115 
 
Guidance for Industry 
 
 
Safety Studies for Veterinary Drug Residues 
in Human Food: Reproduction Toxicity 
Testing VICH GL22 
 
FINAL GUIDANCE 
 
(This version of the guidance replaces the version that was made available in April 
21, 2004. This guidance document has been revised to correct the contact 
information in regard to this document.) 
 
 
This document provides guidance on reproduction safety studies for veterinary drug 
residues in human food.  
 
Comments and suggestions regarding the document should be submitted to Dockets 
Management Branch (HFA-305), Food and Drug Administration, 5630 Fishers Lane, Rm. 
1061, Rockville, MD  20852. Submit electronic comments to http://www.regulations.gov.   
All comments should be identified with the Docket No. 00D-1630.   
 
For questions regarding this document, contact the Division of Human Food Safety, 
Center for Veterinary Medicine, (HFV-150), Food and Drug Administration, 7500 Standish 
Place, Rockville, MD 20855, 240-276-8211. 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Veterinary Medicine 
July 27, 2006 
 
   
 
VICH GL22 (SAFETY: REPRODUCTION) 
June 2001 
For implementation at Step 7 
 
 
 
 
 
 
 
 
S T E T S  
TUDIES  O  VALUATE  HE  AFETY
O R O V  
  F  ESIDUES  F  ETERINARY 
D I H F :  
RUGS  N  UMAN  OOD  
REPRODUCTIVE TOXICITY TESTING 
 
 
 
 
 
 
Recommended for Implementation  
at Step 7 of the VICH Process 
on June 2001 
by the VICH Steering Committee 
 
 
 
 
 
 
THIS GUIDANCE HAS BEEN DEVELOPED BY THE APPROPRIATE VICH EXPERT WORKING GROUP AND HAS 
BEEN SUBJECT TO CONSULTATION BY THE PARTIES, IN ACCORDANCE WITH THE VICH PROCESS.  AT STEP 7 
OF THE PROCESS THE FINAL DRAFT IS RECOMMENDED FOR ADOPTION TO THE REGULATORY BODIES OF THE 
EUROPEAN UNION, JAPAN AND USA. 
 
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STUDIES TO EVALUATE THE SAFETY OF RESIDUES OF VETERINARY 
DRUGS IN HUMAN FOOD: REPRODUCTION TOXICITY TESTING 
 
 
 
 
 
1. INTRODUCTION....................................................................................................4 
1.1.  Objective of the guidance...........................................................................4 
1.2.  Background................................................................................................4 
1.3.  Scope of the guidance................................................................................5 
1.4.  General Principles......................................................................................5 
2. GUIDANCE.............................................................................................................5 
2.1.  Number of species.....................................................................................5 
2.2.  Number of generations...............................................................................6 
2.3.  Number of litters per generation.................................................................6 
2.4.  Recommended study protocol....................................................................6 
3.   REFERENCES.....................................................................................................7 
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This final guidance represents the agency's current thinking and does not create 
or confer any rights for or on any person and does not operate to bind FDA or the 
public. An alternative method may be used as long as it satisfies the requirements 
of the applicable statutes and regulations. 
 
1. INTRODUCTION 
1.1.  Objective of the guidance 
In order to establish the safety of veterinary drug residues in human food, a number of toxicological 
evaluations are recommended, including the assessment of any risks to reproduction. The objective 
of this guidance is to ensure international harmonization of reproduction toxicity testing, which is 
appropriate for the evaluation of risks to reproduction from long-term, low-dose exposures, such as 
may be encountered from the presence of veterinary drug residues in food. 
1.2.  Background 
There  has  been  considerable  overlap  in  the  reproduction  and  developmental  toxicity  testing 
requirements of the EU, Japan and the US, for establishing the safety of veterinary drug residues in 
human  food.  Although  each  region  differed  on  some  aspects  of  detail,  all  recommended  a 
multigeneration study in at least one rodent species, dosing beginning with the first parental (P ) 
0
group and continuing through at least two subsequent (F  and F ) generations. All three regions also 
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recommended developmental toxicity (teratology) studies. Developmental toxicity studies are the 
subject of a separate guidance (see VICH GL-32) and will not be further addressed here, except to 
note that it is no longer recommended that a developmental toxicity phase be included as part of a 
multigeneration study. 
  
