Federal Register, Volume 62 Issue 64 (Thursday, April 3, 1997)

[Federal Register Volume 62, Number 64 (Thursday, April 3, 1997)]
[Notices]
[Pages 16026-16030]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 97-8554]

[[Page 16025]]

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Part V

Department of Health and Human Services

_______________________________________________________________________

Food and Drug Administration

_______________________________________________________________________

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

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

[[Page 16026]]

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.

[[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
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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]
BILLING CODE 4160-01-F}}}