Guidance for Industry

S2B Genotoxicity: A Standard

Battery for Genotoxicity Testing of 

Pharmaceuticals

July 1997

ICH
Guidance for Industry

S2B Genotoxicity: A Standard

Battery for Genotoxicity Testing of

Pharmaceuticals

Additional copies are available from:

 the Drug Information Branch (HFD-210),

Center for Drug Evaluation and Research (CDER),

5600 Fishers Lane, Rockville, MD 20857 (Tel) 301-827-4573

http://www.fda.gov/cder/guidance/index.htm

or

Office of Communication,

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Center for Biologics Evaluation and Research (CBER)

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http://www.fda.gov/cber/guidelines.htm

(Fax) 888-CBERFAX or 301-827-3844

(Voice Information) 800-835-4709 or 301-827-1800

U.S. Department of Health and Human Services

Food and Drug Administration

Center for Drug Evaluation and Research (CDER)

Center for Biologics Evaluation and Research (CBER)

July 1997

ICH
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Table of Contents

I. INTRODUCTION (1) .................................................. 1

II. GENERAL PURPOSE OF GENOTOXICITY TESTING (2) .................... 1

III. THE STANDARD TEST BATTERY FOR GENOTOXICITY (3) ................ 2

IV. MODIFICATIONS OF THE 3-TEST BATTERY (4) .......................... 3

A. Limitations to the Use of Bacterial Test Organisms (4.1) .................. 4

B. Compounds Bearing Structural Alerts for Genotoxic Activity (4.2) ........... 4

C. Limitations to the Use of Standard in Vivo Tests (4.3) .................... 4

D. Additional Genotoxicity Testing in Relation to the Carcinogenicity Bioassay (4.4) 4

V. STANDARD PROCEDURES FOR IN VITRO TESTS (5) ...................... 5

VI. NOTES (6) .......................................................... 6

VII. GLOSSARY (7) ...................................................... 9
 This guidance was developed within the Expert Working Group (Safety) of the International Conference on 1

Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) and has been

subject to consultation by the regulatory parties, in accordance with the ICH process. This document has been endorsed

by the ICH Steering Committee at Step 4 of the ICH process, July 1997. At Step 4 of the process, the final draft is

recommended for adoption to the regulatory bodies of the European Union, Japan and the United States. This guidance

was published in the Federal Register on November 21, 1997 (62 FR 62472), and is applicable to drug and biological

products. This guidance represents the Agency’s current thinking on a recommended standard battery for genotoxicity

testing of a pharmaceutical. It does not create or confer any rights for or on any person and does not operate to bind

FDA or the public. An alternative approach may be used if such approach satisfies the requirements of the applicable

statute, regulations, or both.

GUIDANCE FOR INDUSTRY1

S2B Genotoxicity: A Standard Battery for 

Genotoxicity Testing of Pharmaceuticals

I. INTRODUCTION (1)

Two fundamental areas in which harmonization of genotoxicity testing for pharmaceuticals is

considered necessary are the scope of this guidance: (1) Identification of a standard set of tests to

be conducted for registration. (2) The extent of confirmatory experimentation in in vitro

genotoxicity tests in the standard battery. Further issues that were considered necessary for

harmonization can be found in the ICH guidance S2A Specific Aspects of Regulatory Genotoxicity

Tests for Pharmaceuticals. The two ICH guidances on genotoxicity complement each other

and therefore should be used together as ICH guidance principles for testing of a

pharmaceutical for potential genotoxicity.

II. GENERAL PURPOSE OF GENOTOXICITY TESTING (2)

Genotoxicity tests can be defined as in vitro and in vivo tests designed to detect compounds that

induce genetic damage directly or indirectly by various mechanisms. These tests should enable a

hazard identification with respect to damage to DNA and its fixation. Fixation of damage to DNA

in the form of gene mutations, larger scale chromosomal damage, recombination and numerical

chromosome changes is generally considered to be essential for heritable effects and in the

multistep process of malignancy, a complex process in which genetic changes may play only a

part. Compounds which are positive in tests that detect such kinds of damage have the potential

to be human carcinogens and/or mutagens, i.e., may induce cancer and/or heritable defects.

