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Guidance for Industry

Studies to Evaluate the Safety

of Residues of Veterinary

Drugs in Human Food:

Genotoxicity Testing

VICH GL23(R)

DRAFT GUIDANCE

This guidance document is for comment purposes only

Submit comments on this draft revised guidance by the date provided in the Federal 

Register notice announcing the availability of the draft guidance. Submit written 

comments to the Division of Dockets Management (HFA-305), Food and Drug 

Administration, 5630 Fishers Lane, Rm. 1061, Rockville, MD 20852. Submit 

electronic comments to http://www.regulations.gov. You should identify all 

comments with Docket number FDA-2000-D-0598. (Formally Docket No. 00D1631). 

For further information regarding this document, contact Tong Zhou, Center 

for Veterinary Medicine (HFV-153), Food and Drug Administration, 7500 

Standish Place, Rockville, MD 20855, 240-276-8120. 

Additional copies of this draft guidance document may be requested from the 

Communications Staff (HFV-12), Center for Veterinary Medicine, Food and Drug 

Administration, 7519 Standish Place, Rockville, MD 20855, and may be viewed on 

the Internet at either http://www.fda.gov/AnimalVeterinary/default.htm or 

http://www.regulations.gov. 

U.S. Department of Health and Human Services

Food and Drug Administration

Center for Veterinary Medicine

March 5, 2013
Contains Non-Binding Recommendations

Draft – Not for Implementation 

2 

VICH GL23(R) (SAFETY - GENOTOXICITY) 

December 2012

Revision at Step 9

For consultation at Step 4

STUDIES TO EVALUATE THE SAFETY

OF RESIDUES OF VETERINARY

DRUGS IN HUMAN FOOD: 

GENOTOXICITY TESTING 

Revision at Step 9

Recommended for Consultation

at Step 4 of the VICH Process in December 2012

by the VICH Steering Committee

THIS GUIDANCE HAS BEEN DEVELOPED BY THE APPROPRIATE VICH EXPERT WORKING

GROUP AND IS SUBJECT TO CONSULTATION BY THE PARTIES, IN ACCORDANCE WITH 

THE VICH PROCESS. AT STEP 7 OF THE PROCESS THE FINAL DRAFT WILL BE

RECOMMENDED FOR ADOPTION TO THE REGULATORY BODIES OF THE EUROPEAN 

UNION, JAPAN AND USA.
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TABLE OF CONTENTS

1. INTRODUCTION .............................................................................................................. 4

1.1. Objective of the guidance ............................................................................................ 4

1.2. Background ................................................................................................................. 4

1.3. Scope of the guidance ................................................................................................. 5

2. STANDARD BATTERY OF TESTS ................................................................................ 5

2.1. A test for gene mutation in bacteria. ........................................................................... 5

2.2. A cytogenetic test for chromosomal damage (the in vitro metaphase chromosome 

aberration test or in vitro micronucleus test), or an in vitro mouse lymphoma tk gene 

mutation assay ........................................................................................................................ 6

2.3. An in vivo test for chromosomal effects using rodent haematopoietic cells. .............. 6

3. MODIFICATIONS TO THE STANDARD BATTERY ..................................................... 6

3.1. Antimicrobials ............................................................................................................. 6

3.2. Metabolic activation .................................................................................................... 7

4. THE CONDUCT OF TESTS .............................................................................................. 7

4.1. Bacterial Test............................................................................................................... 7

4.2. In Vitro Test for Chromosomal Effects in Mammalian Cells ..................................... 7

4.3. In Vitro Test for Gene Mutation in Mammalian Cells ................................................ 7

4.4. In Vivo Test for Chromosomal Effects ........................................................................ 8

ASSESSMENT OF TEST RESULTS ........................................................................................ 9

REFERENCES .......................................................................................................................... 9

GLOSSARY ............................................................................................................................. 10
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STUDIES TO EVALUATE THE SAFETY OF RESIDUES OF 

VETERINARY DRUGS IN HUMAN FOOD:

GENOTOXICITY TESTING

This draft guidance, when finalized, will represent the Food and Drug Administration’s 

(FDA’s) current thinking on this topic. It does not create or confer any rights for or on 

any person and does not operate to bind FDA or the public. You can use an alternative 

approach if the approach satisfies the requirements of the applicable statutes and 

regulations. If you want to discuss an alternative approach, contact the appropriate 

FDA staff. If you cannot identify the appropriate FDA staff, call the appropriate 

number listed on the title page of this guidance. 

