[Federal Register Volume 59, Number 183 (Thursday, September 22, 1994)]
[Unknown Section]
[Page 0]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 94-23377]


[[Page Unknown]]

[Federal Register: September 22, 1994]


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Part VII





Department of Health and Human Services





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Food and Drug Administration



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International Conference on Harmonisation, Draft Guideline on Specific 
Aspects of Regulatory Genetoxicity Tests; Notice
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DEPARTMENT OF HEALTH AND HUMAN SERVICES

Food and Drug Administration
[Docket No. 94D-0324]

 

International Conference on Harmonisation; Draft Guideline on 
Specific Aspects of Regulatory Genotoxicity Tests; Availability

AGENCY: Food and Drug Administration, HHS.

ACTION: Notice.

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SUMMARY: The Food and Drug Administration (FDA) is publishing a draft 
guideline entitled ``Notes for Guidance on Specific Aspects of 
Regulatory Genotoxicity Tests.'' This guideline was prepared by the 
Safety Expert Working Group of the International Conference on 
Harmonisation of Technical Requirements for Registration of 
Pharmaceuticals for Human Use (ICH). This draft guideline is intended 
to provide guidance on genotoxicity testing.
DATES:  Written comments by December 6, 1994.

ADDRESSES: Submit written comments on the draft guideline to the 
Dockets Management Branch (HFA-305), Food and Drug Administration, rm. 
1-23, 12420 Parklawn Dr., Rockville, MD 20857. Copies of the draft 
guideline are available from the CDER Executive Secretariat Staff (HFD-
8), Center for Drug Evaluation and Research, Food and Drug 
Administration, 7500 Standish Pl., Rockville, MD 20855.

FOR FURTHER INFORMATION CONTACT: 
    Regarding the draft guideline: Alan Taylor, Center for Drug 
Evaluation and Research (HFD-502), Food and Drug Administration, 5600 
Fishers Lane, Rockville, MD 20857, 301-443-2544.
    Regarding the ICH: Janet Showalter, Office of Health Affairs (HFY-
20), Food and Drug Administration, 5600 Fishers Lane, Rockville, MD 
20857, 301-443-1382.

SUPPLEMENTARY INFORMATION: In recent years, many important initiatives 
have been undertaken by regulatory authorities and industry 
associations to promote international Harmonisation of regulatory 
requirements. FDA has participated in many meetings designed to enhance 
Harmonisation and is committed to seeking scientifically based 
harmonized technical procedures for pharmaceutical development. One of 
the goals of Harmonisation is to identify and then reduce differences 
in technical requirements for drug development.
    ICH was organized to provide an opportunity for tripartite 
Harmonisation initiatives to be developed with technical input from 
both regulatory and industry representatives. FDA also seeks input from 
consumer representatives and others. ICH is concerned with 
Harmonisation 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 Industry 
Associations, the Japanese Ministry of Health and Welfare, the Japanese 
Pharmaceutical Manufacturers Association, 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.
    At a meeting held on March 10, 1994, the ICH Steering Committee 
agreed that a guideline entitled ``Notes for Guidance on Specific 
Aspects of Regulatory Genotoxicity Tests'' 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 Expert Working Group. Ultimately, FDA intends to adopt 
the ICH Steering Committee's final guideline.
    The draft guideline is to be applied in conjunction with existing 
guidelines in the United States, Japan, and Europe. No required battery 
of genetic toxicology tests has been adopted by FDA for pharmaceutical 
development in the United States, pending completion of ICH 
negotiations on this topic. The test battery recommended by FDA's 
Center for Food Safety and Applied Nutrition (58 FR 16536, March 29, 
1993) for food additives, however, is currently preferred for the 
evaluation of pharmaceuticals and is recommended to those seeking 
initial guidance in this area. This battery is similar to that endorsed 
previously by the Environmental Protection Agency's Office of 
Pesticides Program (Environmental and Molecular Mutagenesis, 21:38-45, 
1993).
    The draft guideline recommends methods of testing pharmaceuticals 
for genetic toxicity. These recommendations are based on historical 
information from the international pharmaceutical industry, regulatory 
agencies in the European Community, Japan, and the United States, and 
scientific literature. In general, while there may be cause for concern 
for mutagenic potential of certain biological products, the currently 
recommended tests should not be routinely applied to such products. 
When there is cause for concern, specific endpoints should be 
identified and relevant tests should be performed.
    In the past, guidelines have generally been issued under 
Sec. 10.90(b) (21 CFR 10.90(b)), which provides for the use of 
guidelines to state procedures or standards of general applicability 
that are not legal requirements but are acceptable to FDA. The agency 
is now in the process of revising Sec. 10.90(b). Therefore, if the 
agency issues this guideline in final form, the guideline would not be 
issued under the authority in current Sec. 10.90(b), and would not 
create or confer any rights, privileges, or benefits for or on any 
person, nor would it operate to bind FDA in any way.
    Interested persons may, on or before December 6, 1994, submit 
written comments on the draft guideline to the Dockets Management 
Branch (address above). Two copies of any comments are to be submitted, 
except that individuals may submit one copy. Comments are to be 
identified with the docket number found in brackets in the heading of 
this document. The draft guideline and received comments may be seen in 
the office above between 9 a.m. and 4 p.m., Monday through Friday.
    The text of the draft guideline follows:

