most frequent routes of administration for
pesticides are diet, drinking water, or gavage.  The  latter,  however,
may result in marked differences in kinetics following the bolus admin-
istration  of a high  dose relative to  more frequent intakes  of small
amounts.  Thus benomyl is teratogenic when administered by  gavage  but
not when administered via the diet [70].  This is believed to be due to
the short-term high plasma levels resulting from gavage administration,
compared to the much lower sustained levels which result  from  dietary

administration.  Effects following gavage administration are not always
more  severe than those resulting from dietary inclusion.  For example,
thalidomide administered to rats by gavage provides  essentially  nega-
tive results whereas administration in the diet in a reproduction study
induces  almost complete embryolethality.   Comparative pharmacokinetic
studies  are useful and  often essential for  relating the findings  in
teratology studies to human dietary exposure.

    The species most commonly used in teratology studies are  the  rat,
the mouse, and the rabbit.  More than one species is generally utilized
in  attempting to assess  teratogenic potential because  of the  varia-
bility  in species sensitivity.   Species differences arise  because of
variations in metabolism, types of placentation, and in the  rates  and
patterns  of fetal development.  As  with other toxicity studies,  more
weight  should  be given  to results in  species that give  the closest
approximation  to humans  in terms  of kinetics,  dynamics,  and  other
relevant parameters.

    The choice of dose levels in teratology studies has recently become
a  point of major concern.  In several publications [108; 109; 110], it
has  been stated  that maternal  toxicity is  associated with  species-
specific  patterns of malformations.  These associations have often led
to false presumptions of cause and effect and, further, to the presump-
tion  or implication that  embryonic effects associated  with  maternal
toxicity  are unimportant.  However,  in such associations  it is  more
probable that effects on the embryo and dam are independent or mutually
interactive.   In practical terms the conceptus and dam are indivisible
and  are  best  considered as a unit.  A presumed and even proven cause
and effect relationship provides only an explanation of  the  mechanism
of  action, it does  not necessarily preclude  the risk.  For  example,
effects  induced by  alcohol, lead,  or methylmercury  show that,  even
though these effects occur at doses that induce maternal toxicity, they
remain of relevance for making decisions regarding safety. In consider-
ing the choice of the highest dose level in teratology studies,  it  is
important  to note that: (a) maternal toxicity can and does occur with-
out  inducing malformations, and  (b) malformations can  occur  without
maternal  toxicity being induced.  Thus, at the present state of knowl-
edge, it may be prudent to continue to utilize high dose  levels  which
induce  minimal maternal toxicity.  Further research regarding the role
of  maternal  stress  in the  induction  of  developmental toxicity  is
recommended.

    In  interpreting the significance of malformations and other struc-
tural variants, it is important to consider the stage of development of
the fetus at examination.  Under routine experimental  conditions,  the
offspring are removed from the mother 12 to 24 hours before anticipated
parturition  to avoid the possibility of cannibalization.  However, the
accuracy  of estimating the age of the offspring at the time of removal
is  questionable, since vaginal smears are normally taken only once per
day,  thereby reducing the accuracy  of the estimation of  the onset of
pregnancy.  Furthermore, delays in the rate of development  may  occur.
For most malformations this is relatively unimportant.   The  incidence
of  minor variants (e.g., ossification variants) may, however, be mark-
edly altered, especially if the compound affects the rate  of  develop-
ment.

    Dose-related minor changes should not be ignored, since they are of
considerable  value in  assessing whether  a low  incidence of  malfor-
mation  is compound-related or  coincidental.  The association  between
changes  in the pattern of  minor anomalies and malformations  has been
amply illustrated in the past. However, considerable variability exists
among  laboratories in both the  reporting and the assessment  of these
minor  structural  deficiencies,  which renders  interpretation of some
studies  extremely  difficult.   A  consistently  higher  standard   of
reporting  of minor anomalies is  encouraged.  Minor anomalies are  not
necessarily of great concern, however, in the absence of other manifes-
tations of developmental toxicity.

    Hydroureter  and  hydronephrosis  are  frequently  associated  with
delayed opening of the ureter at the point of entry into  the  bladder,
with a subsequent hydrostatic effect.  Even when the incidence of these
conditions is high in pre-partum fetuses, they may not be  apparent  in
4-day-old post-partum pups [179]. Further research is encouraged in the
development  of protocols for the postnatal assessment of developmental
toxicity.

8.3.3.3     Screening studies in teratology

    Studies  in  which non-mammalian  species  or mammalian  organs and
tissue cultures are used to attempt to predict teratogenicity  in  mam-
malian  systems  are not  generally of value  in safety assessments  at
present.  Although some tests may be useful as preliminary  screens  to
prioritize compounds for further investigation, none of  the  available
techniques  can  be  considered definitive  studies.  These techniques,
however, are of value in follow-up studies to determine  the  mechanism
of action of compounds demonstrating positive effects in  standard    in
 vivo  studies, or as support studies.

    Further discussion of screening teratology studies will be found in
references [91] and [174].

8.3.3.4     Principles

1.  Tests  in reproducing animals are essential for the complete safety
    evaluation of a pesticide.

2.  A  well designed and  conducted multigeneration study  providing no
    evidence  that the pesticide  exerts a selective  effect on  repro-
    duction or enhancement of general toxic effects should be  given  a
    high weighting towards establishing its safety.

3.  The  detection of effects  in a multigeneration  study may  require
    further studies if the protocol has a limited ability  for  charac-
    terizing a specific effect.

4.  The  limited sensitivity of some end-points of a reproduction study
    needs  to be borne in mind.  Discrete responses such as pregnant or
    non-pregnant,  "fertile" or "infertile" are  quite insensitive,
    since  objective discrimination between  groups is governed  by the
    same laws of statistical probability as are applicable to other low
    frequency events such as malformations.

5.  Support  studies such  as examination  of sperm  motility and  mor-
    phology  may provide a sensitive  end-point that can allow  further
    characterization of effects observed in reproduction studies.

6.  Histopathological  examination of gonads performed  in chronic tox-
    icity studies may also provide valuable supplementary information.

8.3.4.  Neurotoxicity studies

8.3.4.1     Delayed neurotoxicity

    JMPR  first reviewed feeding studies in hens, with subsequent exam-
ination  of  brain,  spinal  cord  and  sciatic  nerves, in  1967  when
dimethoate was evaluated [41].  In 1968, the first study  using  single
doses  in hens was reviewed,  when dioxathion was evaluated  [43].  The
question  of delayed neurotoxicity was first considered in 1974 [53] in
response  to requests from various countries for guidance on the intro-
duction  and use of the organophosphate leptophos.  This was considered
further at the 1975 JMPR, the report stating:

    "A  major toxicological problem  long recognized to  be associated
with such organophosphate esters as tri- O -cresylphosphate  (TOCP), and
more recently brought to the attention of the Meeting in the evaluation
of  leptophos is that  known commonly as  `delayed neurotoxicity' . . .
The delayed neurotoxicity syndrome affects only certain animal species,
including  man.  The most  susceptible animal for  laboratory  bioassay
procedures, the adult hen, is not susceptible before 3-4 months of age.
While  the adult hen  is the animal  of choice for  laboratory testing,
cats,  dogs, calves, and sheep have been shown to be susceptible.  Some
sub-human primates and rodents are resistant to both the  clinical  and
the  histological lesions. In contrast, man has been shown to be highly
susceptible  to the syndrome, as suggested by studies where occurrences
of  paralysis have been reported . . .  There are no known antidotes to
delayed  neurotoxicity,  and  recovery  from  ataxia  is  predominantly
through development of collateral nerve pathways and  physical  therapy
to develop muscles not served by affected nerves." [54, p. 11-12].

    A  certain degree of peripheral nerve regeneration also occurs, but
regeneration is not observed in the central nervous system (CNS) axons.
Therefore,  the ataxia is clinically "irreversible" although the pic-
ture  changes  from  a flaccid  paralysis  (peripheral  nerve plus  CNS
lesions) to a spastic paralysis (CNS lesions only).

    Reference has been made in some studies [14; 22; 104] to the induc-
tion  of neurotoxicity by  certain organophosphorus compounds  used  as
pesticides  and  drugs.  The  dose  administered  in  most experimental
studies  is high, and  atropine has been  used to protect  animals from
acute signs of poisoning to allow time for the  neurotoxicity  syndrome
to  develop.   While atropine  protects  against the  short-term  acute
cholinergic signs of poisoning,it is ineffective against delayed neuro-
toxicity occurring 8-14 days after treatment.

    A  key factor in the problem of delayed neurotoxicity, discussed by
the  1975  Joint Meeting  and endorsed by  the 1976 [55]  and 1978 [59]
Meetings, is the dose response.  "The Meeting concluded  that  delayed

neurotoxicity appears to follow a dose-response relationship  and  that
it  is therefore possible to estimate a no-effect level following acute
or  chronic exposure in a susceptible species.  With an adequate margin
of safety an ADI for man can be allocated with a sufficient  degree  of
assurance  as far as pesticide  residues in food are  concerned." [54,
p. 13].

    The  1982 JMPR, in  discussing acceptable protocols  for  pesticide
toxicology  studies,  indicated  that "multiple  dosing  (single  oral
doses, 21 days apart) was required for studies of delayed neurotoxicity
of organophosphorus compounds." [67, p. 2].

    The 1984 Meeting noted that:

    "Some  OPs  (organophosphates)  induce both  acute  reversible and
delayed irreversible neurotoxicity; the latter relates to inhibition of
another enzyme called neuropathy target esterase (NTE)  [115].  Organo-
phosphorus-delayed  neurotoxicity is believed to be initiated by a two-
step mechanism: a high level of NTE inhibition and `aging' of the phos-
phoryl enzyme complex [105]."

    "Inhibition of NTE within 24-48 hours after dosing correlates with
the  clinical and morphological  effects of delayed  neurotoxicity seen
10-20 days  later. This test model  was found to be  valid for all  OPs
known to cause delayed neuropathy in man." [147].

    The JMPR recommended that delayed neurotoxicity testing need not be
done  for monomethylcarbamates, phosphinates, or sulfonates.  They also
recommended  that  TOCP  be used as a positive control only for OPs and
that the NTE assay be included in the assessment of OPs [72].

    The  most recent comments  on delayed neurotoxicity  relate to  the
optical isomers of organophosphorus esters.  The 1987 JMPR stated:

    "Recent  evidence  suggests that  when  racemic mixtures  of phos-
phonates are used in test animals, the optical isomers might  show  the
same  phosphonylating ability  for NTE  but the  rates of  aging  might
differ.   Consequently only the optical  isomer which forms an  ageable
protein-phosphonyl complex will cause delayed polyneuropathy.  This was
the  case for EPN, an OP no longer in use.  Therefore, whenever OPs are
mixtures  of  optical isomers  the  delayed neurotoxic  potential might
depend on the chirality." [78, p. 11].