Because the relationship between exposure to particular chemicals and carcinogenesis is

established for man, while a similar relationship has been difficult to prove for heritable diseases,

genotoxicity tests have been used mainly for the prediction of carcinogenicity. Nevertheless,

because germ line mutations are clearly associated with human disease, the suspicion that a

compound may induce heritable effects is considered to be just as serious as the suspicion that a 
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compound may induce cancer. In addition, the outcome of such tests may be valuable for the

interpretation of carcinogenicity studies.

III. THE STANDARD TEST BATTERY FOR GENOTOXICITY (3)

Registration of pharmaceuticals requires a comprehensive assessment of their genotoxic potential.

It is clear that no single test is capable of detecting all relevant genotoxic agents. Therefore, the

usual approach should be to carry out a battery of in vitro and in vivo tests for genotoxicity. Such

tests are complementary rather than representing different levels of hierarchy.

The general features of a standard test battery can be outlined as follows:

(i) It is appropriate to assess genotoxicity in a bacterial reverse mutation test. This

test has been shown to detect relevant genetic changes and the majority of

genotoxic rodent carcinogens.

(ii) DNA damage considered to be relevant for mammalian cells and not adequately

measured in bacteria should be evaluated in mammalian cells. Several mammalian

cell systems are in use: systems that detect gross chromosomal damage (in vitro

tests for structural and numerical chromosomal aberrations), systems that detect

primarily gene mutations (see Note 1), and a system that detects gene mutations

and clastogenic effects (mouse lymphoma tk assay) (see Note 2). The information

given in Notes 3 and 4 demonstrates that with appropriate test protocols (see

section V (5)) the various in vitro tests for chromosomal damage and the mouse

lymphoma tk assay yield results with a high level of congruence for compounds

that are regarded as genotoxic but yield negative results in the bacterial reverse

mutation assay. Therefore, these systems are currently considered interchangeable

when used together with other genotoxicity tests in a standard battery for

genotoxicity testing of pharmaceuticals, if these test protocols are used.

(iii) An in vivo test for genetic damage should usually be a part of the test battery to

provide a test model in which additional relevant factors (absorption, distribution,

metabolism, excretion) that may influence the genotoxic activity of a compound

are included. As a result, in vivo tests permit the detection of some additional

genotoxic agents (see Note 5). An in vivo test for chromosomal damage in rodent

hematopoietic cells fulfills this need. This in vivo test for chromosomal damage in

rodents could be either an analysis of chromosomal aberrations in bone marrow

cells or an analysis of micronuclei in bone marrow or peripheral blood

erythrocytes.
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 (i) A test for gene mutation in bacteria.

 (ii) An in vitro test with cytogenetic evaluation of chromosomal damage with mammalian

cells or an in vitro mouse lymphoma tk assay.

 (iii) An in vivo test for chromosomal damage using rodent hematopoietic cells.

The following standard test battery is recommended based upon the considerations mentioned

above:

For compounds giving negative results, the completion of this 3-test battery, performed and

evaluated in accordance with current recommendations, will usually provide a sufficient level of

safety to demonstrate the absence of genotoxic activity (see Note 6). Compounds giving positive

results in the standard test battery may, depending on their therapeutic use, need to be tested

more extensively (see ICH S2A Specific Aspects of Regulatory Genotoxicity Tests for

Pharmaceuticals).

The suggested standard set of tests does not imply that other genotoxicity tests are generally

considered inadequate or inappropriate (e.g., tests for measurement of DNA adducts, DNA strand

breaks, DNA repair or recombination). Such tests serve as options in addition to the standard

battery for further investigation of genotoxicity test results obtained in the standard battery.

Furthermore, molecular techniques to study mechanisms of genotoxicity in the standard battery

systems may be useful for risk assessment. Only under extreme conditions in which one or more

tests comprising the standard battery cannot be employed for technical reasons, alternative

validated tests can serve as substitutes. For this to occur, sufficient scientific justification should

be provided to support the argument that a given standard battery test is not appropriate. 

The standard battery does not include an independent test designed specifically to test for

aneuploidy. However, information on this type of damage may be derived from the tests for

chromosomal damage in vitro and in vivo. Elements of the standard protocols that provide such

information are elevations in the mitotic index, polyploidy induction, and micronucleus evaluation.

