International Conference on Harmonisation; Draft Guideline on Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals; Availability

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.

[[Page 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,

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 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

[[Page 16028]]

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

range-finding 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 range-finding

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

[[Page 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) chromosome damage (CA) chromosome damage

mainly structural mainly polyploidy (CA)

positive positive negative

mouse positive 21\1\ 5\1\ 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, p-tert

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, azo-structures, N-nitroso-groups, aromatic nitro-

groups.

(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 relationships among

chemical structure, carcinogenicity and mutagenicity for 301

chemicals tested by the U.S. NTP,'' Mutation Research, 257:229-308.

1991.

Ashby, J., and D. Paton, ``The influence of chemical structure

on the extent and sites of carcinogenesis of 522 rodent carcinogens

and 55 different human carcinogen exposures,'' Mutation Research,

286:3-74, 1993.

Blazak, W. F., F. J. Los, C. J. Rudd, and W. J. Caspary,

``Chromosome analysis of small and large L5178Y mouse lymphoma cell

colonies: Comparison of trifluorothymidine-resistant and unselected

cell colonies from mutagen-treated and control cultures,'' Mutation

Research, 224:197-208, 1989.

El-Tarras, A., J. S. Dubins, J. Warner, C. Hoffman, and R. R.

Cobb, ``Molecular analysis of the TK locus in L5178Y large and small

colony mouse lymphoma cell mutants induced by hycanthone

methanesulfonate,'' Mutation Research, 332:89-95, 1995.

[[Page 16030]]

Galloway, S. M., T. Sofuni, M. D. Shelby, A. Thilagar, V.

Kumaroo, N. Sabharwal, D. Gulati, D. L. Putman, H. Murli, R.

Marshall, N. Tanaka, B. Anderson, E. Zeiger, and M. Ishidate, Jr.,

``A multi-laboratory comparison of in vitro tests for chromosome

aberrations in CHO and CHL cells tested under the same protocols,''

Environmental and Molecular Mutagenesis, (in press) 1996.

Garriott, M. L., D. A. Casciano, L. M. Schechtman, and G. S.

Probst, ``Meeting report: Workshop on mouse lymphoma assay testing

practices and data interpretation,'' Environmental and Molecular

Mutagenesis, 25:162-164, 1995.

Glatt, H. R., ``Comparison of common gene mutation tests in

mammalian cells in culture: A position paper of the GUM Commission

for the development of guidelines for genotoxicity testing,''

Mutation Research, 313:7-20, 1994.

Kinashi, Y., H. Nagasawa, and J. B. Little, ``Molecular

structural analysis of 417 HPRT mutations induced by restriction

endonucleases in Chinese hamster ovary (CHO) cells,'' Mutation

Research, 326:83-92, 1995.

Matsuoka, A., K. Yamakage, H. Kusakabe, S. Wakuri, M. Asakura,

T. Noguchi, T. Sugiyama, H. Shimada, S. Nakayama, Y. Kasahara, Y.

Takahashi, K. F. Miura, M. Hatanaka, M. Ishidate, Jr., T. Morita, K.

Watanabe, M. Hara, K. Odawara, N. Tanaka, M. Hayashi, and T. Sofuni,

``Re-evaluation of chromosomal aberration induction on nine mouse

lymphoma assay ``unique positive'' NTP carcinogens,'' Mutation

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Sofuni, T., H. Honma, M. Hayashi, H. Shimada, N. Tanaka, S.

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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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