16026 Federal Register / Vol. 62, No. 64 / Thursday, April 3, 1997 / Notices

DEPARTMENT OF HEALTH AND

HUMAN SERVICES

Food and Drug Administration

[Docket No. 97D–0112]

International Conference on

Harmonisation; Draft Guideline 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

draft guideline entitled ‘‘Genotoxicity: A

Standard Battery for Genotoxicity

Testing of Pharmaceuticals.’’ The draft

guideline was prepared under the

auspices of the International Conference

on Harmonisation of Technical

Requirements for Registration of

Pharmaceuticals for Human Use (ICH).

The draft guideline identifies a standard

set of genotoxicity tests to be conducted

for pharmaceutical registration, and

recommends the extent of confirmatory

experimentation in in vitro genotoxicity

tests in the standard battery. The draft

guideline complements the ICH

guideline ‘‘Guidance on Specific

Aspects of Regulatory Genotoxicity

Tests for Pharmaceuticals.’’

DATES: Written comments by June 2,

1997.

ADDRESSES: Submit written comments

on the draft guideline to the Dockets

Management Branch (HFA–305), Food

and Drug Administration, 12420

Parklawn Dr., rm. 1–23, Rockville, MD

20857. Copies of the draft guideline 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 guideline: 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 the 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 September 1996, the ICH Steering

Committee agreed that a draft guideline

entitled ‘‘Genotoxicity: A Standard

Battery for Genotoxicity Testing of

Pharmaceuticals’’ should be made

available for public comment. The draft

guideline is the product of the Safety

Expert Working Group of the ICH.

Comments about this draft will be

considered by FDA and the Safety

Expert Working Group.

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 draft

guideline addresses two areas of

genotoxicity testing for pharmaceuticals:

(1) Identification of a standard set of

tests to be conducted for registration,

and (2) the extent of confirmatory

experimentation in in vitro genotoxicity

tests in the standard battery. The draft

guideline is intended to be used

together with the ICH guideline 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.

Although not required, FDA has in

the past provided a 75- or 90-day

comment period for draft ICH

guidelines. However, the comment

period for this guideline has been

shortened to 60 days so that comments

may be received by FDA in time to be

reviewed and then discussed at a July

1997 ICH meeting involving this

guideline.

This guideline 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.

Interested persons may, on or before

June 2, 1997, submit to the Dockets

Management Branch (address above)

written comments on the draft

guideline. 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 draft

guideline 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

guideline is available via Internet by

using the World Wide Web (WWW). To

connect to the CDER home page, type

‘‘http://www.fda.gov/cder’’ and go to

the ‘‘Regulatory Guidance’’ section.

The text of the draft guideline follows:

Genotoxicity: A Standard Battery for

Genotoxicity Testing of Pharmaceuticals

1. Introduction

Two fundamental areas in which

harmonization of genotoxicity testing for

pharmaceuticals is considered necessary are

the scope of this guideline: (I) Identification

of a standard set of tests to 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 guideline ‘‘Guidance

on Specific Aspects of Regulatory

Genotoxicity Tests for Pharmaceuticals,’’ (61

FR 18198, April 24, 1996). The two ICH

guidelines on genotoxicity complement each

other and therefore should be used together

as ICH guidance principles for testing of a

pharmaceutical for potential genotoxicity.
Federal Register / Vol. 62, No. 64 / Thursday, April 3, 1997 / Notices 16027

2. General Purpose of Genotoxicity Testing

Genotoxicity tests can be defined as in

vitro and in vivo tests designed to detect

compounds which 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. In addition, the outcome of

such tests may be valuable for the

interpretation of carcinogenicity studies.

Nevertheless, 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.

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 would

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

initially 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 which detect

gross chromosomal damage (in vitro tests for

chromosomal damage), a system which

detects gene mutations and clastogenic

effects (mouse lymphoma tk assay), and

systems which detect primarily gene

mutations (see Notes 1 and 2).

There has been a debate whether in vitro

tests for chromosomal damage and the mouse

lymphoma tk assay are equivalent for

detection of clastogens. Several studies have

shown that most of the differences reported

are due to differences in the test protocols

employed. The scientific information given

in Notes 3 and 4 demonstrate that with

appropriate test protocols (see section 5) the

various in vitro tests for chromosomal

damage and the mouse lymphoma tk assay

yield results with a high level of congruence.