1. INTRODUCTION

1.1. Objective of the guidance

In order to establish the safety of veterinary drug residues in human foods, a number of 

toxicological evaluations are recommended, including investigation of possible hazard from 

genotoxic activity. Many carcinogens have a genotoxic mode of action and it is prudent to 

regard genotoxicants as potential carcinogens unless there is convincing evidence that this 

is not the case. Additionally, substances causing reproductive and/or developmental toxicity 

may have a mode of action that involves genotoxic mechanisms. The results of genotoxicity 

tests will not normally affect the numerical value of an acceptable daily intake (ADI), but 

they may influence the decision about whether an ADI can be established. 

The objective of this draft guidance is to ensure international harmonisation of genotoxicity 

testing.

FDA’s guidance documents, including this draft revised guidance, do not establish 

legally enforceable responsibilities. Instead, guidances describe the Agency’s current 

thinking on a topic and should be viewed only as recommendations, unless specific 

regulatory or statutory requirements are cited. The use of the word “should” in 

Agency guidances means that something is suggested or recommended, but not 

required.

1.2. Background 

There have been differences in the genotoxicity testing requirements of the EU, Japan and 

the USA, for establishing the safety of veterinary drug residues in human food. 

This draft guidance is one of a series of VICH guidances developed to facilitate the mutual 

acceptance of safety data necessary for the establishment of ADIs for veterinary drug 

residues in human food by the relevant regulatory authorities. It should be read in 

conjunction with the guidance on the overall strategy for the evaluation of veterinary drug 

residues in human food (see VICH GL33). This VICH draft guidance was developed after 

consideration of the existing International Conference on Harmonisation of Technical 
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Requirements for Approval of Pharmaceuticals for Human Use (ICH) guidances for 

pharmaceuticals for human use: “Genotoxicity: A Standard Battery of Genotoxicity 

Testing of Pharmaceuticals” and “Guidance on Specific Aspects of Regulatory 

Genotoxicity Tests for Pharmaceuticals”. Account was also taken of Organisation for 

Economic Co-operation and Development (OECD) Guidances for Testing of Chemicals 

and of national/regional guidances and the current practices for evaluating the safety of 

veterinary drug residues in human food in the EU, Japan, the USA, Australia, New 

Zealand, and Canada. 

1.3. Scope of the draft guidance

This draft guidance recommends a Standard Battery of Tests that can be used for the 

evaluation of the genotoxicity of veterinary drugs. In most cases, the results will give a 

clear indication of whether or not the test material is genotoxic. However, the Standard 

Battery of Tests is not appropriate for certain classes of veterinary drugs. For instance, 

some antimicrobials may be toxic to the tester strains used in the test for gene mutation in 

bacteria. The draft guidance advises on amendments to the basic battery of tests for testing 

such drugs. In some instances the results of the Standard or amended Battery of Tests may 

be unclear or equivocal, so advice is given on the assessment and interpretation of results. 

Additional testing may be recommended in some instances, e.g., substances showing 

potential aneugenic and/or germ cell effects.

In most cases, it is the parent drug substance that is tested, although in some cases it may 

be recommended to also test one or more of the major metabolites that occur as residues in 

food. Instances when testing a metabolite may be recommended include situations in which 

the metabolite has structural alerts that are not present in the molecular structure of the 

parent drug and when the residues in food are mostly in the form of a metabolite that has a 

molecular structure that is fundamentally different from that of the parent drug. Salts,

esters, conjugates and bound residues are usually assumed to have similar genotoxic 

properties to the parent drug, unless the converse can be demonstrated.

2. STANDARD BATTERY OF TESTS

The following battery of three tests is recommended for use as a screen of veterinary 

drugs for genotoxicity:

2.1. A test for gene mutation in bacteria. 

For the bacterial gene mutation test, a very extensive database has been built up for 

bacterial reverse mutation tests for gene mutation in strains of Salmonella typhimurium 

and Escherichia coli. The best-validated strains are Salmonella typhimurium strains 

TA1535, TA1537 (or TA97 or TA97a), TA98 & TA 100. These strains may not detect 

some oxidising mutagens and cross- linking agents, so to correct for this, Escherichia 

coli strains WP2 (pKM101), WP2uvrA (pKM101) or Salmonella typhimurium TA102 

should also be used in the bacterial test. However, the bacterial gene mutation test, 

whilst being an efficient primary screen for detecting compounds with inherent 

potential for inducing gene mutations, does not detect all compounds with mutagenic 

potential. Some clastogenic compounds do not produce mutations in the Salmonella 
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test (e.g., inorganic arsenic compounds). 

2.2. A cytogenetic test for chromosomal damage (the in vitro metaphase chromosome 

aberration test or in vitro micronucleus test), or an in vitro mouse lymphoma tk gene 

mutation assay 

The second test should evaluate the potential of a chemical to produce chromosomal effects.