    Two types of studies are generally conducted on chemicals suspected
of  being  neurotoxic.  The  first is the  use of a  suitable sensitive
species (usually the adult hen), where test substance  is  administered
at  two acute exposures  (separated by 21 days)  to  atropine-protected
animals  at a level at or above the LD50 of  the compound. Observations
on  body weight, ataxia,  and signs of  delayed neurotoxicity are  made
while the animals are alive.  At termination, usually 42 days after the
first dose, histopathological examination of the brain, spinal cord and
proximal  and distal sections  of (usually) the  sciatic nerve is  per-
formed.  Data from this type of test suffer from two  major  drawbacks:
the  evaluation is often  subjective, and a  negative result cannot  be
graded.  The second type of test is the determination of  NTE  activity
[13; 105].   In its simplest form, this involves treatment of the adult

hen  with  a single  maximum tolerated dose  of the test  substance and
subsequent  assay of the brain enzyme after the time of peak inhibition
but  before substantial re-synthesis of  new enzyme has occurred.   The
time of peak inhibition, which can be from 3 to 48 h  post-dosing  (and
is  determined by the pharmacokinetics  of the compound), can  often be
assessed by observation of the time of onset of cholinergic signs.  The
threshold level of NTE inhibition at this early stage, which correlates
with delayed neurotoxicity, is approximately 80%. No clinical signs are
associated  with an inhibition of  60% or less. When  multiple determi-
nations  of NTE are made  during chronic exposures, plateau  levels are
observed  in 2-3 weeks. If  inhibition of NTE  in the brain  and spinal
cord  is less than  50%, delayed neuropathy  does not occur.   However,
inhibition  of  60-70%  in such  studies  might  result in  neuropathic
sequelae  as reported by some authors, while others state that the same
threshold  of NTE inhibition (70-80%)  has to be reached  in single and
repeated exposures.

8.3.4.2     Acute neurotoxicity (acetylcholinesterase inhibition)

    In 1967, the WHO Scientific Group on Procedures  for  Investigating
Intentional  and Unintentional Food  Additives [169] noted  that plasma
and  erythrocyte  cholinesterase  activities were  markedly  reduced by
organophosphorus and carbamate pesticides.  This Group also  noted  the
absence  of a correlation between  blood cholinesterase levels and  the
signs  and symptoms of toxicity.   Thus cholinesterase levels in  blood
"may  be useful as an indication of exposure to a substance with anti-
cholinesterase activity, but not as an invariable guide to  the  degree
of  intoxication  present  or predicted"  [169, p. 17-18].   The Group
indicated  that "although changes in blood cholinesterase  levels  may
be  helpful in  toxicological studies,  it is  important  that  further
research  should  be  done to  relate  the  indices used  as closely as
possible  to the biochemical  changes concerned in  bringing about  the
toxic effects . . . " [169, p. 18].

    Cholinesterase-inhibiting  compounds  have  been evaluated  at vir-
tually every Joint Meeting.  Until 1982, JMPR used inhibition of plasma
cholinesterase,  as well as  erythrocyte and brain  cholinesterase, for
the  purpose of establishing NOELs. In 1982, the status of cholinester-
ase  activity as an  indicator of anticholinesterase  compound toxicity
was reconsidered:

    "In  reviewing some organophosphorus and carbamate pesticides, the
Meeting  noted that previous JMPR  reports have commented on,  and made
recommendations on the basis of, inhibition of plasma cholinesterase as
a  major criterion in the  evaluation of some of  these compounds.  The
present Meeting recognized that most organophosphorus compounds inhibit
butyrylcholinesterase,  known also as plasma  cholinesterase or pseudo-
cholinesterase,  at concentrations lower than  those at which they  in-
hibit  acetylcholinesterase found in  erythrocytes and in  nerve synap-
ses.

    "The function of plasma cholinesterase is not understood but it is
known  that it plays no  role in cholinergic transmission,  the physio-
logical function which is impaired by anticholinesterases. On the other
hand, acetylcholinesterase in erythrocytes, although playing no role in
cholinergic  transmission  itself,  reflects  the  acetylcholinesterase

activity in nerve synapses, since the two enzymes are  considered  bio-
chemically  identical.  Therefore, erythrocyte  cholinesterase activity
may be taken as an indicator of the biochemical effect  of  anticholin-
esterase pesticides." [67, p. 6].

    A  biologically significant reduction in erythrocyte cholinesterase
is normally considered to be a reduction of >20% of pretest  levels  in
the same animals in short-duration studies, or in  concurrent  controls
in longer studies.

    The  1988 Joint Meeting  further considered the  utility of  plasma
cholinesterase  and erythrocyte and brain acetylcholinesterase measure-
ments  [79]. It noted that "the correlation between acetylcholinester-
ase  inhibition in erythrocytes  and in the  nervous system is  usually
unknown"  and  indicated  that "data  on  brain  acetylcholinesterase
inhibition are considered to be of greater value than those on erythro-
cytes in assessing the cholinergic effects of  cholinesterases."   The
Meeting  also noted, however,  that in the  absence of measurements  of
brain  acetylcholinesterase, those of  erythrocyte acetylcholinesterase
serve  as a better indicator  of toxicity than those  of plasma cholin-
esterase activity.  It was noted that  in vitro  kinetic studies  may  be
necessary for pesticides with anti-esterase activity.  Results of these
studies  in  different  species may  be  combined  with  in vivo   study
findings to establish ADIs for these compounds.

    JMPR has drawn attention to the methodology for  measuring  cholin-
esterase  inhibition, stating that "the currently used methods for the
determination  of cholinesterase activity  may lead to  erroneous  con-
clusions  when applied to rapidly reversible cholinesterase inhibitions
(e.g., N -methyl- and N,N -dimethylcarbamates). in vitro  kinetic studies
should be made to elucidate the nature of reversible  inhibition  reac-
tions.  The results obtained in  in vivo  studies should be  interpreted
cautiously  until  more  satisfactory  methods  are  available."  [55,
p. 11].

    In  1983 [70], the problem  of measurement of cholinesterase  inhi-
bition by carbamate pesticides was again addressed by JMPR.  The report
of  this Meeting  states "the  Meeting noted  that in  the reports  of
several studies on carbamate pesticides, the method of determination of
cholinesterase  inhibition  was inadequately  reported and occasionally
data were inconsistent with respect to dose and the degree  of  cholin-
esterase inhibition. Carbamates are considered to be reversible inhibi-
tors of cholinesterase with a short duration of action.  Because of the
reversible  inhibition of the enzyme by dilution, as would occur during
the preparation of the assay, inhibition cannot be accurately measured.
The Meeting stressed that in order to permit evaluation of cholinester-
ase  inhibition  by carbamates  in vivo,  special  care is  required  in
reporting all details of such studies." [70, p. 10]. Carbamate cholin-
esterase  inhibition studies should utilize minimal dilution during the
preparation  of the assay, minimal  incubation times and minimal  times
between blood sampling and assay (e.g., the Ellman method [28]).

8.3.4.3     Chronic neurotoxicity

    The  1972 JMPR [50] noted the work of Murphy & Cheever [144], which
reported  modification of the electroencephalographic  patterns in cer-
tain experimental animals following long-term exposure to low levels of
cholinesterase-inhibiting   compounds.   The  Meeting   indicated  that
"insufficient information was available to permit any conclusion to be
reached  on the  relationship of  these studies  to  the  toxicological
assessment of cholinesterase-inhibiting compounds." [50, p. 8].

    The  1974 Meeting [53]  reiterated the desirability  of determining
the  usefulness of electroencephalographic  criteria for assessing  the
effects  of cholinesterase-inhibiting pesticides.  However,  no further
information or verification of this aspect of cholinesterase-inhibiting
pesticide toxicity has become available to JMPR.

8.3.4.4     Pyrethroid-induced neurotoxicity

    JMPR has evaluated data on pyrethroids during many  meetings  since
1965  [39; 47; 51; 61; 63; 66; 182].   Most pyrethroids  can be divided
into  two  classes:  the  T-syndrome  (tremor)  and   the   CS-syndrome
(coreoathetosis-seizures).   In  general, alpha-cyanopyrethroids  cause
CS-syndrome  neurotoxic effects, and other pyrethroids cause T-syndrome
effects.   The  1984  Meeting [73]  noted  that  the  neurotoxicity  of
pyrethroids originates from their primary action on the sodium channels
of nerve membranes [123].  This interaction is reversible, as  are  the
clinical signs of toxicity.

    Morphological  changes  in  peripheral  nerves  are  produced  as a
secondary  effect of the primary interaction only at doses close to the
LD50.    Therefore, considering the reversibility  of pyrethroid neuro-
toxicity  and the  high doses  required to  cause  permanent  secondary
effects,  the neurotoxicity of pyrethroids  is not considered to  be of
great concern in the evaluation of pesticide residues in food.

8.3.4.5     Neurobehavioural toxicity

    A  recent WHO publication [175]  on the Principles and  Methods for
the Assessment of Neurotoxicity Associated with Exposure  to  Chemicals
stated the following:

    "There is ample evidence of real and potential hazards of environ-
mental  chemicals for nervous system function.  Changes or disturbances
in  central nervous function, many  times manifest by vague  complaints
and  alterations in behaviour, reflect on the quality of life; however,
they have not yet received attention.  Neurotoxicological assessment is
therefore an important area for toxicological research. It  has  become
evident,  particularly in the last  decade, that low-level exposure  to
certain toxic agents can produce deleterious neural effects that may be
discovered  only when appropriate procedures are used.  While there are
still  episodes of large-scale  poisoning, concern has  shifted to  the
more subtle deficits that reduce functioning of the nervous  system  in
less obvious, but still important ways, so that  intelligence,  memory,
emotion,  and other complex neural functions are affected.  Information
on neurobehaviour, neurochemistry, neurophysiology, neuroendocrinology,

and  neuropathology is vital for understanding the mechanisms of neuro-
toxicity.   One of the major  objectives of a multifaceted  approach to
toxicological  studies is to  understand effects across  all levels  of
neural  organization.  Such a  multifaceted approach is  necessary  for
confirmation  that  the  nervous system  is  the  target organ  for the
effect.  Interdisciplinary studies are also necessary to understand the
significance  of any behavioural  changes observed and  thus to aid  in
extrapolation  to human beings  by providing specific  neurotoxic  pro-
files.  Concomitant measurements at different levels of neural organiz-
ation can improve the validity of results."

    Since  the publication of this monograph, a number of protocols for
neurobehavioural  toxicity have been  proposed for use  [11].  However,
the  1989 JMPR noted  that the use  of behavioural tests  in laboratory
animals  has not been validated  [183].  The meeting concluded:  "This
failure relates both to the inter-individual and intra-individual vari-
ations  in behaviour and the  difficulty in quantifying these  changes.
In  addition, the biochemistry, electrophysiological  and morphological
correlates  of observed changes are often lacking."  Although much has
been  written on  behavioural teratology  [158; 159], no  data on  this
aspect  of toxicology has been  reviewed by JMPR.  A  discussion of the
utility of these tests will be found in reference [174].

8.3.4.6     Principles

1.  Delayed  neurotoxicity appears to follow  a dose-response relation-
    ship. Thus, with an adequate margin of safety an ADI can  be  allo-
    cated.

2.  Delayed  neurotoxicity  testing  should be  conducted routinely for
    organophosphates.   However, it need  not be done  for  monomethyl-
    carbamates, phosphinates, or sulfonates.