There is also limited experimental evidence that aneuploidy inducers can be detected in the mouse

lymphoma tk assay (see Note 4). In such cases, further testing may be needed.

IV. MODIFICATIONS OF THE 3-TEST BATTERY (4)

The following sections give situations where the standard 3-test battery may need modification.
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A. Limitations to the Use of Bacterial Test Organisms (4.1)

There are circumstances where the performance of the bacterial reverse mutation test does

not provide appropriate or sufficient information for the assessment of genotoxicity. This

may be the case for compounds that are excessively toxic to bacteria (e.g., some

antibiotics) and compounds thought or known to interfere with the mammalian cell

replication system (e.g., topoisomerase inhibitors, nucleoside analogues, or inhibitors of

DNA metabolism). For these cases, usually two in vitro mammalian cell tests should be

performed using two different cell types and of two different endpoints (gene mutation

(see Note 1) and chromosomal damage). Nevertheless, it is still important to perform the

bacterial reverse mutation test (see Note 7); either a full test or a limited (range-finding)

test (see section V (5)) may be appropriate.

B. Compounds Bearing Structural Alerts for Genotoxic Activity (4.2)

Structurally alerting compounds (see Note 8) are usually detectable in the standard 3-test

battery. However, compounds bearing structural alerts that have given negative results in

the standard 3-test battery may necessitate limited additional testing. The choice of

additional test(s) or protocol modification(s) depends on the chemical nature, the known

reactivity, and metabolism data on the structurally alerting compound under question (see

Note 9 and ICH S2A Specific Aspects of Regulatory Genotoxicity Tests for

Pharmaceuticals).

C. Limitations to the Use of Standard in Vivo Tests (4.3)

There are compounds for which standard in vivo tests do not provide additional useful

information. These include compounds for which data from studies on toxicokinetics or

pharmacokinetics indicate that they are not systemically absorbed and therefore are not

available for the target tissues in standard in vivo genotoxicity tests. Examples of such

compounds are some radioimaging agents, aluminum-based antacids, and some dermally

applied pharmaceuticals. In cases where a modification of the route of administration does

not provide sufficient target tissue exposure, it may be appropriate to base the evaluation

only on in vitro testing.

D. Additional Genotoxicity Testing in Relation to the Carcinogenicity

Bioassay (4.4)

1. Evidence for tumor response (4.4.1)

Additional genotoxicity testing in appropriate models may be conducted for

compounds that were negative in the standard 3-test battery but which have shown

effects in carcinogenicity bioassay(s) with no clear evidence for a nongenotoxic

mechanism. To help understand the mechanism of action, additional testing can

include modified conditions for metabolic activation in in vitro tests or can include
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in vivo tests measuring genetic damage in target organs of tumor induction (e.g.,

liver UDS test, P-postlabeling, mutation induction in transgenes, molecular 32

characterization of genetic changes in tumor-related genes).

2. Structurally unique chemical classes (4.2.2)

On rare occasions, a completely novel compound in a unique structural chemical

class will be introduced as a pharmaceutical. When such a compound will not be

tested in chronic rodent carcinogenicity bioassays, further genotoxicity evaluation

may be invoked.

V. STANDARD PROCEDURES FOR IN VITRO TESTS (5)

Reproducibility of experimental results is an essential component of research involving novel

methods or unexpected findings; however, the routine testing of chemicals with standard, widely

used genotoxicity tests need not always be completely replicated. These tests are sufficiently well

characterized and have sufficient internal controls that repetition can usually be avoided if

protocols with built-in confirmatory elements, such as those outlined below, are used.

For both bacterial and mammalian cell gene mutation tests, the results of a range-finding test can

be used to guide the selection of concentrations to be used in the definitive mutagenicity test. By

these means, a range-finding test may supply sufficient data to provide reassurance that the

reported result is the correct one. In bacterial mutagenicity tests, preliminary range-finding tests

performed on all bacterial strains, with and without metabolic activation, with appropriate positive

and negative controls, and with quantification of mutants, may be considered a sufficient

replication of a subsequent complete test. Similarly, a range-finding test may also be a satisfactory

substitute for a complete repeat of a test in gene mutation tests with mammalian cells other than

the mouse lymphoma tk assay (see below) if the range-finding test is performed with and without

metabolic activation, with appropriate positive and negative controls, and with quantification of

mutants (see Note 10).