Therefore these systems may be treated as

equally sensitive and considered

interchangeable for regulatory purposes if

these test protocols are used. Consequently,

for regulatory purposes, a negative result in

an in vitro test with cytogenetic evaluation of

chromosomal damage or in a mouse

lymphoma tk assay gives additional

assurance to the other parts of the standard

battery that the compound tested does not

induce genetic damage. In any event, the

mammalian cells used for genotoxicity

evaluation in vitro should be carefully

selected taking the specific particulars of the

test cells, the test protocol, and the test

compound into account.

(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 may be

deduced from 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. Compounds

giving positive results in the standard test

battery may, depending on their therapeutic

use, need to be tested more extensively (see

ICH ‘‘Guidance on Specific Aspects of

Regulatory Genotoxicity Tests for

Pharmaceuticals’’ (60 FR 18198, April 24,

1996)).

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. 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 a

substitute. 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 numerical chromosome changes, e.g.,

aneuploidy and polyploidy. However,

information on this type of damage should be

derived from the cytogenetic evaluation of

chromosomal damage in vitro and in vivo.

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, nucleosideanalogues, or inhibitors of DNA metabolism).

For these cases, usually two in vitro

mammalian cell tests should be performed

using two different cell types and two

different endpoints (gene mutation (see Note

1) and chromosomal damage). Nevertheless it

is still important to perform the bacterial

reverse mutation test, either a full test or a

limited (range-finding) test (see section 5).

4.2 Compounds bearing structural alerts for

genotoxic activity

Structurally alerting compounds (see Note

6) 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 using

induced rat liver S9 for metabolic activation

as standard in the in vitro tests and using

mouse erythropoietic cells as standard test

cells for the in vivo test may need limited

additional testing. The choice of additional

test(s) or protocol modification(s) depend on

the chemical nature, the known reactivity,

and metabolism data on the structurally

alerting compound under question (see Note

7).

4.3 New/unique chemical structures/classes

On relatively rare occasions, a completely

novel compound in a unique structural or

functional (i.e., potentially DNA-reactive)

chemical class will be introduced as a

pharmaceutical. It may not be easy to

categorize such compounds, e.g., with

respect to alerting structures, metabolism

requirements, or interaction with cell
16028 Federal Register / Vol. 62, No. 64 / Thursday, April 3, 1997 / Notices

replication. In order to gain knowledge on

the genotoxic potential of such compounds it

may be necessary to test them more

comprehensively than in the standard 3-test

battery, e.g., in a further in vitro test with

mammalian cells.

4.4 Genotoxicity testing of pharmaceuticals

using solely in vitro tests

There are compounds for which

conventional in vivo tests do not provide

additional useful information. These include

compounds that are not systemically

absorbed and therefore are not available for

the target tissues in in vivo genotoxicity tests

(i.e., bone marrow or liver). Examples of such

compounds are some radioimaging agents,

aluminum-based antacids, and some

dermally applied pharmaceuticals. In these

cases, a test battery composed solely of in

vitro test models is acceptable which should

consist of a bacterial gene mutation assay, a

gene mutation assay with mammalian cells

(see Note 1), and a test for chromosomal

damage with mammalian cells.

4.5 Considerations for additional

genotoxicity testing in relation to the

carcinogenicity bioassay

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 genotoxic damage in

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

UDS test, 32P-postlabeling, mutation

induction in transgenes).

5. Standard Procedures for In Vitro Tests in

the Standard Battery

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

outlined below are used.

Complete repetition of gene mutation tests

is usually not necessary if the protocol

includes a range-finding test that supplies

sufficient data to provide reassurance that the

reported result is the correct one. For

example, 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

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 if the rangefinding test is performed with and without

metabolic activation, with appropriate

positive and negative controls, and with

quantification of mutants (see Note 8). For

both bacterial and mammalian cell gene

mutation tests, the results of the rangefinding test should guide the selection of

concentrations to be used in the definitive

mutagenicity test.

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 cell

cycles is needed in case of a negative result

for the short treatment period without

metabolic activation. If severe cell cycle

delay is noted, a prolonged treatment or

sampling time is needed. Negative results in

the presence of a metabolic activation system

may need confirmation on a case-by-case

basis (see Note 9). 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.

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 24 hours is advisable

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 9). In any case, the conduct of a mouse

lymphoma tk assay involves 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 define test

results as clearly negative or clearly positive.