3.  TOCP  is  recommended  as a  positive  control  substance only  for
    organophosphates.

4.  The NTE assay should be included in the data base  for  organophos-
    phate evaluations.

5.  Data on brain acetylcholinesterase are of greater value  in  safety
    assessment than are data on erythrocyte acetylcholinesterase.

6.  Plasma  cholinesterase  (butyrylcholinesterase)  inhibition is  not
    considered to be an adverse toxicological effect.

8.3.5.  Genotoxicity studies

    The topic of mutagenicity (now generally referred to by the broader
term, genotoxicity) and its relevance to the evaluation of  the  safety
of  pesticide residues has  been repeatedly considered  by JMPR.   Most
recently,  the 1983 Meeting recognized  the uncertainty of the  associ-
ation between mutagenic and carcinogenic activity, and  indicated  that
data  from long-term carcinogenicity studies must override any possible
concerns  raised by mutagenicity studies.   In considering mutagenicity
tests per  se, the 1983 JMPR was  unable to determine the  relevance of

the results of such tests to possible human health hazards.  It  there-
fore  indicated such data cannot be utilized directly in the assessment
of the ADI [70].

    A recent publication [5] surveying 222 chemicals tested in mice and
rats  (NCI/NTP bioassays) has  indicated a strong  association  between
structure/activity,  mutagenicity in  Salmonella strains, and the extent
and  sites  of  rodent tumourigenicity.   When  structure/activity  and
 Salmonella tests   were considered and utilized as an index of genotox-
icity,  the use of such  an index indicated two  groups of carcinogens:
those that are genotoxic and those that are  apparently  non-genotoxic.
In  examining sites of action, some 16 tissues were susceptible to car-
cinogenic effects with genotoxins only (accounting for 31% of the indi-
vidual  chemical/tissue reports), whereas the remaining 13 tissues were
affected  by both groups of  carcinogens, the most frequently  affected
tissue  being the mouse  liver (24% of  all individual  chemical/tissue
reports).   Furthermore, chemicals active  as carcinogens in  both rats
and  mice,  or in  two or more  tissues, showed a  70% correlation with
positive  Salmonella tests,  whereas  single  species or  single  tissue
carcinogens showed only 39% correlation.  The study also confirmed that
many  in vitro  genotoxins were not carcinogenic (possibly due to malab-
sorption, metabolism  in vivo,  or the supposedly greater sensitivity of
the  in vitro   tests).   Mouse  liver-specific  carcinogens  were  also
 Salmonella positive   in only 30% of  the cases, indicating that  mouse
liver  tumour induction may  be mechanistically independent  of  inter-
action of the test chemical with DNA.

    These  results  support  the position  that  rodent carcinogenicity
tests are required for all pesticide evaluations (see section 8.3.4.1),
since  without such studies it cannot be determined that a pesticide is
a trans-species, multiple-tissue rodent carcinogen.

8.3.5.1     Principles

1.  Mutagenicity is utilized only as supplementary information  in  the
    weight-of-the-evidence determination for carcinogenicity.

2.  Mutagenicity  tests, especially mammalian  in vivo  tests, which are
    indicative of compound-induced alterations in DNA are of  value  in
    assisting in the determination of the mechanism of action  of  some
    carcinogens.

3.  Genotoxicity testing is also potentially useful in  the  prediction
    of the risk of heritable defects.

4.  Protocols  that are sensitive,  practical, and predictive  of heri-
    table human risk remain to be developed.

8.3.6.  Immunotoxicity studies

8.3.6.1     Background

    In  1967, progressive haemolytic  anaemia was observed  in  monkeys
exposed to dieldrin [41].  However, it was not recognized at  the  time
that this anaemia resulted from antibodies produced in the animal which
were directed against dieldrin bound to the erythrocytes [89]. The 1976

JMPR "noted the first observation in a group of animals of a pesticide
(pirimicarb)  causing a  haemolytical reaction  which might  be  of  an
immuno-reactive  nature.  In the case observed, the phenomenon occurred
only  with relatively high  doses in a  closed, inbred colony  of dogs.
However, it is possible that, by prolonged and constant use of  such  a
pesticide, hypersensitivity may be built up which could eventually lead
to  an immunological reaction  of a haematological  or other  nature."
[55, p. 14].  In 1978, JMPR [59] again considered pirimicarb, and noted
that  haemolytic changes occurred in a second strain of dogs but not in
monkeys or in rodent species.  The effect was therefore  considered  to
be species specific.

8.3.6.2     Current position

    Immunotoxicology  has been defined as the discipline concerned with
the study of events that can lead to undesired effects as a  result  of
the interaction of test substances with the immune system.  These unde-
sired effects may be a consequence of:

 *  direct  and/or indirect action  of the test  substance (and/or  its
    biotransformation product) on the immune system;

 *  an immunologically-based host response to the compound  and/or  its
    metabolites;

 *  host antigens modified by the compound or its metabolite(s).

    Zbinden  [181] has indicated that  chemicals may affect the  immune
system  immediately and preferentially, but they may also act either by
injury  to other organs or  by creating a general  deterioration of the
health  of the animal,  resulting in a  secondary effect on  the immune
system.  Consequently, as with  any aspect of  toxicology,  immunotoxi-
cology must be considered in the light of all available  toxicity  data
and not as an entity independent of other factors.

    In  mammals  the  primary  lymphoid  tissues  comprise  the thymus,
spleen, lymph nodes, bone marrow and diffuse lymphoid  tissues  associ-
ated with the gastrointestinal and respiratory systems [117; 165]. Pro-
genitor  cells produced in the  bone marrow and other  lymphoid tissues
undergo maturation in early life via residence in the thymus to produce
the  T-cell  series (which  are  mainly responsible  for  cell-mediated
immunity), and via development in peripheral lymphoid tissues to become
members  of the B-cell series,  which form the basis  of humoral (anti-
body-mediated)  immunity.  Throughout life,  the development of  immune
reactions and defenses involves interactions between several  types  of
T- and  B-cells and soluble factors  produced by early stages  of these
cells, phagocytic cells, and polymorphs.

    Chemically-induced immune alterations may be detectable from patho-
logical  changes  (quantitative  and qualitative)  in  lymphoid organs.
Thus,  changes in the  weight of the  thymus, spleen, and  lymph nodes,
combined  with histopathological changes in these organs can be import-
ant  in assessing the potential immunotoxicity of a chemical.  Further-
more,  examination of mucosa-associated lymphoid  tissue (e.g., Peyer's
patches) may indicate immunotoxic potential. Examination of bone marrow
is  essential in any immunotoxic assessment, as is consideration of the
resistance to infection of the living animal.

    Atrophy  and lymphocytic depletion in the thymic cortex, hypoplasia
or  hypercellularity  of  the paracortical  areas  of  the lymph  node,
changes  in the  numbers of  lymphoid follicles,  changes  in  germinal
centres  and  plasma  cells in lymph nodes and the spleen, and the size
and cellularity of the marginal zone of the spleen may all  be  indica-
tive  of immunotoxicity.  However,  other factors also  induce some  of
these  effects (e.g.,  thymic atrophy  due to  stress or  weight  loss)
[165].

    Haematological studies of serial blood samples for total  and  dif-
ferential leucocyte counts and platelet numbers can provide a potential
indicator  of certain autoimmune processes.  Similarly, measurements of
body temperature and serum chemistry to determine cortisol and fibrino-
gen  levels may suggest consequences of certain types of immunotoxicity
[121].

    The  recognition  that  an increased  tumour  incidence (especially
lymphomas)  can be associated with immunosuppression indicates that the
immune  system may be involved in controlling neoplastic changes.  This
involvement  is supported by  in vivo  evidence of tumour immunogenicity
(e.g.,  transplant rejection; lymphoid  cell transfer experiments),  by
the  promising use of  monoclonal antibodies as  therapeutic agents  in
cancer therapy, and by many laboratory demonstrations of  cellular  and
humoral responses to neoplasms.

    A  number of agents (e.g., tricothecene mycotoxins), known to occur
as  contaminants  in  food, can be shown to affect the immune system of
laboratory animals. These mycotoxins (nivalenol, deoxynivalenol, etc.),
which  are unaffected by heating or baking, occur on cereal crops grown
in temperate climates.  Information on the potential of pesticide resi-
dues to interact with such immunosuppressive agents would be  of  value
in the safety assessment of pesticides.

    It  is becoming apparent that  immune dysfunctions induced by  test
substances  sometimes have severe  and diverse health  effects  ranging
from  autoimmune diseases or hypersensitivity reactions to the possible
induction  of cancer. In the past, this area has received little atten-
tion because of the lack of basic knowledge of suitable  test  methods.
The complexity of the mechanisms of action of the immune  system  makes
it difficult to decide on appropriate studies.  Some potential probably
exists  for the general identification of immunotoxicants from standard
toxicological  protocols, but full identification  of immunotoxicity is
likely  to  require  further  ancillary  studies.   The  development of
additional methods relevant to the safety assessment of pesticide resi-
dues is to be encouraged in the hope that sets of tests, suitably vali-
dated, will permit evaluation of this important aspect  of  toxicology.
A  collaborative  study, sponsored  by the IPCS  and CEC, is  currently
underway  to examine and validate test methodologies for the assessment
of immunotoxicity.

8.3.6.3     Principles

1.  Immune  dysfunctions  induced  by  test  substances  can  result in
    serious health effects and should be considered in  the  evaluation
    of pesticide residues in food.

2.  Validation of a tiered approach to immunotoxicity tests relevant to
    safety assessment is to be encouraged.

8.3.7.  Absorption, distribution, metabolism, and excretion

8.3.7.1     Background

    The  meeting held in 1961 to consider Principles Governing Consumer
Safety in Relation to Pesticide Residues [32] indicated that  the  pro-
cedures  to be followed in generating data for the safety evaluation of
a  pesticide "must be  determined by . . . its toxicological  and bio-
chemical  actions, as they  are discovered during  the progress of  the
investigation."   The report  also cited  the second  and fifth  JECFA
reports  [31; 33],  indicating that  the  procedures detailed  in these
reports should be followed when a new pesticide is being investigated.

    The  second JECFA report  addressed biochemical and  other  special
investigations. It indicates that "the aspects of metabolic  and  bio-
chemical  activity that might be  profitably studied include the  route
and rate of absorption of the test material, the levels of  storage  in
tissues and the subsequent fate of the stored material.  Studies of the
metabolism  of the material,  together with the  identification of  the
metabolites, might be extended to include balance experiments, in which
an  attempt is made to account for the administered dose as metabolites
excreted or material stored in the body" [31, p. 13]. The report indi-
cated  that studies should be  performed initially at high  dose levels
and  later they should be extended to investigate lower dose levels. It
also indicated that examination of enzyme processes and  studies  using
pharmacodynamic techniques may be useful in specific cases.

    The  1963 JMPR stated that "it is important to know whether a sub-
stance  is absorbed, its distribution in the body after absorption, its
mechanism of action including its influence on enzyme systems,  how  it
is metabolized, and the routes of final elimination.  The toxicity of a
pesticide may be altered at any of these stages." [35, p. 8].

