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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 

STUDIES TO EVALUATE THE SAFETY OF RESIDUES OF VETERINARY DRUGS IN HUMAN FOOD: 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 (P0) 

group and continuing through at least two subsequent (F1 and F2) generations. All three regions also 

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 Products”1 

and its Addendum, “Toxicity to Male Fertility”2

, 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. Onegeneration 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.