This approach to reproduction and developmental toxicity testing of veterinary products differs in 
some respects from that adopted by the International Conference on Harmonisation of Technical 
Requirements  for  Registration  of  Pharmaceuticals  for  Human  Use  (ICH).1,2  The  ICH  guidance 
advocates a combination of three studies, in which dosing extends for shorter periods to cover adult 
fertility  and  early  embryonic  development,  pre-  and  postnatal  development  and  embryo-fetal 
development. While such an approach should be considered appropriate for most human medicines, 
exposure to veterinary drug residues in human food may be long-term, including exposure throughout 
life. For long-term, low-dose exposure, a multigeneration study, in which dosing extends through 
more than one generation is considered more appropriate. This guidance provides harmonized 
guidance  on  the  core  recommendation  for  a  multigeneration  study  for  the  safety  evaluation  of 
veterinary drug residues in human food. 
 
The current guidance is one of a series of guidances developed to facilitate the mutual acceptance of 
safety data necessary for the determination of Acceptable Daily Intakes (ADIs) for veterinary drug 
residues in human food by the relevant regulatory authorities. This guidance should be read in 
conjunction with the guidance on the overall strategy for the safety evaluation of veterinary residues 
in human food (see VICH GL-33). It was developed after consideration of the existing ICH guidance 
for pharmaceuticals for human use on Detection of Toxicity to Reproduction for Medicinal Products1 
and its Addendum, Toxicity to Male Fertility2, in conjunction with the current practices for evaluating 
veterinary drug residues in human food in the EU, Japan, the US, Australia, New Zealand and 
Canada. 
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1.3.  Scope of the guidance 
This document provides guidance on the core recommendation for a multigeneration study for those 
veterinary medicinal products that leave residues in human food.  However, it does not seek to limit 
the studies that may be performed to establish the safety of residues in human food with respect to 
reproductive function. Neither does it preclude the possibility of alternative approaches that may offer 
an equivalent assurance of safety, including scientifically-based reasons as to why such data may 
not  need  to  be  provided.  This  guidance  is  not  intended  to  cover  the  information  that may be 
recommended to establish the safety of a veterinary product with respect to reproduction in the target 
species. 
1.4. General Principles 
The  aim  of  a  multigeneration  reproduction  toxicity  study  is  to  detect  any  effect  of  the  parent 
substance or its metabolites on mammalian reproduction. These include effects on male and female 
fertility, mating, conception, implantation, ability to maintain pregnancy to term, parturition, lactation, 
survival, growth and development of the offspring from birth through to weaning, sexual maturation 
and the subsequent reproductive function of the offspring as adults. While multigeneration studies 
are not specifically designed to detect developmental abnormalities because malformed offspring 
may be destroyed by the dams at birth, such studies may provide an indication of developmental 
toxicity if litter size at birth, birth weight or survival in the first few days after birth are reduced. 
 
The study of more than one generation allows detection not only of any effects on adult reproduction, 
but also any effects on subsequent generations due to exposure in utero and early postnatally. 
Critical aspects of development, which affect adult reproductive capacity, take place prenatally and 
early postnatally. Adverse effects of sex hormones and their analogues administered during this 
critical period on reproductive tract development and function in males and females are well known. 
More recently, studies of other chemicals with endocrine disrupting potential have illustrated the 
critical role of exposure during the early developmental period on subsequent reproductive function in 
adult  life.  This  can  result  in  much  greater  effects  on  the  reproductive  capacity  of  subsequent 
generations compared with the original parental generation.  Studies of more than one generation 
may also allow detection of reproductive effects due to bioaccumulation of the test substance. 
Interference with the developing reproductive tract or bioaccumulation may manifest themselves via 
increasing degree or severity of adverse effects in successive generations.  
 
The design of the study should be such that where any effects on reproduction are detected, the 
dose(s) at which they occur and the dose(s) giving rise to no adverse effects are clearly identified. 
Some observations may indicate further studies to fully characterize the nature of the response or of 
the dose-response relationship. 
2. GUIDANCE 
2.1.   Number of species 
A multigeneration test in one species is normally sufficient. In practice, the majority of multigeneration 
studies for all classes of chemical have been conducted in the rat and the rat will undoubtedly 
continue to be the species of choice for most future studies. Provided strains with good fecundity are 
used, rats generally give more consistent reproductive performance than mice. There is also a much 
larger historical database available for rats. Reference can also be made, if necessary, to the results 
of other kinetic, metabolic and toxicity tests on rats within the overall test battery for the compound.  
However, studies on compounds originally used for other purposes but later proposed for veterinary 
use have sometimes been conducted in mice for historical reasons. Or there may be good scientific 
reason to conduct a study in mice (e.g. if there is known metabolic similarity to humans). Provided 
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reproductive performance is satisfactory, there is no general reason why the mouse should not also 
be an acceptable test species.  
 