For the cytogenetic evaluation of chromosomal damage in vitro, the test protocol includes the

conduct of tests with and without metabolic activation, with appropriate positive and negative

controls, where the exposure to the test articles is 3 to 6 hours and a sampling time of

approximately 1.5 normal cell cycles from the beginning of the treatment. A continuous treatment

without metabolic activation up to the sampling time of approximately 1.5 normal cell cycles is

needed in case of a negative result for the short treatment period without metabolic activation.

Certain chemicals may be more readily detected by longer treatment or delayed sampling times,

e.g., some nucleoside analogues or some nitrosamines. Negative results in the presence of a

metabolic activation system may need confirmation on a case-by-case basis (see Note 11). In any

case, information on the ploidy status should be obtained by recording the incidence of polyploid

cells as a percentage of the number of metaphase cells. An elevated mitotic index or an increased 
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incidence of polyploid cells may give an indication of the potential of a compound to induce

aneuploidy. In such cases, further testing may be needed.

For the mouse lymphoma tk assay, the test protocol includes the conduct of tests with and

without metabolic activation, with appropriate positive and negative controls, where the exposure

to the test articles is 3 to 4 hours. A continuous treatment without metabolic activation for

approximately 24 hours is needed in case of a negative result for the short treatment without

metabolic activation (see Note 4). Negative results in the presence of a metabolic activation

system may need confirmation on a case by case basis (see Note 11). In any case, an acceptable

mouse lymphoma tk assay includes (i) the incorporation of positive controls, which induces

mainly small colonies and (ii) colony sizing for positive controls, solvent controls, and at least one

positive test compound dose (should any exist), including the culture that gave the greatest

mutant frequency.

Following such testing, further confirmatory testing in the case of clearly negative or positive test

results is not usually needed.

Ideally, it should be possible to declare test results as clearly negative or clearly positive.

However, test results sometimes do not fit the predetermined criteria for a positive or negative

call and therefore are declared "equivocal." The application of statistical methods aids in data

interpretation, however, adequate biological interpretation is of critical importance. Nonetheless,

further testing is usually indicated for equivocal results.

VI. NOTES (6)

(1) Test approaches currently accepted for the assessment of mammalian cell gene mutation

involve the tk locus using mouse lymphoma L5178Y cells or human lymphoblastoid TK6

cells, the hprt locus using CHO cells, V79 cells, or L5178Y cells, or the gpt locus using

AS52 cells.

(2) The molecular dissection of mutants induced at the tk locus shows a broad range of

genetic events including point mutations, deletions, translocations, recombinations, etc.

Small colony mutants have been shown to predominantly lack the tk allele as a b

consequence of structural or numerical alterations or recombinational events. There is

some evidence that other loci, such as hprt or gpt are also sensitive to large deletion

events. However, due to the X-chromosomal origin of the hprt gene which is probably

flanked by essential genes, large scale deletion events or numerical alterations often do not

give rise to mutant colonies, thus limiting the sensitivity of this genetic locus relative to the

tk locus for the detection of a wide range of genetic changes. 

(3) With respect to the cytogenetic evaluation of chromosomal damage, it is not uncommon

for the systems currently in use, i.e., several systems with permanent mammalian cells in

culture and human lymphocytes either isolated or in whole blood, to give different results
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for the same test compound. However, there is evidence that some of the differences

observed have been due to protocol differences. This may be minimized by using the

procedures described in section V (5).

For the great majority of presumptive genotoxic compounds that were negative in a

bacterial reverse mutation assay, the data on chromosomal damage in vitro and mouse

lymphoma tk results are in agreement. Several reliable studies indicate that the mouse

lymphoma tk assay is able to detect compounds that induce structural and numerical

chromosomal damage. For safety testing of pharmaceuticals, the mouse lymphoma tk

assay is considered an acceptable alternative to the direct analysis of chromosomal damage

in vitro. Although colony sizing is an essential element of the mouse lymphoma tk assay

test protocol, it gives only limited information on the type of damage induced in mutant

colonies. Further mechanistic investigations may be used to assess the nature of

cytogenetic changes induced by clastogens and aneuploidy inducers in the mouse

lymphoma tk assay. Such information could be provided by studies to demonstrate the loss

of the tk gene or the loss of the chromosome carrying the tk gene.