Notes for Guidance on Specific Aspects of Regulatory Genotoxicity Tests

Introduction

    Guidelines for the testing of pharmaceuticals for genetic 
toxicity exist in the European Community (EC, 1987) and Japan 
(Japanese Ministry of Health and Welfare, 1989). FDA's Centers for 
Drug and Biologics Evaluation and Research (CDER and CBER) currently 
consider the guidance on genetic toxicity testing provided by FDA's 
Center for Food Safety and Applied Nutrition (Federal Register 
notice, March 29, 1993) to be applicable to pharmaceuticals.
    The following notes for guidance should be applied in 
conjunction with existing guidelines in the three ICH regions. The 
recommendations below are derived from considerations of historical 
information from the international pharmaceutical industry, the 
three regulatory bodies, and the scientific literature. Where 
relevant, the recommendations from the latest review of the 
Organization for Economic Cooperation and Development guidelines and 
the International Workshop on Standardization of Genotoxicity Test 
Procedures held in Melbourne, Australia (February 1993) have been 
considered. Information from the survey carried out by the Centre 
for Medicines Research on current practices and strategies used by 
the pharmaceutical industry for genotoxicity testing (Purves, et 
al., 1994) has also been considered.

A. The Base Set of Strains Used in Bacterial Mutation Assays

    Current guidelines for the detection of bacterial mutagens call 
for the inclusion of several strains to detect base substitution and 
frameshift point mutations. The Salmonella typhimurium strains cited 
in guidelines (normally TA1535, TA1537, TA98, and TA100) will detect 
such changes at G-C sites within target histidine genes. It is clear 
from the literature that some mutagenic carcinogens attack A-T base 
pairs preferentially (e.g., Levin, 1982; Wilcox, et al., 1990; also 
see note 1). Therefore, the standard set of strains used in 
bacterial mutation assays should include strains that will detect 
point mutations at A-T sites, such as S. typhimurium TA102, which 
possesses a mutation at an A-T site within multiple copies of hisG 
genes or E. coli WP2 uvrA, which possesses an A-T mutational site in 
the trpE gene or the same strain carrying the plasmid (pKM101), 
which carries mucAB genes that enhance error prone repair.
    In conclusion, the following base set of bacterial strains 
should be used for routine testing: the strains cited below are all 
S. typhimurium isolates, unless specified otherwise.
    1. TA98; 2. TA100; 3. TA1535; 4. TA1537 or TA97 or TA97a (see 
note 2); 5. TA102 or E. coli WP2 uvrA or E. coli WP2 uvrA (pKM101).