This can be evaluated using one of the following three tests: (1) an in vitro chromosomal 

aberrations test using metaphase analysis, which detects both clastogenicity and 

aneugenicity; (2) an in vitro mammalian cell micronucleus test, which detects the activity 

of clastogenicity and aneugenicity; or (3) a mouse lymphoma test, which, with 

modification, can detect both gene mutation and chromosomal damage.

2.3. An in vivo test for chromosomal effects using rodent haematopoietic cells.

A third test has been added to the Standard Battery of Tests in order to give added 

assurance that the Standard Battery of Tests will detect all potential mutagens. The 

VICH was aware that, for the testing of some classes of chemicals, some authorities 

recommend the use of an initial battery of mutagenicity tests that consists solely of in 

vitro tests, with in vivo testing recommended only if the in vitro battery gives a positive 

or equivocal result. The VICH considered this approach but chose to include an in vivo 

test in its basic battery of tests in order to achieve harmony with the requirements of 

ICH for testing human drugs for genotoxicity. This could be either a micronucleus test 

or a cytogenetics test.

3. MODIFICATIONS TO THE STANDARD BATTERY 

For most substances the Standard Battery of Tests should be sufficient, but in a few 

instances modifications to the choice of tests or to the protocols of the individual tests 

undertaken may be appropriate. The physicochemical properties of a substance (e.g., 

volatility, pH, solubility, stability, etc.) can sometimes make standard test conditions 

inappropriate. It is recommended that this be given due consideration before tests are 

conducted. Modified protocols should be used where it is evident that standard 

conditions will give a false negative result. The OECD Guidances for Testing of 

Chemicals for the genotoxicity tests give some advice on the susceptibility of the 

individual tests to the physical characteristics of the test material and they give some 

advice on compensatory measures that can be taken. Drugs tested using alternative 

batteries of genotoxicity tests should be considered on a case-by-case basis. A scientific 

justification should be given for not using the Standard Battery of Tests.

3.1. Antimicrobials

Some antimicrobial substances are excessively toxic to bacteria and therefore difficult 

to test in bacterial tests. In this case, it would be appropriate to perform a bacterial 

test using concentrations up to the limit of cytotoxicity and to supplement the 

bacterial test with an in vitro test for gene mutation in mammalian cells.
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3.2. Metabolic activation 

The in vitro test should be performed in the presence and absence of a metabolic activation 

system. The most commonly used metabolic activation system is S9 mix from the livers 

of rats treated with an enzyme inducing agent (Aroclor 1254 or a combination of 

phenobarbital and beta- naphthoflavone). However, other systems can be used. A 

scientific rationale should be given to justify the choice of an alternative metabolic 

activation system.

4. THE CONDUCT OF TESTS

4.1. Bacterial Test

A bacterial reverse mutagenicity test should be performed according to the protocol set out 

in OECD Test Guidance 471

1

.

4.2. In Vitro Test for Chromosomal Effects in Mammalian Cells 

Chromosome aberration tests should be performed according to OECD Test Guidance

473

2

. These cytogenetic tests should detect clastogenicity and may also detect 

heteroploidy. To detect induction of polyploidy, longer (e.g., 3 normal cell cycles) 

continuous treatment can give higher sensitivity. Limited information on potential 

aneugenicity can be obtained by recording the incidences of hyperploidy, polyploidy 

and/or modification of mitotic index in the cytogenetic test. If there are indicators of 

aneugenicity (e.g., induction of polyploidy) then this should be confirmed using 

appropriate staining procedures such as FISH (fluorescence in situ hybridisation) or 

chromosome painting. As apparent loss of chromosomes can occur artifactually, only 

hyperploidy should be regarded as a clear indication of induced aneuploidy. 

An in vitro mammalian micronucleus test (OECD Test Guidance 4876

) can be performed as 

part of the initial battery of genotoxicity tests as a substitute for the chromosome aberration 

tests described in OECD Test Guidance 4732. The in vitro mammalian cell micronucleus 

test can detect both clastogenicity and aneugenicity, and can simultaneously detect mitotic 

delay, apoptosis, chromosome breakage, and chromosome loss.

If the mouse lymphoma tk test is conducted, it should be with a protocol amended to 

include measurements of both small and large colonies. The protocol should conform to 

the criteria set out in OECD Test Guidance 476

5

and should include the use of 

appropriate positive controls (clastogens). 