    A  WHO Scientific Group  in 1967 also  indicated the importance  of
metabolism studies, stating that:

    "The  detailed study of metabolism at the molecular level has been
applied  to many problems and this has special relevance to toxicology.
Modification of substances in the course of their metabolism  may  sig-
nificantly affect their toxicity; chemicals may alter  enzyme  activity
and  some  substances  may  stimulate  the  production  of metabolizing
enzymes. Hence for a full understanding of the effects of a chemical on
biological  systems, it is necessary to have as much knowledge as poss-
ible  about the relationship between the chemical (and its derivatives)
and the complex pattern of enzymes in living organisms." [169, p. 4].

    In  the section of  the report that  addressed enzyme studies,  the
Scientific Group stated:

    "It has become more and more apparent that, among  the  mechanisms
of  action of toxic  substances, those of  a biochemical nature  are of
prime  importance.  In this  connection, the basic  enzyme systems  are
certainly among the first sites of action to merit careful study, since

their inhibition often constitutes the causal biochemical  lesion  that
determines,  at  least in  part, the nature  of toxic effects."  [169,
p. 14].

    In  1975, JMPR [54] re-emphasized the principle that tissue distri-
bution and the mode and rate of metabolism and excretion can profoundly
influence the toxicity of a compound. It noted, however, that such data
were usually based on single-dose studies.  In proposing the  need  for
multiple-dose studies, the Meeting noted that biliary  excretion,  with
the potential for enterohepatic circulation, and the problems  of  dis-
tribution  and storage of highly lipophilic substances in fat deposits,
as  well  as potential  accumulation  of slowly  metabolized compounds,
would not be adequately addressed by single-dose studies.

8.3.7.2     Current position

    In  discussing  doses  in  toxicity  studies  and  extrapolation to
humans, the 1987 JMPR indicated that comparative metabolism of the test
material in the experimental animal and man were basic to the choice of
dose levels [78].  The Meeting recognized the rarity of such  data  and
the  ethical problems involved  in obtaining the  required data in  the
required  sequence (i.e., experiments in man prior to completion of all
animal studies).  In addition, the following points were made:

1.  "The processes involved in absorption, distribution, biotransform-
    ation,  and excretion are  dependent upon many  factors,  including
    physico-chemical  properties, extent of protein  binding, bioavail-
    ability, and dose. Some of these processes are saturable.  Products
    of biotransformation may be formed at different rates and  in  dif-
    ferent quantities, or by different pathways at high doses (e.g., 2-
    phenylphenol) . . .  It  is valid to extrapolate animal data to man
    only if the biotransformation pathways of the chemical  are  ident-
    ical or very similar between species, and if the doses do  not  ex-
    ceed  the capacity of the pathways being compared. If this capacity
    is exceeded, different metabolites may be produced.

2.  "Kinetic  data are  useful in  the design  of studies  and in  the
    interpretation  and extrapolation of the data.  For example, if the
    test material is not absorbed, the need for one or  more  long-term
    studies would be obviated.

3.  "Extrapolation of animal data to man may be compromised by differ-
    ences between species in the movement of the chemical after absorp-
    tion.   For example, the  administration of high  doses of  certain
    chemicals  may result in increased enterohepatic circulation of the
    chemical and/or its metabolites. This is an important system in the
    rat, but less so in man.

4.  "The proper design of definitive long-term studies should be based
    on comparative data on absorption, distribution, biotransformation,
    excretion,  and appropriate kinetic considerations of the test sub-
    stance." [78, p. 3-4].

    Some explanation of specific points in the above quote are required
to  clarify the intent of  JMPR.  Point 1 emphasizes the  importance of
obtaining comparative metabolism and pharmacokinetic data in humans and
the  species in  which a  toxic effect  is observed.   Although in  the

absence of such data it is assumed that biotransformation in humans and
the  test species is  similar, only comparative  metabolic studies  can
confirm the validity of the extrapolation.  In point 2, species differ-
ences  regarding absorption should  be considered.  The  variability in
gut microflora between species and the possible effects  of  intestinal
breakdown products require consideration. Similarly, the use of kinetic
data to determine whether a "steady state" has been  achieved  (i.e.,
the achievement of a state of equilibrium between intake and excretion)
is important in protocol design.

    From  the above discussion, it  is apparent that data  on pharmaco-
kinetics,  pharmacodynamics, biotransformation, and studies  on enzymes
are basic to many considerations in toxicology.  Since  toxic  activity
depends on the interaction of a chemical and a target site  (or  sites)
in  the intact animal, some  knowledge of the identity  and quantity of
the material and/or its metabolites reaching the target site is needed.
The 1986 JMPR stressed the importance of understanding  the  mechanisms
that  result in the expression  of toxicity.  It noted  that: "Current
knowledge  of mechanisms of toxicity is limited, but there is already a
sufficient  understanding in some cases  to permit better design,  per-
formance,  and  interpretation  of toxicological  studies.  Mechanistic
studies  are therefore encouraged,  since a knowledge  of mechanism  of
action is likely to result in a more rational assessment of the risk to
man." [76, p. 2].

    The  material absorbed may be  the administered chemical(s), or  it
may  be metabolites and/or reaction products of the administered chemi-
cal.   Variations in absorption  occur because of  species  differences
(especially  when  specialized  transport mechanisms  are  involved  in
absorption,  such  as those  encountered  with metals),  differences in
intestinal  flora  (discussed  extensively in  reference  [176]),  age,
nutritional  status,  dietary  fibre  content,  and  factors  affecting
motility. The identity of the absorbed material may also  differ  mark-
edly  from that administered,  due to acid-mediated  hydrolysis in  the
stomach, breakdown by gastrointestinal enzymes (e.g., splitting of pep-
tides),  chemical reactions between food  components (e.g., nitrosamine
formation  by reaction  between nitrite  and secondary  amines  in  the
stomach), and the activity of the intestinal flora.  Secondary  absorp-
tion  may also  occur, arising  from biliary  excretion and  subsequent
reabsorption  of the excreted material, either in its original excreted
form or following hydrolysis in the intestine.

    Information  about the site of  absorption of the test  material is
also  important, since this may  alter the overall metabolism  and thus
the  toxicological profile of the test substance.  If absorption occurs
in the buccal cavity, the oesophagus, or the stomach, it is  likely  to
be  distributed  widely  throughout the body in the form in which it is
absorbed. If absorption is from the small intestine, the transportation
of the absorbed material will be via the hepatic portal system  to  the
liver.  Within the liver, it may be metabolized, resulting  in  distri-
bution of metabolites rather than of parent compound.  This  factor  is
of  major importance  when considering  routes of  exposure other  than
those by the oral route.  Resolution of these potential problems can be
achieved by adequate pharmacokinetic and metabolic data.

    Once absorbed from the gastrointestinal tract, distribution depends
on a variety of factors, which may differ between and  within  species.
For  example, the age of the animal, the rate of metabolism, the degree
of  previous exposure, and  the amount and  rate of blood  flow through
different  organs  may  all affect  the  eventual  distribution of  the
absorbed  material. The distribution and, ultimately, the concentration
at  the  receptor level,  is greatly influenced  by the ability  of the
chemical  to penetrate biological membranes such as the placenta, glom-
erular membrane, and the blood/brain barrier. This, in turn, is primar-
ily a function of lipophilicity, molecular size, and extent  of  ioniz-
ation (pKa).

    The  metabolism of the absorbed material depends on an equally wide
range of variables:

 *  the degree of enzyme development is dependent on age;

 *  enzymes may vary between species, both qualitatively and quantitat-
    ively;

 *  Michaelis-Menten  kinetics indicate that saturation  of enzyme sys-
    tems may occur at some level, either increasing the  importance  of
    secondary mechanisms of metabolism or resulting in  greater  plasma
    levels of parent compound;

 *  the  site of  metabolic activity  may differ  among species  (e.g.,
    microbial metabolism in the rodent stomach, which is  not  observed
    in humans, primates, or dogs);

 *  the  rate of metabolism may  differ within and between  species and
    between different tissues and cells;

 *  interaction among test substances may occur, or metabolism  may  be
    affected  by other test substances (e.g., enzyme inhibition, stimu-
    lation, or induction);

 *  duration  of exposure (acute  or chronic) may  modify the rate  and
    pathways of metabolism.

    Both the route and rate of excretion of a test substance  may  vary
between  species.  The pharmacokinetic parameters of clearance and bio-
logical  half-life are considered to be indicators of the potential for
accumulation.   However, rapid elimination of a chemical and its metab-
olites clearly does not necessarily equate to a lack of toxicity.

    Use of radioactive labelling or heavy isotope  techniques  provides
data  on absorption, distribution, and excretion.  These studies assist
in the identification of sites of covalent binding, and  are  virtually
indispensable  in the study  of metabolism and  pharmacokinetics.  Data
from  such studies, in  conjunction with analytical  determinations  of
excreted  products,  provide the  basis  for determining  the  probable
metabolic pathways for administered compounds.  It must  be  remembered
that  in interpreting studies involving radiolabelling techniques, con-
sideration must be given to the site of the label on the  molecule  and

the  stability (mobility) of the radiolabel.  Thus, an organic molecule
containing  several  different  ring structures  may  require  multiple
studies,  with radiolabelling at  different sites in  the molecule,  to
ensure the determination of all metabolic products.

    The above list of factors is incomplete, but nevertheless serves to
indicate  the complexity of the problems associated with studies of ab-
sorption,  distribution, metabolism, and excretion of a test substance.
For useful publications covering these issues, the reader  is  referred
to  the comprehensive texts  which have been  published on the  subject
(e.g., reference [107]).

8.3.7.3     Principles

1.  Studies  on absorption, distribution, metabolism, and excretion are
    essential  in the evaluation of  the safety of a  pesticide.  These
    studies  provide a foundation for  the interpretation of all  other
    toxicology studies.

2.  Ethically  conducted  comparative  metabolic  and   pharmacokinetic
    studies  in humans and animal test species may permit more accurate
    extrapolation of animal data to humans.

9.  EVALUATION OF DATA

9.1.  Extrapolation of Animal Data to Humans

    The  objective of the  safety evaluation of  pesticide residues  in
food  is to determine  the maximum daily  intake of the  pesticide that
will  not result in adverse effects at any stage in the human lifespan.
Since,  in the  majority of  cases, data  on humans  are inadequate  to
permit  such a determination, effects observed in other species must be
extrapolated  to humans. Ideally, data on comparative pharmacokinetics,
metabolism, and mechanism of action should be utilized in the extrapol-
ation.   However, such data are not available in the majority of cases.
The  use of relevant biomarkers of exposure and effect such as the for-
mation of adducts to DNA or blood proteins like haemoglobin  in  humans
and  test  animals  may also  be  useful  in the  extrapolation  across
species.  Further research in this area is to be encouraged.

    Three  basic approaches are now generally used in the extrapolation
of the results of studies in experimental animals to humans: the use of
safety factors, the use of pharmacokinetic extrapolation  (widely  used
in the safety evaluation of pharmaceuticals), or the use of linear low-
dose extrapolation models.

    JMPR  has not utilized the  third approach (the use  of linear low-
dose extrapolation models).  A number of these models have been used to
determine  the "virtually safe dose" (VSD) of carcinogens for humans.
One major drawback of these models is the lack of consideration of many
of  the biological factors which should be taken into account. Further-
more, the various mathematical models available (Probit, Wiebel, etc.),
when applied to the same data, can result in VSD values which  vary  by
orders  of magnitude.  There is no agreement among toxicologists on the
"best" mathematical model available today, nor on whether these math-
ematical models have any biological meaning at all.

