[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



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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.
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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