(4) The detection of a number of different nucleoside analogues and base analogues is

enhanced for the mouse lymphoma tk assay when the treatment protocol for both agar and

microtitre methods includes a 24-hour treatment regimen in the absence of an exogenous

metabolic activation system. Similarly, the detection of aneuploidy inducers is enhanced if

a 24-hour treatment regimen is used with the microtitre method. Currently, there is no

evidence to support this conclusion for the soft agar method. The specificity of the test

protocol, i.e., to obtain correct test results for presumptive nongenotoxic compounds,

does not change significantly using a 24-hour treatment in the microtitre method. For the

soft agar method, there appears to be a reduction in specificity under the same treatment

regimen. Based on this information, the microtitre method is recommended for use in the

standard battery. 

(5) There are a small but significant number of genotoxic carcinogens that are reliably

detected by the bone marrow tests for chromosomal damage that have yielded

negative/weak/conflicting results in the pairs of in vitro tests outlined in the standard

battery options, e.g., bacterial reverse mutation plus one of a selection of possible tests

with cytogenetic evaluation of chromosomal damage or bacterial mutation plus the mouse

lymphoma tk assay. Carcinogens such as procarbazine, hydroquinone, urethane and

benzene fall into this category.

(6) The continuing evolution of short-term tests and test methodologies will afford new, more

sensitive, more practical, more expeditious, and more economical techniques for detection

of genotoxic compounds. Some of these may ultimately replace the genotoxicity tests

used for regulatory purposes. Among the more promising tests, the in vitro micronucleus

test appears to offer potential for screening purposes.
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(7) Some antibacterial agents, albeit highly toxic to the tester strains, are detected as

genotoxic at very low, sublethal concentrations in the bacterial reverse mutation test (e.g.,

nitrofuran antibiotics).

(8) Certain structurally alerting molecular entities are recognized as being causally related to

the carcinogenic and/or mutagenic potential of chemicals. Examples of structural alerts

include alkylating electrophilic centers, unstable epoxides, aromatic amines, azostructures, N-nitroso-groups, aromatic nitro-groups.

(9) For some classes of compounds with specific structural alerts, it is established that specific

protocol modifications/additional tests are necessary for optimum detection of

genotoxicity (e.g., molecules containing an azo-group, glycosides, compounds such as

nitroimidazoles requiring nitroreduction for activation, compounds such as phenacetin

requiring another rodent S9 for metabolic activation). The additional testing needed when

the chosen 3-test battery yields negative results for a structurally alerting test compound

could consist of such modifications.

(10) The dose range-finding study should (i) give information on the shape of the toxicity doseresponse curve if the test compound exhibits toxicity, (ii) include highly toxic

concentrations, and (iii) include quantification of mutants in the cytotoxic range. If a

compound is not toxic, then mutants should nevertheless be quantified.

(11) A repetition of a test using the identical source and concentration of the metabolic

activation system is usually not necessary. A modification of the metabolic activation

system may be indicated for certain chemical classes where knowledge is available on

specific requirements of metabolism. This would usually invoke the use of an external

metabolizing system which is known to be competent for the metabolism/activation of the

class of compound under test.
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VII. GLOSSARY (7)

Cytogenetic evaluation: Chromosome structure analysis in mitosis or meiosis by light

microscopy.

DNA adduct: (Covalent) binding of chemicals to DNA.

DNA repair: Reconstitution of damaged DNA sequence.

DNA strand breaks: Single or double strand scissions in the DNA.

Numerical chromosome changes: Chromosome numbers different from the original haploid or

diploid set of chromosomes; for cell lines, chromosome numbers different from the modal

chromosome set.

Recombination: Breakage and balanced or unbalanced rejoining of DNA.

Transgene: An exogenous or foreign gene inserted into the host genome, into either somatic

cells or germ line cells.