**116**

**Guidance for Industry**

**Studies to Evaluate the Safety of Residues of Veterinary Drugs in
Human Food:**

**Genotoxicity Testing**

**VICH GL23**

**FINAL GUIDANCE**

This final guidance recommends a basic battery of tests that can be used
for the evaluation of the genotoxicity of veterinary drugs submitted for
approval to the European Union, Japan, and the United States.

Comments and suggestions regarding this 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.fda.gov/dockets/ecomments>. All comments should be
identified with the Docket No. 00D-1631.

For questions regarding this final document, contact Louis T. Mulligan,
Center for Veterinary Medicine, (HFV-153), Food and Drug Administration,
7500 Standish Place, Rockville, MD 20855, 301-827-6984, e-mail:
[lmulliga@cvm.fda.gov.](mailto:imulliga@cvm.fda.gov)

######### U.S. Department of Health and Human Services

######### Food and Drug Administration

**Center for Veterinary Medicine**

**January 3, 2002**

**VICH GL23 [(Safety: Genotoxicity)]{.smallcaps}**

**June 2001**

**For implementation at Step 7**

Studies To Evaluate The Safety

Of Residues Of Veterinary

Drugs In Human Food:

Genotoxicity 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]{.smallcaps} USA.

# STUDIES TO EVALUATE THE SAFETY OF RESIDUES OF VETERINARY DRUGS IN HUMAN FOOD: GENOTOXICITY TESTING {#studies-to-evaluate-the-safety-of-residues-of-veterinary-drugs-in-human-food-genotoxicity-testing .WW-Heading}

STUDIES TO EVALUATE THE SAFETY OF RESIDUES OF VETERINARY DRUGS IN HUMAN
FOOD: GENOTOXICITY TESTING
[3](#studies-to-evaluate-the-safety-of-residues-of-veterinary-drugs-in-human-food-genotoxicity-testing)

1\. INTRODUCTION [4](#introduction)

1.1. Objective of the guidance [4](#objective-of-the-guidance)

1.2. Background [4](#background)

1.3. Scope of the Guidance [4](#scope-of-the-guidance)

2\. STANDARD BATTERY OF TESTS [5](#standard-battery-of-tests)

3\. MODIFICATIONS TO THE STANDARD BATTERY
[5](#modifications-to-the-standard-battery)

3.1. Antimicrobials [6](#antimicrobials)

3.2. Metabolic Activation [6](#metabolic-activation)

4\. THE CONDUCT OF TESTS [6](#the-conduct-of-tests)

4.1. Bacterial Test [6](#bacterial-test)

4.2. In Vitro Test for Chromosomal Effects in Mammalian Cells
[6](#in-vitro-test-for-chromosomal-effects-in-mammalian-cells)

4.3. In Vitro Test for Gene Mutation in Mammalian Cells
[6](#in-vitro-test-for-gene-mutation-in-mammalian-cells)

4.4. In Vivo Test for Chromosomal Effects
[6](#in-vivo-test-for-chromosomal-effects)

ASSESSMENT OF TEST RESULTS [6](#assessment-of-test-results)

REFERENCES [7](#references)

GLOSSARY [8](#glossary)

> ***This final guidance represents the agency\'s current thinking on
> this subject 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
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
genotoxins 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 harmonisation
of genotoxicity testing.

## 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 (VICH Guidance on General Testing Approach
will be made available at a later time). This VICH guidance was
developed after consideration of the existing International Conference
on Harmonisation of Technical 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 European Union (EU), Japan, the USA, Australia and
New Zealand.

## 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 necessary to also test one or more of the major
metabolites that occur as residues in food. Instances when the need to
test 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:

**I. 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), WP2*uvr*A (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).

**II. An in vitro test for chromosomal effects in mammalian cells**

The second test should evaluate the potential of a chemical to produce
chromosomal effects. In the EU, the *in vitro* cytogenetic test using
metaphase analysis is preferred, which detects both clastogenicity and
aneugenicity. In the USA, the mouse lymphoma test is preferred, which,
with modification, can detect both gene mutation and chromosomal damage.
Either test is acceptable in Japan.

**III. 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 may 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.

## 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 may 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.

## 4.2. *In Vitro* Test for Chromosomal Effects in Mammalian Cells

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.

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 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.

## 4.4. *In Vivo* Test for Chromosomal Effects 

Either a mammalian erythrocyte micronucleus test (OECD Test Guidance
474) or a mammalian bone marrow chromosome aberration test (OECD Test
Guidance 475) may be performed as part of the initial battery of
genotoxicity tests. The mammalian erythrocyte micronucleus test may 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.

#  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-by-case basis.

In the case of other positive or equivocal results in the Standard
Battery of Tests the need for further tests should be decided on a
case--by-case basis.

# 

# REFERENCES

OECD. 1997. Test Guidance 471. Bacterial Reverse Mutation Test. In: OECD
Guidance for Testing of Chemicals. Paris, Organization for Economic
Cooperation & Development.

OECD. 1997. Test Guidance 473. *In Vitro* Mammalian Chromosome
Aberration Test. In: OECD Guidance for Testing of Chemicals. Paris,
Organization for Economic Cooperation & Development.

OECD. 1997. Test Guidance 474. Mammalian Erythrocyte Micronucleus Test.
In: OECD Guidance for Testing of Chemicals. Paris, Organization for
Economic Cooperation & Development.

OECD. 1997. Test Guidance 475. Mammalian Bone Marrow Chromosome
Aberration Test. In: OECD Guidance for Testing of Chemicals. Paris,
Organization for Economic Cooperation & Development.

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.

# 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 may 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
may 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.