62472 Federal Register / Vol. 62, No. 225 / Friday, November 21, 1997 / Notices

1This 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.

DEPARTMENT OF HEALTH AND

HUMAN SERVICES

Food and Drug Administration

[Docket No. 97D–0112]

International Conference on

Harmonisation; Guidance on

Genotoxicity: A Standard Battery for

Genotoxicity Testing of

Pharmaceuticals; Availability

AGENCY: Food and Drug Administration,

HHS.

ACTION: Notice.

SUMMARY: The Food and Drug

Administration (FDA) is publishing a

guidance entitled ‘‘S2B Genotoxicity: A

Standard Battery for Genotoxicity

Testing of Pharmaceuticals.’’ The

guidance was prepared under the

auspices of the International Conference

on Harmonisation of Technical

Requirements for Registration of

Pharmaceuticals for Human Use (ICH).

The guidance identifies a standard set of

genotoxicity tests that should be

conducted for pharmaceutical

registration and recommends the extent

of confirmatory experimentation in in

vitro genotoxicity tests in the standard

battery. The guidance complements the

ICH guidance ‘‘Guidance on Specific

Aspects of Regulatory Genotoxicity

Tests for Pharmaceuticals’’ (S2A).

DATES: Effective November 21, 1997.

Submit written comments at any time.

ADDRESSES: Submit written comments

on the guidance to the Dockets

Management Branch (HFA–305), Food

and Drug Administration, 12420

Parklawn Dr., rm. 1–23, Rockville, MD

20857. Copies of the guidance are

available from the Drug Information

Branch (HFD–210), Center for Drug

Evaluation and Research, Food and

Drug Administration, 5600 Fishers

Lane, Rockville, MD 20857, 301–827–

4573.

FOR FURTHER INFORMATION CONTACT:

Regarding the guidance: Robert E.

Osterberg, Center for Drug

Evaluation and Research (HFD–

520), Food and Drug

Administration, 9201 Corporate

Blvd., Rockville, MD 20850, 301–

827–2123.

Regarding ICH: Janet J. Showalter,

Office of Health Affairs (HFY–20),

Food and Drug Administration,

5600 Fishers Lane, Rockville, MD

20857, 301–827–0864.

SUPPLEMENTARY INFORMATION: In recent

years many important initiatives have

been undertaken by regulatory

authorities and industry associations to

promote international harmonization of

regulatory requirements. FDA has

participated in many meetings designed

to enhance harmonization and is

committed to seeking scientifically

based harmonized technical procedures

for pharmaceutical development. One of

the goals of harmonization is to identify

and then reduce differences in technical

requirements for drug development

among regulatory agencies.

ICH was organized to provide an

opportunity for tripartite harmonization

initiatives to be developed with input

from both regulatory and industry

representatives. FDA also seeks input

from consumer representatives and

others. ICH is concerned with

harmonization of technical

requirements for the registration of

pharmaceutical products among three

regions: The European Union, Japan,

and the United States. The six ICH

sponsors are the European Commission,

the European Federation of

Pharmaceutical Industries Associations,

the Japanese Ministry of Health and

Welfare, the Japanese Pharmaceutical

Manufacturers Association, the Centers

for Drug Evaluation and Research and

Biologics Evaluation and Research,

FDA, and the Pharmaceutical Research

and Manufacturers of America. The ICH

Secretariat, which coordinates the

preparation of documentation, is

provided by the International

Federation of Pharmaceutical

Manufacturers Associations (IFPMA).

The ICH Steering Committee includes

representatives from each of the ICH

sponsors and the IFPMA, as well as

observers from the World Health

Organization, the Canadian Health

Protection Branch, and the European

Free Trade Area.

In the Federal Register of April 3,

1997 (62 FR 16026), FDA published a

draft tripartite guideline entitled

‘‘Genotoxicity: A Standard Battery for

Genotoxicity Testing of

Pharmaceuticals’’ (S2B). The notice gave

interested persons an opportunity to

submit comments by June 2, 1997.

After consideration of the comments

received and revisions to the guidance,

a final draft of the guidance was

submitted to the ICH Steering

Committee and endorsed by the three

participating regulatory agencies on July

16, 1997.

In accordance with FDA’s Good

Guidance Practices (62 FR 8961,

February 27, 1997), this document has

been designated a guidance, rather than

a guideline.