    Pharmacokinetic  extrapolation requires human pharmacokinetic data,
which  are rarely available for pesticides.  The method involves a com-
parison  of  pharmacokinetics in  human  and experimental  animals. The
relative  sensitivity of receptor  sites must also  be taken into  con-
sideration.

    The  JMPR approach has generally  been limited to the  first of the
three approaches, that is the use of safety factors.  These are applied
to the NOAEL determined from the experimental animal data,  or  prefer-
ably, from data in humans, if available.

9.2.  Safety Factors

9.2.1.  Background

    The  1963 JMPR adopted the commonly used empirical approach for the
extrapolation  of data  to man,  i.e. "the  maximum no-effect  dietary
level  obtained in animal experiments,  expressed in mg/kg body  weight
per day, was divided by a `factor', generally 100." [35, p. 11].  This
concept  appears  to have  been adopted from  the report of  the second

JECFA  Meeting which states that  ". . . a dosage level can  be estab-
lished that causes no demonstrable effects in the animals used.  In the
extrapolation of this figure to man, some margin of safety is desirable
to allow for any species differences in susceptibility,  the  numerical
differences between the test animals and the human  population  exposed
to the hazard, the greater variety of complicating disease processes in
the  human population, the difficulty  of estimating the human  intake,
and  the possibility of synergistic action among food additives." [31,
p. 17].  The Committee then stated that the 100-fold margin  of  safety
applied  to  the  maximum ineffective  dose  (expressed  in mg/kg  body
weight per day) was believed to be an adequate factor.

    The  1965 JMPR [36] discussed  the concept of the  acceptable daily
intake  and safety factors.  It noted that the 100-fold factor could be
modified according to circumstances (e.g., reduction to 10  or  20 fold
when  human data are available  or in the case  of well-studied organo-
phosphates).

    The  1966 JMPR indicated that  when a temporary ADI  was allocated,
the  margin of safety  applied to the  NOAEL derived from  experimental
animal  data should be increased [38]. These principles were applied by
the  1966 Joint Meeting when establishing a temporary ADI for pyrethrin
(safety factor of 250) [39].

    A  WHO Scientific Group  considered safety factors  in 1967  [169].
This  Group  noted that  safety factors could  be varied and  described
circumstances  where increased safety  factors should be  used.   These
included toxicological data gaps and when it was necessary to establish
temporary  ADIs.  Decreasing  the margin  of safety  was proposed  when
pertinent  biological data indicates uniform species response, when the
initial  effect  is clear-cut  and  reversible, or  when cholinesterase
inhibition  or adaptive liver enlargement is the initial effect. Other-
wise a 100-fold safety factor was considered to be a useful guide.

    The 1968 JMPR [42] indicated that, where human data  comprised  the
basis  for the  NOAEL used  in determining  the ADI,  a smaller  safety
factor  might be utilized.   This statement was  amplified by the  1969
JMPR [44] to include human biochemical as well as toxicological data as
justification for reducing safety factors.

    The  1975  JMPR, in  addressing the question  of safety factors  in
toxicological evaluation, stated that:

    "It  should be  emphasized that  the magnitude  of the  margin  of
safety  applied in each individual  case is based on  the evaluation of
all available data. In consideration of any information that gives rise
to  particular concern, the magnitude  of the margin of  safety will be
increased. Where the data provide an assurance of safety, the magnitude
may  be decreased. Therefore, it is impossible to recommend fixed rules
for the margin of safety to be applied in all instances." [54, p. 9].

    In  1977, the  JMPR "wished  to clarify  the  situation  regarding
safety factors in arriving at ADIs for man.  The establishment  of  the
ADI  for man is not a simple arithmetic exercise based on the no-effect

level,  as  the  safety factor  may  vary  widely from  one compound to
another.   Although safety factors are determined empirically, they are
dependent on the nature of the compound, the amount, nature and quality
of the toxicological data available, the nature of the toxic effects of
the compound, whether the ADI or TADI for man is established,  and  the
nature of any further data required." [57, p. 4].

    During  a discussion on  general principles used  by the JMPR,  the
1984 Meeting [72] stressed the degree of uncertainty that accompanies a
toxicological evaluation, and stated:

    "The  use of variable safety factors by the JMPR in the estimation
of  ADI values reflects this uncertainty, and underlines the complexity
of  assessing the human health hazards of pesticides.  No hard and fast
rules can be made with regard to the magnitude of this  safety  factor,
since  many aspects have to be considered, such as species differences,
individual variations, incompleteness of available data, and  a  number
of  other  matters such  as considerations of  the fact that  pesticide
residues  may be ingested  by people of  all ages throughout  the whole
life-span, that they are eaten by the sick and the healthy as  well  as
children,  and that  there are  wide variations  in individual  dietary
patterns." [72, p. 3].

    The  original  concept of  the use of  100-fold safety factors  was
based on interspecies and intraspecies variations [114].   Included  in
this  consideration were variations between strains, provision for sen-
sitive  human population sub-groups,  and possible synergistic  effects
due to exposure to more than one chemical.

    The  100-fold safety factor can  be viewed as two  10-fold factors,
one for inter- and one for intra-species variability [111]. While these
safety  factors appear, on the basis of experience, to provide adequate
margins  of safety in the  extrapolation of data to  man, they may,  of
course, be questioned. Some experimental support for safety factors was
published  by Dourson & Stara [26] in 1983. This paper also proposed an
additional 10-fold factor for extrapolating sub-chronic data,  and  for
converting  lowest-observed-adverse-effect levels to NOAELs (factors of
1-10,  depending upon the severity  and concern raised by  the observed
effect).   Additional clinical and epidemiological research may improve
the  characterization of  the variation  in response  within the  human
population to various pesticides and may allow a more  accurate  deter-
mination of safety factors.

9.2.2.  Principles

    When  determining ADIs, the 100-fold  safety factor is used  as the
starting point for extrapolating animal data to man and may be modified
in  the  light  of the data that are available and the various concerns
that arise when considering these data. Some of these are given below:

1.  When  relevant human  data are  available, the  10-fold factor  for
    inter-species variability may not be necessary. However, relatively
    few  parameters are studied in  man in the assessment  of pesticide
    safety, and data on oncogenicity, reproduction, and chronic effects

    are  rarely available.  Thus, even  if the  parameter  measured  in
    humans is the same as the most sensitive adverse  effects  measured
    in  the experimental animal  (e.g., erythrocyte cholinesterase  de-
    pression),  uncertainty still remains with respect to the potential
    effects on other parameters. This usually necessitates an increased
    safety factor. Consequently, JMPR rarely utilizes safety factors as
    low as 10-fold.

2.  The  quality of the  data supporting the  NOAELs determined in  the
    animal experiments (and also in human experiments)  influences  the
    choice of the safety factor.  Unfortunately, toxicity  studies  are
    rarely  perfect in all respects.  While a study may serve to answer
    a basic question, the degree of certainty with which  the  question
    is  answered may be reduced by, for example, increased mortality in
    all  groups  in an  oncogenicity  study, resulting  in  marginally-
    acceptable  data being available at  the termination of the  study.
    When  a  request  for a  repeat  study  is not  fully justified, an
    increased safety factor may be utilized under such circumstances.

3.  The  quality of the total data base may affect the choice of safety
    factor.   Significant data deficits may warrant an increased safety
    factor due to increased uncertainty.

4.  The type and significance of the initial toxic response  may  alter
    the  safety factor.  Thus a response which is reversible may result
    in a reduced safety factor.

5.  The  limited numbers of animals used in oncogenicity studies limits
    the sensitivity of the study in the identification of  a  threshold
    dose.   When evidence of neoplasia has been identified, safety fac-
    tors may be increased depending on the available ancillary data and
    the establishment of an NOAEL.

6.  The shape of the dose/response curve (in those cases where data are
    adequate  to permit derivation  of such a  curve) may also  be con-
    sidered in assessing safety factors.

7.  Metabolic  considerations may influence  the choice of  the  safety
    factor.   Thus, saturation of metabolic pathways resulting in toxic
    manifestations,  biphasic metabolic patterns, and  data on compara-
    tive metabolism may all affect the magnitude of the safety factor.

8.  Knowledge  of the comparative mechanism  of toxic action in  exper-
    imental animals and man may influence the choice of safety factor.

    Several of the factors cited above may apply in  the  consideration
of  any one compound.  Certain factors may serve to increase and others
to  decrease the choice of the final safety factor.  Therefore, it must
be stressed that the total weight of evidence has to be  considered  in
determining  the appropriate  safety factor  to be  used and  that  the
determination  of safety factors must  be considered on a  case-by-case
basis.

9.3.  Allocating the ADI

9.3.1.  Background

    The FAO/WHO Joint Meeting on Principles Governing  Consumer  Safety
in  Relation to Pesticide Residues indicated that the assessment of the
amount of pesticide to which man can be exposed daily for  a  lifetime,
without  injury, was the  primary aim of  toxicological investigations.
The  Meeting indicated that  "when the (toxicological)  investigations
are completed, it is possible, by the use of scientific  judgement,  to
name  the  acceptable daily  intake."  [32, p. 9].  The  meeting  also
defined the ADI as follows:

    "The  daily dosage of a chemical which, during an entire lifetime,
appears  to  be  without appreciable risk on the basis of all the facts
known  at the time.   `Without appreciable risk'  is taken to  mean the
practical certainty that injury will not result even after  a  lifetime
of exposure.  The acceptable daily intake is expressed in milligrams of
the  chemical,  as  it appears in the food, per kilogram of body weight
(mg/kg)." [32, p. 5].

    The first JMPR adopted this definition and discussed the concept of
the ADI.  The Meeting stated that the following information  should  be
available in order to arrive at an ADI:

(a) "the chemical nature of the residue. Pesticides may undergo chemi-
    cal changes and are frequently metabolized by the tissues of plants
    and  animals which have been treated with them.  Even when a single
    chemical  has been applied, the residues may consist of a number of
    derivatives with distinct properties, the exact nature of which may
    differ in animals and plants and in different crops and products.

(b) the  toxicities of the chemicals  forming the residues from  acute,
    short-term and long-term studies in animals. In addition, knowledge
    is  required of the  metabolism, mechanism of  action and  possible
    carcinogenicity of residue chemicals where consumed.

(c) A  sufficient knowledge of the effects of these chemicals in man."
    [35, p. 6].

    The  Meeting also noted that  the identity of the  food bearing the
chemical  should theoretically be immaterial;  that the ADI was  an ex-
pression of opinion, which carried no guarantee of "absolute" safety;
that  new knowledge or  data could always  lead to re-evaluation  of an
ADI;  and  that  JMPR would confine itself to proposing a single set of
ADI  figures for pesticides.   Finally, the Meeting  stated that  "The
proposed  levels (of  ADIs) could  normally be  regarded as  acceptable
throughout  life; they are not set with such precision that they cannot
be  exceeded  for  short periods  of  time."  [35, p. 7] (see  section
9.3.3).