B. Acceptable Bone Marrow Tests for the Detection of Clastogens In Vivo

    Tests measuring chromosomal aberrations in nucleated bone marrow 
cells in rodents can detect a wide spectrum of changes in 
chromosomal integrity. These changes almost all result from breakage 
of one or more chromatids as the initial event. Breakage of 
chromatids can result in micronucleus formation if an acentric 
fragment is produced; therefore, assays detecting either chromosomal 
aberrations or micronuclei are acceptable for detecting clastogens 
(see note 3). Micronuclei can also result from dislocation of 
chromosomes from the mitotic spindle, and, thus, micronucleus tests 
have the potential to detect aneugenic compounds.
    In conclusion, the analysis of either chromosomal aberrations or 
micronuclei in bone marrow cells in vivo is acceptable for the 
detection of clastogens.
    The acceptability of the peripheral blood micronucleus assay as 
a substitute for the bone marrow micronucleus assay is being 
actively considered (see note 4).

C. Guidance on the Further Evaluation of Compounds Giving In Vitro 
Positive Results

    Comparative trials have shown conclusively that each in vitro 
test system generates both false negative and false positive results 
in relation to predicting rodent carcinogenicity. The test battery 
approach is designed to reduce the risk of false negative results. 
However, the genotoxicity test batteries as described will only 
detect carcinogens that act primarily via a mechanism involving 
direct genetic damage, such as the majority of known human 
carcinogens. According to the results of the National Toxicology 
Program (Haseman, et al., 1990) and an analysis of results from 
tests of pharmaceuticals and industrial chemicals in Japan (Shimada, 
1993), approximately 15 percent of carcinogens are not detected by 
the commonly used batteries of genotoxicity tests. Therefore, a 
positive result in any assay for genotoxicity does not necessarily 
mean that the test compound poses a genotoxic hazard to man. The 
following points should be considered when assessing in vitro 
positive results.

In vitro

    Positive results that are without apparent biological relevance 
should be excluded. These include the following considerations (this 
list is not exhaustive, but is given as an aid to decisionmaking):
    (i) Is the response clearly reproducible?
    (ii) Is the magnitude of the response regarded as biologically 
significant?
    (iii) For positive responses in the presence of a competent 
metabolic system, is in vitro metabolism similar to in vivo 
metabolism? Are in vitro specific metabolites induced?
     (iv) Can the effect be attributed to extreme culture conditions 
that do not occur in in vivo situations (i.e., extremes of pH; 
osmolality, etc.)?
    (v) Is the effect only seen at extremely low survival levels?
    (vi) Are compounds in this chemical class normally associated 
with positive effects in vitro? Are compounds in this chemical class 
normally associated with negative effects in vivo?

In vivo

    A positive result in an in vitro test that is regarded as 
biologically relevant (see previous paragraph) indicates that the 
test compound has genotoxic potential. An in vitro test measuring 
the same genetic endpoint should be carried out for confirmation. 
Such in vitro tests usually carry more significance than the 
comparable in vitro assays (Ashby, 1983). Thus, for a compound 
showing clastogenic activity in vitro the bone marrow micronucleus 
or chromosomal aberration assay can fulfil this role. It is 
recognized that, at present, there is no validated, widely used in 
vivo system which measures gene mutation. However, in vivo gene 
mutation assays using endogenous genes or transgenes in the rat and 
mouse are at various stages of development and validation. Until 
these tests for mutation become accepted, results from other widely 
used in vivo tests for genotoxicity in tissues other than the bone 
marrow can provide valuable additional data. Flexibility is 
desirable in the choice of a second in vivo assay (see note 5).
    In conclusion, where positive results have been obtained in one 
or more of the established in vitro tests, analysis should take 
place on a case-by-case basis as described above and in note 5.

D. Validation of Negative In Vivo Test Results

    Because in vivo tests have a pivotal role in genotoxicity test 
batteries, it is necessary to prove adequate exposure of the target 
tissue. This can be achieved by a clear biological response in the 
tissue in question or by toxicokinetic data. If adequate exposure 
cannot be achieved (e.g., with compounds showing very poor 
bioavailability, extensive protein binding, etc.), conventional in 
vivo genotoxicity tests may have little value.
    The following recommendations apply to bone marrow cytogenetic 
assays. If other target tissues are used, similar principles should 
be applied.
    For compounds showing positive results in any of the in vitro 
tests employed, validation of in vivo exposure should be made by any 
of the following measurements:
    (i) By measuring a significant change in the proportion of 
immature erythrocytes among total erythrocytes in the bone marrow, 
at the doses and sampling times used in the micronucleus test or by 
measuring a significant reduction in mitotic index for the 
chromosomal aberration assay.
    (ii) Evidence of bioavailability of drug-related material either 
by measuring blood or plasma levels (see note 6).
    (iii) By direct measurement of drug-related material in bone 
marrow.
    (iv) By autoradiographic assessment of tissue exposure.
    For methods (ii) to (iv), assessments should be made 
preferentially at the top dose using the same species/strain and 
dosing route used in the bone marrow assay.
    If in vitro tests do not show genotoxic potential, validation of 
in vivo (systemic) exposure is also needed and can be achieved by 
any of the methods above, but can also be inferred from the results 
of standard absorption, distribution, metabolism, and excretion 
(ADME) studies in rodents.