Genotoxicity tests are in vitro and in

vivo tests designed to detect compounds

that induce genetic damage directly or

indirectly by various mechanisms.

Compounds that are positive in tests

that detect such damage have the

potential to be human carcinogens and/

or mutagens, i.e., may induce cancer

and/or heritable defects. The guidance

addresses two areas of genotoxicity

testing for pharmaceuticals: (1)

Identification of a standard set of tests

that should be conducted for

registration and (2) the extent of

confirmatory experimentation in in vitro

genotoxicity tests in the standard

battery. The guidance is intended to be

used together with the ICH S2A

guidance entitled ‘‘Guidance on Specific

Aspects of Regulatory Genotoxicity

Tests for Pharmaceuticals’’ (61 FR

18198, April 24, 1996) as ICH guidance

principles for testing pharmaceuticals

for potential genotoxicity.

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.

As with all of FDA’s guidances, the

public is encouraged to submit written

comments with new data or other new

information pertinent to this guidance.

The comments in the docket will be

periodically reviewed, and, where

appropriate, the guidance will be

amended. The public will be notified of

any such amendments through a notice

in the Federal Register.

Interested persons may, at any time,

submit written comments on the

guidance to the Dockets Management

Branch (address above). Two copies of

any comments are to be submitted,

except that individuals may submit one

copy. Comments are to be identified

with the docket number found in

brackets in the heading of this

document. The guidance and received

comments may be seen in the office

above between 9 a.m. and 4 p.m.,

Monday through Friday. An electronic

version of this guidance is available on

the Internet (http://www.fda.gov/cder/

guidance.htm).

The text of the guidance follows:

S2B Genotoxicity: A Standard Battery for

Genotoxicity Testing of Pharmaceuticals1

1. Introduction

Two fundamental areas in which

harmonization of genotoxicity testing for
Federal Register / Vol. 62, No. 225 / Friday, November 21, 1997 / Notices 62473

pharmaceuticals is considered necessary are

the scope of this guidance: (I) Identification

of a standard set of tests that should be

conducted for registration. (II) 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 ‘‘Guidance on 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.

2. General Purpose of Genotoxicity Testing

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 compound may induce

cancer. In addition, the outcome of such tests

may be valuable for the interpretation of

carcinogenicity studies.

3. The Standard Test Battery for

Genotoxicity

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 5 of this document) 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.

The following standard test battery is

recommended based upon the considerations

mentioned above:

(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.

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 ‘‘Guidance on

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.

4. Modifications of the 3-Test Battery

The following sections give situations

where the standard 3-test battery may need

modification.

4.1 Limitations to the Use of Bacterial Test

Organisms

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 5 of this document) may be

appropriate.

4.2 Compounds Bearing Structural Alerts for

Genotoxic Activity

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

‘‘Guidance on Specific Aspects of Regulatory

Genotoxicity Tests for Pharmaceuticals’’).

4.3 Limitations to the Use of Standard In

Vivo Tests

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.
62474 Federal Register / Vol. 62, No. 225 / Friday, November 21, 1997 / Notices

4.4 Additional Genotoxicity Testing in

Relation to the Carcinogenicity Bioassay

4.4.1 Evidence for Tumor Response

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 in

vivo tests measuring genetic damage in target

organs of tumor induction (e.g., liver UDS

test, 32P-postlabeling, mutation induction in

transgenes, molecular characterization of

genetic changes in tumor-related genes).

4.4.2 Structurally Unique Chemical Classes

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.

5. Standard Procedures for In Vitro Tests

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 rangefinding test can be used to guide the selection

of concentrations to be used in the definitive

mutagenicity test. By these means, a rangefinding 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 rangefinding 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 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.

6. Notes

(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 tkb allele as a

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 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 5 of this document.

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,
Federal Register / Vol. 62, No. 225 / Friday, November 21, 1997 / Notices 62475

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.

(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, azo-structures, N-nitrosogroups, 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 dose-response 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.

7. Glossary

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.

Dated: November 15, 1997.

William K. Hubbard,

Associate Commissioner for Policy

Coordination.

[FR Doc. 97–30706 Filed 11–20–97; 8:45 am]

BILLING CODE 4160–01–F