Generally,  it  is  recommended  that  a  study  in  a  single  rodent  species,  preferably  the  rat,  be 
conducted. 
2.2. Number of generations 
Studies in one generation only have been the normal testing recommended for pharmaceuticals for 
human use, where the main concerns are exposure during short-term dosing periods. However, 
multigeneration studies of two or three generations have long been the usual recommendation for 
food  additives  and  food  contaminants  such  as  pesticides  and  veterinary  drug  residues.  One-
generation studies, in which treatment should be terminated when the first generation of offspring is 
weaned, do not permit assessment of the reproductive performance of animals that have been 
exposed to the test substance prenatally through to puberty. A study of more than one generation is 
therefore considered appropriate (see 1.4.).  
 
A study of more than one generation should also allow confirmation of any effects seen in the first 
generation or clarification of equivocal effects seen at any stage in the test. It may also give an 
indication of the effects due to bioaccumulation. 
 
The minimum number of generations appropriate to give clear and interpretable results in most cases 
is  considered  to  be  two.  While  early  multigeneration  test  protocols  for  some  chemical  classes 
recommended a third generation in certain cases, it is now generally considered that effects which 
are clear in the third generation should also be adequately detected in the second generation.  
 
Generally, it is recommended that a study of two generations be conducted. 
2.3.  Number of litters per generation 
A study with one litter per generation may be sufficient if the results clearly show either absence of 
any effects or presence of adverse effects with a well-defined no-adverse-effect level. Under certain 
circumstances however, it may be appropriate to extend the study to produce second litters and it is 
recommended that results from the study be closely monitored to enable such a decision to be taken, 
if appropriate. The value of second litters is that they may help to clarify the significance of any 
apparently dose-related or equivocal effects in first litters, which may be either the result of treatment, 
or due to chance, or to poor reproductive performance unrelated to treatment. Poor reproductive 
performance  in  controls  should  be  minimized  by  avoidance  of  nutritional  problems  and  other 
disturbances, ensuring the weight variation of the parental (P0) generation animals is not too great, 
and by not mating animals when they are too young or too old. 
  
It is therefore recommended that in general a study with one litter per generation be conducted. It 
may be appropriate, under certain circumstances mentioned above, to extend the study by producing 
second litters. 
2.4.   Recommended study protocol 
The OECD Test Guideline 416 Two-Generation Reproduction Toxicity Study 3 is an appropriate 
reference method for a multigeneration study to establish the safety of any veterinary residues in 
human food. This OECD Test Guideline includes discussion of the selection of test animals, selection 
of doses, timing of commencement of treatment, timing of mating, observations and reporting of 
results, all of which are relevant for the testing of veterinary products for the safety evaluation of 
residues in human food. It is noted that this Test Guideline is currently being updated. The Revised 
Draft  Guideline  416  (1999  et  seq.)4,  in  addition  to  the  usual  observations  included  in  a 
multigeneration study conducted according to the 1983 Test Guideline 4163, also includes evaluation 
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of adult sperm parameters, sexual maturation of offspring and provision for functional investigations 
of offspring, if such investigations are not included in other studies. The inclusion of these additional 
parameters is considered appropriate for the testing of veterinary products to modern standards. 
 
3.   REFERENCES 
1. ICH. 1993. ICH Harmonised Tripartite Guideline S5A. Detection of Toxicity to Reproduction for 
Medicinal Products. International Conference on Harmonisation of Technical Requirements for 
Registration of Pharmaceuticals for Human Use. 
 
2. ICH. 1995. ICH Harmonised Tripartite Guideline S5B. Toxicity to Male Fertility: An Addendum to 
the ICH Tripartite Guideline on Detection of Toxicity to Reproduction for Medicinal Products. 
International  Conference  on  Harmonisation  of  Technical  Requirements  for  Registration  of 
Pharmaceuticals for Human Use. 
 
3. OECD. 1987. Test Guideline 416. In: Guidelines for the Testing of Chemicals. Two-Generation 
Reproduction Toxicity Study. Paris, Organisation for Economic Cooperation & Development. 
 
4. OECD. 1999. Test Guideline 416. Two-Generation Reproduction Toxicity Study. Revised Draft 
Guideline 416, August 1999. Paris, Organisation for Economic Cooperation & Development.   
 
 
 
 
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