4.3. In Vitro Test for Gene Mutation in Mammalian Cells

When an in vitro mammalian cell gene mutation test is used, it should be performed 

according to OECD Test Guidance 476

5

.
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4.4. In Vivo Test for Chromosomal Effects

Either a mammalian erythrocyte micronucleus test (OECD Test Guidance 474

3

) or a 

mammalian bone marrow chromosome aberration test (OECD Test Guidance 475

4

) can be 

performed as part of the initial battery of genotoxicity tests. The mammalian erythrocyte

micronucleus test can be conducted by analysis of either bone marrow or peripheral blood. 

If it is conducted using peripheral blood, the test species should be the mouse and not the 

rat, as the spleen of the latter removes circulating micronucleated erythrocytes.

These tests are designed to give a qualitative answer to the question of whether or 

not a substance may express genotoxicity in vivo, not to establish no-effect levels.
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ASSESSMENT OF TEST RESULTS

The assessment of the genotoxic potential of a compound should take into account the 

totality of the findings and acknowledge the intrinsic values and limitations of both in vitro 

and in vivo tests.

Clearly negative results for genotoxicity in a series of tests, including the Standard Battery 

of Tests, should usually be taken as sufficient evidence of an absence of genotoxicity.

If a substance gives clearly positive result(s) for genotoxicity in vitro but a clearly negative 

result in the in vivo genotoxicity test(s) performed using bone marrow, another in vivo 

genotoxicity test using a target tissue other than bone marrow should be used to confirm 

whether it’s genotixic or not. The most appropriate test should be decided on a case-bycase basis.

In the case of other positive or equivocal results in the Standard Battery of Tests the 

decision of whether further tests are warranted should be made on a case–by-case basis.

REFERENCES

1. OECD. 1997. Test Guidance 471. Bacterial Reverse Mutation Test. In: OECD Guidance 

 for Testing of Chemicals. Paris, Organization for Economic Cooperation & Development. 

2. OECD. 1997. Test Guidance 473. In Vitro Mammalian Chromosome Aberration 

Test. In: OECD Guidance for Testing of Chemicals. Paris, Organization for 

Economic Cooperation & Development. 

3. OECD. 1997. Test Guidance 474. Mammalian Erythrocyte Micronucleus Test. In: 

OECD Guidance for Testing of Chemicals. Paris, Organization for Economic 

Cooperation & Development.

4. OECD. 1997. Test Guidance 475. Mammalian Bone Marrow Chromosome Aberration 

 Test. In: OECD Guidance for Testing of Chemicals. Paris, Organization for Economic 

 Cooperation & Development.

5. OECD. 1997. Test Guidance 476. In Vitro Mammalian Cell Gene Mutation Test. In: 

OECD Guidance for Testing of Chemicals. Paris, Organization for Economic 

Cooperation & Development.

6. OECD. 2010. Test Guidance 487. In Vitro Mammalian Cell Micronucleus Test. In: 

OECD Guidance for the Testing of Chemicals. Paris, Organization for Economic 

Cooperation & Development. 
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GLOSSARY 

Aneugenicity: The ability to cause aneuploidy.

Aneuploidy: Numerical deviation of the modal number of chromosomes in a cell or 

organism, other than an extra or reduced number of complete sets of 

chromosomes. 

Clastogen: An agent that produces structural changes of chromosomes, usually 

detectable by light microscopy.

Clastogenicity: The ability to cause structural changes of chromosomes 

(chromosomal aberrations). 

Cytogenetics: Chromosome analysis of cells, normally performed on dividing cells when 

chromosomes are condensed and visible with a light microscope after 

staining.

Gene mutation: A detectable permanent change within a single gene or its regulating 

 sequences. The change could be a point mutation, insertion, deletion, etc. 

Genotoxicity: A broad term that refers to any deleterious change in the genetic 

material regardless of the mechanism by which the change is induced. 

Heteroploidy: Any abnormal number of chromosomes in a cell or organism. This is a 

general term that covers polyploidy, aneuploidy, hyperploidy, etc.

Hyperploidy: An increase over the normal number of chromosomes in a cell or organism. 

Micronucleus: Particle in a cell that contains microscopically detectable nuclear DNA; it 

might contain a whole chromosome(s) or a broken centric or acentric part(s) 

of chromosome(s). The size of a micronucleus is usually defined as less 

than 1/5 but more than 1/20 of the main nucleus. 

Mutagenicity: The capacity to cause a permanent change in the amount or structure of the 

genetic material in an organism or cell that may result in change in the 

characteristics of the organism or cell. The alteration can involve changes 

to the sequence of bases in the nucleic acid (gene mutation), structural 

changes to chromosomes (clastogenicity) and/or changes to the number of 

chromosomes in cells (aneuploidy or polyploidy). 

Polyploidy: An extra or reduced number of complete sets of chromosomes.