But test results sometimes do not fit into the

criteria for a positive or negative call and

therefore have to be defined as ‘‘equivocal.’’

In these circumstances, the application of

statistical methods can aid in data

interpretation. Since the use of statistical

methods is not always satisfying for some of

the standard genotoxicity tests, adequate

biological interpretation is of critical

importance. The criteria for declaration of a

test result as positive or negative must in part

be based on the experience and standards of

the laboratory carrying out the test.

Equivocality then, for example, encompasses

test results which lack a dose-related increase

of the effect in an appropriate dose range

and/or test results which exceed the

concurrent negative control values but may

lie within historical negative control data.

Further testing is usually indicated in the

case of results that have to be called

equivocal even if the results are obtained

with protocols such as outlined above.

6. Notes

(1) Test systems seen currently as

appropriate for the assessment of mammalian

cell gene mutation include the L5178Y

tk∂/¥‰ tk¥/¥ mouse lymphoma assay

(mouse lymphoma tk assay), the HPRT-tests

with CHO-cells, V79-cells, or L5178Y cells,

or the GPT-(XPRT) test with AS52 cells, and

the human lymphoblastoid TK6 test.

(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. (e.g., Applegate et al., 1990). Small

colony mutants have been shown to

predominantly lack the tkb allele as a

consequence of structural or numerical

alterations or recombinational events (Blazak

et al., 1989; El-Tarras et al., 1995). There is

some evidence that other loci, such as hprt

or gpt are also sensitive to large deletion

events (Glatt, 1994; Kinashi et al., 1995).

However, due to the X-chromosomal origin of

the hprt gene which is probably flanked by

essential genes, large scale chromosomal

damage (e.g., deletion) or numerical

alterations often do not give rise to mutant

colonies, thus limiting the sensitivity of this

test. Therefore, the mouse lymphoma tk assay

has advantages in comparison to other gene

mutation assays and it may be recommended

to conduct the mouse lymphoma tk assay as

the gene mutation test. A positive result in

the mouse lymphoma tk assay may constitute

a case for further investigation of the type

and/or mechanism of genetic damage

involved.

(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, a recently

conducted multilaboratory comparison of in

vitro tests with cytogenetic evaluation of

chromosomal damage gave conclusive

evidence that the differences observed are

most often due to protocol differences

(Galloway et al., 1996).

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. A

recently conducted mouse lymphoma tk

collaborative study reinforced this view.

Under cooperation of the Japanese Ministry

of Health and Welfare and the Japanese

Pharmaceutical Manufacturers Association, a

collaborative study on the mouse lymphoma

tk assay (MLA) was conducted by 45

Japanese and 7 other laboratories in order to

clarify how well the MLA can detect in vitro

clastogens and polyploidy (aneuploidy)

inducers and how well the in vitro tests with

cytogenetic evaluation of chromosomal

damage can detect compounds that were

thought to act exclusively in the MLA. On

the basis of published data, 40 compounds

were selected, which were negative in

bacterial reverse mutation assays, but

positive either in in vitro tests with

cytogenetic evaluation of chromosomal

damage (30 compounds) or in the MLA (9
Federal Register / Vol. 62, No. 64 / Thursday, April 3, 1997 / Notices 16029

compounds). These compounds were

examined by the microwell method using

L5178Y tk∂/¥ 3.7.2C cells or were

reexamined in CHL/IU cells for induction of

chromosomal aberrations. Various aspects of

this study are currently in the process of

publication (Matsuoka et al., 1996; Sofuni et

al., 1996).

The table below gives the results of

this major attempt to compare the

results of in vitro tests with cytogenetic

evaluation of chromosomal damage in

different cells (human lymphocytes,

CHO, V79 and CHL cells) and the mouse

lymphoma tk assay:

chromosome damage

(CA) mainly structural

chromosome damage

(CA) mainly polyploidy

chromosome damage

(CA)

positive positive negative

mouse positive 211 51 2

lymphoma inconcl./equiv. 3 2 1

tk assay negative 2 1 3

1 7 compounds (colchicine, 2′-deoxycoformycin, dideoxycytidine, phenacetin, p-tert butylphenol, theophylline, thiabendazole) yielded clearly

positive results in the MLA when the cells were treated in the absence of S–9 mix for 24 hours instead of 4 hours.

Of 34 CA (carcinogen) positive chemicals,

3 (9 percent) were negative in the MLA.