    Although the ADI can be exceeded for short periods of time,  it  is
not possible to make generalization on the duration of the  time  frame
which  may cause concern.   The induction of  detrimental effects  will

depend upon factors which vary from pesticide to pesticide.   The  bio-
logical half-life of the pesticide, the nature of the toxicity, and the
amount by which the exposure exceeds the ADI are all crucial.

    The  large safety factors generally involved in establishing an ADI
also  serve to provide  assurance that exposure  exceeding the ADI  for
short  time periods is  unlikely to result  in any deleterious  effects
upon health.  However, consideration should be given to the potentially
acute  toxic effects that are not normally considered in the assessment
of an ADI.

    The  principles discussed above  were adopted by  subsequent  Joint
Meetings  but, as would be  expected, have been further  developed with
time.   Thus the 1968 JMPR [42] indicated that metabolites would, under
certain conditions, be considered to be included in the ADI. Generally,
if the metabolites in food commodities are qualitatively and quantitat-
ively  the same as those  observed in laboratory test  species, the ADI
would  apply  to  the parent compound as well as to metabolites. If the
metabolites  are not  identical or  not present  at the  same order  of
magnitude,  separate studies on the metabolites may be necessary.  When
one  or  several  pesticides  are  degradation  products   of   another
pesticide,  a single ADI may  be appropriate for the  pesticide and its
metabolites,  e.g.,  oxydemeton-methyl, demeton- S -methyl   sulfone and
demeton- S -methyl  [183].

    In  1973, when considering  the accuracy with  which ADIs or  TADIs
could  be estimated,  JMPR recommended  that ADIs  should be  expressed
numerically  using only one  significant figure [52].  The use of  more
than one significant figure might be taken to imply a greater degree of
accuracy than that which can be achieved when assessing the hazard from
the wide range of factors that influence toxicity.

9.3.2.  Temporary ADIs

    Use  of the TADI,  first proposed by  the Scientific Group  on Pro-
cedures  for Investigating Intentional  & Unintentional Food  Additives
[169], was adopted by JMPR in 1966.  Criteria were set that had  to  be
met  prior to the  establishment of the  TADI. These included  the con-
sideration of each chemical on its own merits, the establishment of the
TADI  for a fixed period (usually 3-5 years), and the subsequent review
of  original and new  data prior to  the expiration of  the provisional
period.

    The  establishment  of  a TADI  has  always  been accompanied  by a
requirement for further work by a specified date and by the application
of  an increased safety factor.  The 1972 JMPR considered the course of
action to be taken if requested data were not forthcoming and indicated
that, under these circumstances, the TADI would be withdrawn. It empha-
sized,  however, that such an  action "did not necessarily  indicate a
potential  health  hazard, but  only  that insufficient  information is
available  at the time of  review to permit the  Meeting to state  with
reasonable  certainty that there is no likelihood of adverse effects on
health resulting from ingestion over a prolonged period." [50, p. 7].

    In  1986 [76], JMPR  indicated that the  previously utilized  terms
"Further  work or information required"  or "Further work or  infor-
mation  desirable" were being  replaced, the former  by the  statement
"Studies without which the determination of an ADI is impracticable",
and  the latter by  the statement "Studies  which will provide  infor-
mation valuable to the continued evaluation of the  compound."   These
new  statements not only reflect the actual work performed by JMPR much
more  clearly than the  previous terms "Required"  and "Desirable",
but they also reflect the Meeting's increasing reluctance  to  allocate
temporary  ADIs  as  well as the desire to continue the evaluation of a
compound even after an ADI has been allocated.

    In 1988 [79], JMPR recommended that TADIs should not  be  allocated
for  new  compounds  and that  an ADI  should not  be allocated  in the
absence  of an adequate data base. The Meeting intended that monographs
be  published  for  all chemicals  which  are  reviewed, regardless  of
whether an ADI is allocated, and that data requirements will be clearly
specified for those chemicals with an inadequate data base.

    The concept of the "conditional acceptable daily intake", adopted
by the 1969 JMPR [44], was limited to those compounds for which the use
was at that time considered essential but for which  the  toxicological
data base was incomplete. This concept, which is unacceptable, has been
abandoned.

9.3.3.  Present position

    The  minimum data base normally utilized in determining an ADI com-
prises  short-term feeding studies, long-term feeding studies, carcino-
genicity  studies, multigeneration reproduction studies, teratogenicity
studies,  and acute and repeated exposure metabolic, toxicokinetic, and
toxicodynamic data.  Where deemed necessary, additional special studies
may also be required, e.g., genotoxicity studies.

    The  NOAEL from the most appropriate study divided by the appropri-
ate  safety factor determines the ADI.  The lowest NOAEL is not necess-
arily the basis for the ADI (see section 8.2.1).  Thus, even though the
NOAEL from a chronic toxicity study may be less than that from a repro-
duction study, the latter may serve as the basis for assessing the ADI,
because  of the potential  use of a  higher safety factor  (see section
9.2). On this basis, the entire age range of the population is normally
covered by the ADI. The present procedure therefore provides an accept-
able  margin of safety  to the entire  population for those  pesticides
with complete data bases.  The advantage of providing separate ADIs for
different age (or physiological) groups of the population, would there-
fore be limited to indicating those groups who may be in a reduced-risk
category, rather than indicating those at increased risk.

    A document entitled "Guidelines for Predicting Dietary  Intake  of
Pesticide Residues" was published by WHO in 1989 [177].  This document
provides guidance on the prediction of the dietary intake  of  residues
of  a pesticide for the purpose of comparison with the ADI allocated by

JMPR.  The document recommends a step-wise approach to  predicting  in-
take,  considering average consumption of the treated commodities and a
number  of factors (such  as processing, variations  in residues  level
with time and the percentage of a given commodity that is treated) that
usually have the effect of providing a more accurate prediction of real
pesticide  residue intake.  An  example of dietary  intake calculations
for  a hypothetical pesticide is given in Chapter 3 of "Guidelines for
Predicting Dietary Intake of Pesticide Residues."

10.  EVALUATION OF MIXTURES

10.1.  Introduction

    Survey data indicate that residues of more than one  pesticide  may
be detected in food.  This gives rise to concern over  the  possibility
of  unanticipated interactions between such residues leading to adverse
toxicological  effects.   There is,  of  course, a  virtually unlimited
number  of combinations of pesticides on various crops. There is also a
very large number of combinations of foods containing  pesticide  resi-
dues.

10.2.  Background

    The possibility of pesticide interaction was recognized as early as
1961 when the FAO/WHO Meeting on Principles Governing  Consumer  Safety
in  Relation to Pesticide  Residues recognized that  "different pesti-
cides  and  other chemicals  are  often absorbed  simultaneously during
occupational  use,  or in  food, by man  or animals" [32, p. 10].  The
first  JMPR [35]  also noted  the possibility  of interactions  between
chemicals in discussions on the shortcomings of the ADI. It  was  indi-
cated that ADI values were calculated on the assumption that  the  diet
was  contaminated by a single  residue, hence additive and  synergistic
effects were not considered.  An extensive review of  the  significance
of interactions of pesticides was performed by the 1967 JMPR [40].  The
1981  Joint Meeting gave  further consideration to  interaction between
pesticide residues and concluded that:

1.  "Not  only could pesticides interact,  but so could all  compounds
    (including those in food) to which man could be exposed. This leads
    to  unlimited possibilities, and there is no special reason why the
    interactions of pesticide residues (which are at very  low  levels)
    should be highlighted as being of particular concern;

2.  "Very little data on these interactions are available;

3.  "The data obtained from acute potentiation studies are  of  little
    value in assessing ADIs for man." [62, p. 12].

10.3.  Principle

    The  consideration of  mixtures of  residues does  not require  any
change in the general principles for estimating ADIs. However, there is
a need for further data on interactions of pesticides with  each  other
and with other common contaminants of food (e.g.,  metals,  mycotoxins)
to  ensure that, at the very low levels of pesticide exposure likely to
occur  via dietary residues,  and over the  prolonged time periods  in-
volved in such human exposure, no adverse effects are likely to occur.

11.  RE-EVALUATION OF PESTICIDES

    In  1961, the Meeting on  Consumer Safety in Relation  to Pesticide
Residues  stated that "of  necessity early views  of the amount  (ADI)
will be estimated and subject to revision as  experience  accumulates"
[32, p. 9]. Thus, from its inception, the provisional nature of the ADI
has  been recognized [35].   The 1965 Meeting  [36] re-examined the  37
pesticides  reviewed in 1963 [35].  Changes in the ADIs were instituted
for  16 of these pesticides,  based on additional information  that had
become available.

    The need for a full re-evaluation of the toxicity data base on some
pesticides was identified by the 1981 JMPR [65], based on concerns over
the validity of previously submitted data (see section 5.1).  The first
of these re-evaluations was undertaken in 1982 [64]. The development of
new  methods for  investigating toxicity  has also  caused  concern  in
relation to pesticides for which ADIs have been established [52].

    The  use of the  TADI [40] ensures  re-evaluation of the  data base
pertaining to specific compounds, since one of the criteria for setting
a  TADI  is  that identified  data  are  required for  evaluation  by a
specific  time.  However, a more systematic method of re-evaluation has
been  suggested  such  as  the  automatic  re-evaluation  of  chemicals
reviewed more than 10 years previously [72, p. 8].

    Establishing  a priority order  for the re-evaluation  of compounds
requires input from a number of sources including the  Codex  Committee
on Pesticide Residues (CCPR). This Committee has initiated this process
for pesticides evaluated prior to 1976 [30].

12.  BIOTECHNOLOGY

    Biotechnology  comprises a number  of different approaches  to pest
control.  Three areas are of emerging concern: the production of chemi-
cals  of  biological origin  with  pest-control activity  (e.g., hydro-
prene);  the use  of microbial  pest control  agents  (e.g.,  bacteria,
fungi, viruses, and protozoa); and the development and use  of  geneti-
cally altered (bioengineered) organisms for specific purposes.

    At  the present time,  JMPR has no  experience with these  types of
pest-control  products other than limited  experience with biologically
derived  chemicals (e.g., pyrethrin). The following comments are there-
fore proposals leading to approaches which may be feasible in assessing
the safety of such products.

    First, with regard to the so-called "biorational" products, these
chemicals are derived from or are synthesized to be identical  to  nat-
urally occurring pesticidal agents.  The fact that they  are  naturally
occurring  does not necessarily  mean that they  are safe.  Thus,  such
chemicals  should be investigated  in the same  way as other  synthetic
chemicals used as pesticides. In certain instances, it is possible that
justification  for reducing the  necessary toxicological data  base may
exist.

    In  dealing  with  microbial pest-control  agents and bioengineered
organisms,  two factors are of primary importance to human health - the
infectivity of the residual organism and the ability of the organism to
produce  toxins which occur as residues.  In the case of viruses, their
ability to incorporate into the cell genome should also be considered.

