#116 
 
Guidance for Industry 
 
Studies to Evaluate the Safety 
of Residues of Veterinary 
Drugs in Human Food: 
Genotoxicity Testing 
 
VICH GL23(R) 
 
Submit comments on this revised guidance at any time.  Submit electronic 
comments to http://www.regulations.gov.   Submit written comments to the Division 
of Dockets Management (HFA-305), Food and Drug Administration,  5630 Fishers 
Lane, Rm. 1061, Rockville,  MD 20852.  All written comments should  be identified 
with Docket No. FDA-2000-D-0598. 
 
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, 240-402-0826,  email: 
tong.zhou@fda.hhs.gov. 
 
Additional  copies of this guidance document may be requested from the Policy and 
Regulations Staff (HFV-6), 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 
May 2015 
    Contains Non-Binding Recommendations 
VICH GL23 (R) (SAFETY) - GENOTOXICITY 
October 2014 
Revision at Step 9 
For consultation at Step 7 
 
 
 
 
 
 
 
STUDIES TO  EVALUATE THE SAFETY 
OF RESIDUES OF  VETERINARY 
DRUGS IN HUMAN FOOD: 
GENOTOXICITY TESTING 
 
 
 
 
 
 
 
Revision at Step 9 
 
Adopted at Step 7 of the VICH Process by the VICH Steering Committee  in September  2014 
for implementation  by October 2015. 
 
 
 
 
 
 
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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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 
5.  ASSESSMENT OF TEST RESULTS ........................................................................... 8 
REFERENCES................................................................................................................... 9 
GLOSSARY..................................................................................................................... 10 
   
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  Contains Non-Binding Recommendations 
Guidance for Industry 
 
STUDIES TO EVALUATE THE SAFETY OF RESIDUES OF 
VETERINARY  DRUGS IN HUMAN FOOD: 
 GENOTOXICITY  TESTING 
 
This guidance represents the Food and Drug Administrations (FDA or Agency) 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 FDA staff responsible for implementing this guidance 
using the contact information 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 and/or mutagens 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 guidance is to ensure international  harmonization  of genotoxicity 
testing. 
 
FDAs guidance documents, including  this revised guidance, do not establish legally 
enforceable responsibilities.  Instead, guidances describe the Agencys 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 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 guidance was developed after 
consideration  of the  International Conference on Harmonisation  of Technical Requirements 
for Registration of Pharmaceuticals for Human Use (ICH) guidances for pharmaceuticals for 
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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 guidance 
 
This 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 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 & TA100. 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  test (e.g., 
inorganic arsenic compounds). 
 
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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 Mutation Test 
 
A bacterial reverse mutagenicity  test should be performed according to the protocol set out in 
1
OECD Test Guidance 471 . 
 
4.2.  In Vitro Test for Chromosomal Effects in Mammalian Cells 
 
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Chromosome aberration tests should be performed according to OECD Test Guidance 473 . 
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 
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the criteria set out in OECD Test Guidance 476 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 
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according to OECD Test Guidance 476 . 
   
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4.4.  In Vivo Test for Chromosomal Effects 
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Either a mammalian erythrocyte micronucleus  test (OECD Test Guidance 474 ) or a 
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mammalian  bone marrow chromosome aberration test (OECD Test Guidance 475 ) 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. 
 
5.  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 its genotixic  or not.  The most appropriate test should be decided on a case-by-case 
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 caseby-case basis. 
   
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REFERENCES 
 
1.  OECD. 1997. Test Guidance 471.  Bacterial Reverse Mutation Test.  In: OECD Guidance 
for Testing of Chemicals. Paris, Organisation for Economic Cooperation and 
Development. 
 
2.  OECD. 1997. Test Guidance 473.  In Vitro Mammalian Chromosome Aberration 
Test.  In: OECD Guidance for Testing of Chemicals. Paris, Organisation for 
Economic Cooperation and Development. 
 
3.  OECD. 1997. Test Guidance 474. Mammalian Erythrocyte Micronucleus  Test.  In: 
OECD Guidance for Testing of Chemicals. Paris, Organisation  for Economic 
Cooperation and Development. 
 
4.  OECD. 1997. Test Guidance 475. Mammalian Bone Marrow Chromosome Aberration 
Test. In: OECD Guidance for Testing of Chemicals. Paris, Organisation for Economic 
Cooperation and Development. 
 
5.  OECD. 1997. Test Guidance 476. In Vitro Mammalian  Cell Gene Mutation Test.  In: 
OECD Guidance for Testing of Chemicals. Paris, Organisation  for Economic 
Cooperation and Development. 
 
6.  OECD. 2010. Test Guidance 487.  In Vitro Mammalian Cell Micronucleus Test. In: 
OECD Guidance for the Testing of Chemicals. Paris, Organisation  for Economic 
Cooperation and 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. 
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