These results suggest that while the MLA

may detect most clastogens and polyploidy

inducers, there may be some it cannot detect

(bromodichloromethane, isophorone,

tetrachloroethane). Tetrachloroethane

induced polyploidy only, whereas

bromodichloromethane and isophorone were

only weakly clastogenic.

Reinvestigation of 9 of 10 mouse

lymphoma unique positive carcinogens that

were reported by the NTP (National

Toxicology Program) (Zeiger et al., 1990)

showed that only 3 were negative in CHL/IU

cells using the comprehensive protocol as

outlined in section 5. The same nine

compounds were reexamined in the present

MLA study and two of the three CA-negative

compounds were positive (trichloroethylene

and cinnamylanthranilate). These data

indicate that the number of MLA unique

positive compounds may be quite limited,

i.e., at the moment, in the absence of

reinvestigation of other NTP reported mouse

lymphoma tk uniquely positive compounds,

only trichloroethylene and

cinnamylanthranilate are known.

Comparison with published data and data

in regulatory files show that many MLA and

CA positive compounds were negative in the

HPRT assay in which large-scale DNA

rearrangements could not be detected.

Only a few more clastogenic compounds

giving negative results in the usual mouse

lymphoma tk assay with 3 to 4 hours of

treatment can be found in the published

literature (Garriott et al., 1995). In

conclusion, it is perceived that, from the

aspect of safety testing for pharmaceuticals,

the mouse lymphoma tk assay is an

acceptable alternative for the direct analysis

of chromosomal damage in vitro. Colony

sizing gives only limited information on the

type of damage induced in mutant colonies

in the mouse lymphoma tk assay (see Note

2). Therefore, a positive result in a mouse

lymphoma tk assay may need to be

investigated further to examine the type of

genetic damage that was induced.

(4) Recent results from a number of

different compounds give evidence that the

ability of the mouse lymphoma tk assay to

detect some clastogens/aneuploidy inducers

is enhanced when the treatment protocol

includes a 24 hour treatment regimen in the

absence of an exogenous metabolic activation

system. Compounds such as colchicine,

vincristine, diethylstilbestrol, caffeine, 2′-

deoxycoformycin, dideoxycytidine,

thiabendazole, theophylline, phenacetin, ptert butylphenol, and azidothymidine gave

negative or only weakly positive results in a

standard mouse lymphoma tk assay with 3 or

4 hours of treatment (absence of S–9 mix) but

were tested clearly positive with 24 hours of

exposure to the test substance.

(Azidothymidine and caffeine are the

compounds which were tested in the agar

version of the mouse lymphoma tk assay

whereas the data on 24 hours of treatment on

the other compounds are generated with the

microwell method.)

(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) Certain structurally alerting molecular

entities are recognized as being causally

related to the carcinogenic and/or mutagenic

potential of chemicals (Ashby and Tennant,

1988; Ashby and Tennant, 1991; Ashby and

Paton, 1993). Examples of structural alerts

include alkylating electrophilic centers,

unstable epoxides, aromatic amines, azostructures, N-nitroso-groups, aromatic nitrogroups.

(7) 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). Such modifications could form

the additional testing needed when the

chosen 3-test battery yields negative results

for a structurally alerting test compound.

(8) 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; (iii) include quantification of

mutants in the cytotoxic range. Even if a

compound is not toxic, mutants should

nevertheless be quantified.

(9) A repetition of a test using the identical

source and concentration of the metabolic

activation system is usually not necessary.

However, 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 involve 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. References to Notes

Applegate, M. L., M. M. Moore, C. B.

Broder, A. Burrell, G. Juhn, K. L. Kasweck,

P. Lin, A. Wadhams, and J. C. Hozier,

‘‘Molecular dissection of mutations at the

heterozygous thymidine kinase locus in

mouse lymphoma cells,’’ Proceedings of the

National Academy of Sciences of the USA,

87:51–55, 1990.

Ashby, J., and R. W. Tennant, ‘‘Chemical

structure, Salmonella mutagenicity and

extent of carcinogenicity as indicators of

genotoxic carcinogenesis among 222

chemicals tested in rodents by the U.S. NCI/

NTP,’’ Mutation Research, 204:17–115, 1988.

Ashby, J., and R. W. Tennant, ‘‘Definitive

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Dated: March 29, 1997.

William K. Hubbard,

Associate Commissioner for Policy

Coordination.

[FR Doc. 97–8554 Filed 4–2–97; 8:45 am]

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