    The determination of the safety of microorganisms should  follow  a
tiered approach, tier 1 being the determination of infectivity and tox-
icity  based on  acute administration.  If measurable  survival of  the
microorganisms  in the test animal is still apparent several days after
administration, short-term feeding studies may be deemed to  be  appro-
priate. If exotoxins are produced by the microorganisms, then the toxin
should be isolated, identified, and subjected to tests similar to those
for  any other chemical utilized as a pesticide. Similarly, if an endo-
toxin  is produced and  there is evidence  that this material  could be
released, the endotoxin should also be subjected to standard toxicology
testing,  as required for  other chemical pesticides.  In the event  of
both endo- and exo-toxin having potential access to humans or  to  dom-
estic animals, consideration should be given to  simultaneous  adminis-
tration  of the two  compounds in toxicity  studies.  If  circumstances
exist that would indicate the possibility of waiving any of the routine
toxicity   tests,  scientifically  supported  evidence  indicating  the
absence of need for such tests must be provided.

13.  SPECIAL CONSIDERATIONS FOR INDIVIDUAL CLASSES OF PESTICIDES

13.1.  Organophosphates - Ophthalmological Effects

    In  1972, JMPR noted published reports suggesting that certain oph-
thalmological  effects may be induced  by exposure to some  organophos-
phate insecticides [50]. Insufficient information was available at that
time  to permit a toxicological  assessment of the significance  of the
reports.  In 1979, additional reports were considered by JMPR [52]. The
Meeting  again concluded that insufficient information was available to
permit an evaluation.  No additional information has been considered by
JMPR.

13.2.  Organophosphates - Aliesterase (carboxylesterase) Inhibition

    The 1967 JMPR [40], in considering interactions between pesticides,
noted  that "some  of the  aliesterases are  more sensitive  than  the
cholinesterases  to  inhibition  by certain  organophosphorus compounds
[83].   Furthermore,  those organophosphates  that  are more  active as
aliesterase inhibitors than as cholinesterase inhibitors appear  to  be
the  most effective in potentiating  the toxicity of other  organophos-
phates [27].  Also, the aliesterases participate in the detoxication of
many  of the  organophosphates and  probably other  chemicals to  which
humans  may be exposed.  For these reasons,  it is suggested  that con-
sideration  be  given to  the use of  no-effect levels for  aliesterase
inhibition  rather than no-effect levels for cholinesterase inhibition,
as a basis for estimating the daily acceptable intakes of those organo-
phosphorus  insecticides to which the aliesterase systems are more sen-
sitive than are the cholinesterases" [40, p. 38].

    In  1972, JMPR noted  that short-term feeding  studies demonstrated
the  fact that in  the case of  many organophosphate pesticides,  inhi-
bition of liver and serum carboxylesterases was a more  sensitive  par-
ameter than inhibition of cholinesterases [122; 151]. However, it noted
that "the physiological significance of carboxylesterase inhibition is
still  unknown" [50, p. 8].  Since carboxylesterase inhibition appears
to  be  a factor  in potentiation, JMPR  indicated the desirability  of
further work in this area.  The 1974 Meeting [53] reiterated  the  need
for information to determine the usefulness of  aliesterase  inhibition
in assessing the safety of organophosphate compounds.

    Carboxylesterases  mainly  hydrolyze  aliphatic esters,  but  their
substrate specificity is not absolute. They can also hydrolyze aromatic
esters  at measurable rates.  There is evidence for marked variation in
humans,  which is genetically  determined. Since there  is such  varia-
bility,  yet no data on  the toxicological significance of  these rela-
tively  non-specific enzymes, they are unlikely to be used to determine
NOAELs  in the evaluation  of organophosphorus compounds.   It  should,
however,  be  noted that  some  organophosphate impurities  are  potent
carboxylesterase  inhibitors and hence markedly potentiate the toxicity
of pesticides that are detoxified by these enzymes (e.g., malathion).

13.3.  The Need for Carcinogenicity Testing of Organophosphates

    In 1986, JMPR noted that organophosphate compounds tend not to show
genotoxicity  in vivo  or to induce carcinogenic responses in laboratory
animals  [76]. It was recommended that careful evaluation of all avail-
able  data  should be  performed  to determine  whether carcinogenicity
tests  are required for  individual organophosphate pesticides.   It is
also  recommended that the possible structure-activity relationships of
the non-phosphate ester moiety of the pesticide should be considered.

13.4.  Ocular Toxicity of Bipyridilium Compounds

    The pyridilium herbicides diquat and paraquat were  first  reviewed
by JMPR in 1970 [77].  The studies on diquat demonstrated the induction
of  lens opacities in rats,  dogs, and cows.  Studies  on paraquat, for
the  same  duration  and at the same dose levels as for diquat, did not
demonstrate ocular effects in any species tested.

    Additional  data on both compounds were evaluated in 1972 [51].  At
that  time, it was demonstrated that prolonged administration of diquat
was required to induce cataracts. The type of cataract induced differed
structurally  from those observed due to physical or disease processes.
Again,  no evidence of compound-related ocular damage was noted in rats
or mice treated with paraquat.

    In 1977, additional data on diquat showed that although  the  inci-
dence  of cataracts  was no  higher than  that of  control animals,  an
earlier  appearance  was  observed [58].  Because  the  data  base  for
paraquat  had been generated  by Industrial Biotest  Laboratories, most
studies  of this  chemical were  repeated.  These  repeat studies  were
evaluated  by  the  1986 JMPR [77].  No cataracts were induced in a one
year  study in dogs or in a long-term feeding study in mice.  Cataracts
were  observed in Fisher  (but not Wistar)  rats in long-term  studies.
Microscopic  examination of these cataracts showed that, in contrast to
diquat-induced cataracts, there was a close similarity  to  age-related
cataracts  in control animals.  There is, therefore, some evidence that
paraquat may cause some ophthalmological toxicity, even though this has
only  been  observed in  one strain of  one species, and  even then the
lesions  noted are similar in type to age-related lesions.  However, it
would be advisable to perform careful ophthalmological studies  on  any
bipyridilium compounds that may be developed as future herbicides.

13.5.  Goitrogenic Carcinogens

    A  probable mechanism  for this  class of  compounds  is  described
below.

    Diets  low in iodine, causing  chronic iodine deficiency in  exper-
imental  animals, lead to hypertrophy, hyperplasia, and follicular cell
neoplasia  of the thyroid gland and pituitary gland adenomas [6; 8; 80;
81; 145].  These effects have also been observed with subtotal thyroid-
ectomy  [24], splenic transplantation of  thyroid tissue [10], and  the
transplantation  of pituitary tumours that  secrete thyroid-stimulating
hormone  (TSH) [24; 90; 148].  The fact that none of these experimental

techniques introduced exogenous agents, other than transplanted tissue,
into  the animal's internal  environment indicated that  the  causative
oncogenic  mechanism must reside within the animal and must be mediated
through  the intimate interrelationship  of the pituitary  and  thyroid
glands.

    This  led to a  concept, subsequently supported  with  experimental
data,  of a  negative feedback  system which  maintained a  homeostatic
balance between the pituitary and thyroid gland secretions.  Later, the
hypothalamus  was added to this  system when it was  discovered that it
exerted  some  control  over  pituitary  gland  secretions.  Subsequent
research  indicated that, while the hypothalamus is essential to normal
pituitary  and thyroid gland functioning, the receptors residing within
the  pituitary/thyroid axis are  of primary importance  in  controlling
thyroid and pituitary hormonal balance [86; 97].  Disturbance  of  this
balance  has significant physiological and morphological effects on the
glands as well as on the well-being of the animal [118].

    Exogenous   physical   and   chemical  agents   can   also   induce
thyroid/pituitary  hypertrophy,  hyperplasia, and  neoplasia by causing
hormonal  imbalance [9; 102; 180].  The chemical  goitrogens were first
discovered  in animal and human  food items [15; 88; 149].  Since  then
many  chemically  defined  substances  have  been  reported  to  induce
thyroid/pituitary   hypertrophy,  hyperplasia,  and,   after  prolonged
exposure, neoplasia. Radioactive iodine and x-rays can produce the same
effects [17; 25; 96; 98].  The mechanisms by which these substances and
the  non-agent  experimental  techniques produce  their pharmacological
(goitrogenic)  and neoplastic effects are  well known, even though  the
precise  triggering event for  the transformation from  hyperplasia  to
neoplasia  is still uncertain  [9; 85; 87]. The most  common mechanisms
are  interference with the  thyroid iodide transport  system or  inter-
ference  with peroxidases essential to  the synthesis and secretion  of
competent thyroid hormone [15; 18; 95; 138; 149; 156].

    The  sequence of events triggered by this interference is also well
understood.   As the circulation of  competent thyroid hormone (TH)  is
reduced,  the receptors in the hypothalamus and pituitary gland receive
a signal for secretion of TSH.  Receptors within the  thyroid  receive,
through TSH, a signal for increased TH production and secretion and the
gland responds, at first, with functional hypertrophy  [86; 97].   Thus
far these events may remain within the normal operation of the feedback
system.   At this stage, if the cause of the thyroid hormone deficiency
is removed or corrected, the circulation of competent TH increases to a
critical level and TSH secretion is reduced. As homeostasis is reestab-
lished, the thyroid gland returns to normal.  However, under conditions
of chronic TH deficiency and the failure of the feedback  mechanism  to
restore  hormonal balance,  both glands  continue to  respond to  their
respective signals and enter hypertrophic and hyperplastic states.  The
pituitary  continues to secrete TSH, to which the thyroid responds, but
the  thyroid cannot signal for TSH shut off because of its inability to
secrete competent TH.  Eventually this relationship results in hormonal
imbalance that induces thyroid gland follicular cell neoplasia and fre-
quently pituitary gland neoplasia.

    The  hypothesis  that  thyroid/pituitary hormonal  imbalance is the
oncogenic  mechanism is supported by evidence that follicular cell neo-
plasia can be prevented by the simultaneous administration  of  goitro-
gens and thyroid hormone.  This has been demonstrated  with  thiouracil
[9]  and thiourea [143], two  members of a class  of potent goitrogens.
The  pharmacological effects, hypertrophy and  hyperplasia, are revers-
ible  upon  removal  of the  goitrogenic  stimulus [4; 9; 85; 106; 140;
153]. Furthermore, NOELs have been demonstrated in several species, for
both the goitrogenic and neoplastic effects of thyroid function inhibi-
tors. These facts coupled with evidence that treatment of  human  hypo-
thyroidism  with goitrogens is without appreciable risk of thyroid neo-
plasia [92], support the concept of a threshold  for  goitrogen-induced
thyroid follicular cell and pituitary neoplasia [137; 138].

    The  weight-of-evidence indicates that goitrogens occupy an unusual
nitch in oncogenesis in that:

 *  their  pharmacological effects and mechanisms of action are reason-
    ably well understood;

 *  their pharmacological effects are reversible;

 *  thresholds,  NOELs, and NOAELs can be established for their pharma-
    cological and neoplastic effects;

 *  pituitary  and  thyroid  neoplasia potentially  induced  by thyroid
    inhibitors  can be prevented by supplying experimental animals with
    competent thyroid hormones during treatment with goitrogens;

 *  a  certain degree of  thyroid inhibition is  accommodated for  pro-
    longed  periods within the homeostatic  control limits of the  nor-
    mally functioning feedback mechanism;

 *  long-term  exposure to excessive  TSH is required  before  hormonal
    imbalance induces thyroid follicular cell neoplasia.

    Increased  TSH secretion is the ultimate common mediator of thyroid
follicular  proliferative lesions induced  by goitrogens, its  level is
moderated  by a feedback mechanism, and its neoplasm-inducing potential
is  subject to mechanisms demonstrating  threshold effects.  Laboratory
animal data demonstrate that there is an ordered linkage of steps: thy-
roid blockade, continuous TSH release, thyroid hypertrophy/hyperplasia,
modularity, and adenoma/carcinoma.  They also show that a threshold for
an  early step automatically becomes a threshold for the whole chain of
steps.   These characteristics should be a major consideration when as-
sessing the human oncogenic potential of thyroid-function inhibitors.



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ANNEX I.  GLOSSARY

I.1  Abbreviations Used in this Document

ADI        Acceptable Daily Intake

CEC        Commission of the European Communities

CNS        Central Nervous System

FAO        Food and Agriculture Organization of the United Nations

GLP        Good Laboratory Practice

IARC       International Agency for Research on Cancer

IPCS       International Programme on Chemical Safety

JECFA      Joint FAO/WHO Expert Committee on Food Additives

JMPR       Joint FAO/WHO Meeting on Pesticide Residues

LD01       Lethal Dose, 1%

LD50       Lethal Dose, median

MRL        Maximum Residue Level

MTD        Maximum Tolerated Dose

NCI        National Cancer Institute (USA)

NOAEL      No-Observed-Adverse-Effect Level

NOEL       No-Observed-Effect Level

NTE        Neurotoxic Esterase

NTP        National Toxicology Program (USA)

OECD       Organization for Economic Cooperation and Development

OP         Organophosphate

SAR        Structure/Activity Relationship

TADI       Temporary Acceptable Daily Intake

TH         Thyroid Hormone

TOCP       Tri- O -Cresyl  Phosphate

VSD        Virtually Safe Dose

TSH        Thyrotropin

WHO        World Health Organization

I.2  Definitions of Terms Used in this Document

Acceptable Daily Intake (ADI): An estimate by JMPR of the amount  of  a
pesticide, expressed on a body weight basis, that can be ingested daily
over  a  lifetime without  appreciable health risk  (standard man =  60
kg).

Codex  Alimentarius Commission:  The Commission  was formed in 1962  to
implement the Joint FAO/WHO Food Standards Programme.   The  Commission
is an intergovernmental body made up of more than  130 Member  Nations,
the  delegates of whom represent their own countries.  The Commission's
work  of harmonizing  food standards  is carried  out  through  various
committees,  one of which is the Codex Committee on Pesticide Residues.
JMPR serves as the advisory body to the Codex  Alimentarius  Commission
on all scientific matters concerning pesticide residues.

Effect: A biological change in an organism, organ, or tissue.

Elimination  (in metabolism):  The  expelling of a  substance or  other
material  from  the  body (or  a  defined  part thereof),  usually by a
process  of  extrusion or  exclusion,  but sometimes  through metabolic
transformation.

Embryo/fetotoxicity: Any  toxic effect on the  conceptus resulting from
prenatal  exposure, including structural or functional abnormalities or
postnatal manifestation of such effects.

JMPR: JMPR is a technical committee of JMPR specialists acting in their
individual  capacities. Each is a separately-constituted committee, and
when either the term "JMPR" or "the Meeting" is used, it  is  meant
to  imply the common policy or combined output of the separate Meetings
over the years.

Long-term toxicity study:  A study in which animals are observed during
the whole life span (or the major part of the life span) and  in  which
exposure  to the test material  takes place over the  whole observation
time or a substantial part thereof.  The term chronic toxicity study is
used sometimes as a synonym for "long-term toxicity study".

Lowest-observed-effect  level (LOEL):  The lowest dose of  a  substance
which  causes  changes distinguishable  from  those observed  in normal
(control) animals.

No-observed-adverse-effect level (NOAEL):  The highest dose of  a  sub-
stance at which no toxic effects are observed.

No-observed-effect level (NOEL):  The highest dose of a substance which
causes no changes distinguishable from those observed in  normal  (con-
trol) animals.

Safety  factor:  A  factor applied  by JMPR  to the  no-observed-effect
level  to derive an  acceptable daily intake  (the no-observed-adverse-
effect  level is divided  by the safety  factor to calculate  the ADI).
The  value of the  safety factor depends  on the nature  of  the  toxic
effect, and the quality of the toxicological information available.

Short-term  toxicity study:  An animal  study (sometimes called a  sub-
acute  or subchronic study) in  which the effects produced  by the test
material,  when administered in repeated doses (or continuously in food
or drinking-water) over a period of about 90 days, are studied.

Temporary ADI:  Used by JMPR as an administrative procedure  to  permit
the  continued acceptance of  the pesticide pending  submission of  new
toxicological data.

Teratogen:   An agent which, when administered prenatally, induces per-
manent abnormalities in structure.

Teratogenicity:   The  property  (or potential)  to  produce structural
malformations or defects in an embryo or fetus.

Threshold dose:  The dose at which an effect just begins to occur, that
is,  at a dose immediately below the threshold dose the effect will not
occur,  and immediately above the threshold dose the effect will occur.
For  a given chemical there can be multiple threshold doses, in essence
one  for each definable effect. For a given effect there may be differ-
ent threshold doses in different individuals. Further, the  same  indi-
vidual may vary from time to time as to his or her threshold  dose  for
any  effect.  However, given  the present state  in the development  of
science, for certain chemicals and certain toxic effects,  a  threshold
dose may not be demonstrable.

    The  threshold dose will fall between the experimentally determined
no-observed-effect  level  (NOEL) and  the lowest-observed-effect level
(LOEL).  Of importance is that when using the NOEL or LOEL,  it  should
be  specified which effect is  being measured, in what  population, and
what is the route of administration. In situations for which the effect
of concern is considered to be adverse, the terminology often  used  is
that  of a no-observed-adverse-effect level (NOAEL) or lowest-observed-
adverse-effect  level (LOAEL), again  specifying the effect,  the popu-
lation,  and the route of  administration.  Both the NOEL  and LOEL (as
well  as the NOAEL  and LOAEL) have  been used by  different scientific
groups as a surrogate for the threshold dose in the performance of risk
assessments.

Toxicity:   The toxicity of a compound is its potential to cause injury
(adverse reaction) to a living organism.


 ANNEX II.  APPROXIMATE RELATION OF PARTS PER MILLION IN THE 
DIET TO MG/KG BODY WEIGHT PER DAYa
----------------------------------------------------------------------------------------------------
                    Weight        Food consumed     Type of     1 ppm in food      1 mg/kg body
                     (kg)           per day (g)      diet       = (mg/kg body      weight per day
Animal                          (liquids omitted)               weight per day)    = (ppm of diet)
----------------------------------------------------------------------------------------------------
Mouse                 0.02             3                            0.150                7

Chick                 0.40            50                            0.125                8

Rat (young)           0.10            10            Dry             0.100               10
                                                    laboratory
Rat (old)             0.40            20            chow            0.050               20
                                                    diets
Guinea-pig            0.75            30                            0.040               25

Rabbit                2.0             60                            0.030               33

Dog                  10.0            250                            0.025               40
----------------------------------------------------------------------------------------------------
Cat                   2              100                            0.050               20

Monkey                5              250            Moist,          0.050               20
                                                    semi-solid
Dog                  10              750            diets           0.075               13

Man                  60             1500                            0.025               40
----------------------------------------------------------------------------------------------------
Pig or sheep         60             2400                            0.040               25

Cow                 500             7500            Relatively      0.015               65
 (maintenance)                                      dry grain
                                                    forage
Cow                 500           15 000            mixtures        0.030               33
 (fattening)

Horse               500           10 000                            0.020               50
----------------------------------------------------------------------------------------------------
a  Lehman, A.J. (1954)  Association of Food and Drug Officials Quarterly Bulletin, 18: 66. The
   values in this table are average figures, derived from numerous sources.

Example:   What  is the value in  ppm and mg/kg body  weight per day  of 
           0.5% substance X mixed in the diet of a rat?

Solution:  I.  0.5% corresponds to 5000 ppm.

           II. From  the table, 1 ppm in the diet of a rat is equivalent
               to  0.050 mg/kg body weight  per day. Consequently,  5000
               ppm  is  equivalent  to  250 mg/kg  body  weight  per day
               (5000 x 0.050).
                                   


INDEX
 
Absorption
Acetylcholinesterase inhibition
Acute studies
ADI, 
    conditional
    temporary
Autolysis
 
Behavioural toxicity
Biochemical studies
Bioengineered organisms
Biological half-life
Biomarkers
Biorational products
Bipyridilium compounds
Body weights
 
Carbamates
Carboxylesterases
Carcinogenic pesticides
Carcinogenicity 
    classification schemes
    genotoxic
    limited evidence of
    organophosphates
    principles
    studies
    testing
Clearance
Clinical chemistry
Codex Committee on Pesticide Residues
Commonly occurring tumours
Comparative metabolic data
Comparative pharmacokinetic data
 
Delayed neurotoxicity
Dietary intakes
Dose/response 
    and safety factors
    from accidental poisonings
    in carcinogenicity
    in delayed neuropathy
    in human volunteers
    relationships
 
Electron microscopic examination
Enterohepatic circulation
Environmental Health Criteria
Epidemiological studies
 

 
FAO
Food intake
 
Goitrogenic carcinogens
Good Laboratory Practices (GLP)
 
Haematological examinations
Half-life
Histopathological examinations
Historical control data
Human 
    Cell lines
    Volunteers
 
Immunotoxicity
Impurities
In utero
In vitro
In vivo
Industrial Bio-Test Laboratories
Inert ingredients
Ingested dose
Intermediates
International Agency for Research on Cancer (IARC)
IPCS
Isomers
 
JECFA
 
Lactation
LD01
LD50
Long-term studies 
    and ADI
    and reproduction
    conduct of
    interpretation of
 
Maternal toxicity
Maximum Tolerated Dose (MTD)
Mechanisms of toxicity
Metabolites 
    (animal)
    (plant)
Michaelis-Menten kinetics
Mixtures
Mouse liver tumours
MRLs
Mutagenicity


 
Neuropathy target esterase (NTE)
Neurotoxicity
Nitrosamines
No-observed-adverse-effect-level (NOAEL)
 
Occupational exposure
Oncogenes
Ophthalmological effects
Organophosphates
 
Peroxisome proliferation
Pharmacokinetic data
Plasma cholinesterase
Poison Control Centres
Proprietary data
Protein binding
Pyrethroids
 
Radioactive labelling techniques
Re-evaluation of pesticides
Reproduction
    and ADI
    dose response
    follow-up studies
    maternally toxic doses
    (multigeneration) study
Routes of exposure
 
Safety factors 
    and ADI
    and TADI
    determination of
    in absence of toxicity
    in carcinogenicity
Satellite groups
Screening teratology studies
Short-term studies
Special stains
Sperm measurements
Stability
Statistical analysis
Structure-activity relationships
 
Task Force of Past Presidents of the Society of Toxicology
Technical grade
Teratogenicity
Tetrachloro-dibenzo-p-dioxin (TCDD)
Threshold
Thyroid 
    Hormone
    Neoplasia
Tolerance
Tumours
    benign
    malignant
 
Urinalysis
 
Validity of data


    See Also:
       Toxicological Abbreviations