E. Definition of the Top Concentration for In Vitro Tests

    (i) High dose for nontoxic compounds
    For freely soluble, nontoxic compounds, the upper treatment 
levels are 5 mg/plate for bacteria and 5 mg/mL or 10 mM for 
mammalian cells.
    (ii) Desired level of cytotoxicity
    Most genotoxic carcinogens are not detectable in in vitro 
mammalian cell genotoxicity assays unless the concentrations tested 
induce some degree of cytotoxicity. It is also apparent that at very 
low survival levels, mechanisms other than direct genotoxicity per 
se can lead to `positive' results that are related to cytotoxicity 
and not genotoxicity (e.g., events associated with apoptosis, 
endonuclease release from lysosomes, etc. (Kirkland, 1992)). Such 
events are likely to occur once a certain concentration threshold is 
reached for a toxic compound.
    To balance these conflicting considerations, the following 
levels of cytotoxicity are currently acceptable for in vitro 
mammalian cell tests:
    The desired level of toxicity for in vitro cytogenetic tests 
using cell lines is defined as greater than 50 percent inhibition of 
cell proliferation or culture confluency. For lymphocyte cultures, 
an inhibition of mitotic index by greater than 50 percent is 
considered sufficient. The desired upper limit of toxicity for 
mammalian cell mutation tests should be at least 80 percent of the 
corresponding control value. Toxicity can be measured either by 
assessment of cloning efficiency immediately after treatment, 
suspension growth immediately after treatment, or by calculation of 
relative total growth.
    (iii) Tests of poorly soluble compounds
    There is some evidence that dose-related genotoxic activity can 
be detected when testing particular compounds in the insoluble 
range. This is always associated with dose-related toxicity (see 
note 7). It is possible that solubilization of a precipitate is 
enhanced by serum in the culture medium or in the presence of S9-mix 
constituents. It is also probable that cell membrane lipid can 
facilitate absorption of lipophilic compounds into cells. In 
addition, some types of mammalian cells are phagocytic (e.g., 
Chinese hamster V79, CHO and CHL cells) and can ingest solid 
particles which may subsequently disperse into the cytoplasm. An 
insoluble compound may also contain soluble genotoxic impurities. It 
should also be noted that some insoluble compounds are administered 
in vivo as suspensions or as particulate materials.
    Heavy precipitates can interfere with scoring the desired 
parameter and render control of exposure very difficult (e.g., where 
a centrifugation step(s) is included in a protocol to remove cells 
from exposure media) (see note 8), or render the test compound 
unavailable to enter cells and interact with DNA.
     The following strategy is recommended for testing relatively 
insoluble compounds. The recommendation refers to the test article 
in the culture medium.
    If no toxicity is observed, the lowest precipitating 
concentration should be used as the top concentration. If dose-
related toxicity or mutagenicity is noted, irrespective of 
solubility, then the top concentration should be based on toxicity 
as described above. It is recognized that the desired levels of 
cytotoxicity may not be achievable if the extent of precipitation 
interferes with the scoring of the test.
    In all cases, precipitation should be evaluated using the naked 
eye.

F. Use of Male/Female Rodents in Bone Marrow Micronucleus Tests

    Extensive studies of the activity of known clastogens in the 
mouse bone marrow micronucleus test have shown that, in general, 
male mice are more sensitive than female mice for micronucleus 
induction (see note 9). Quantitative differences in sensitivity to 
micronucleus induction have been identified between the sexes, but 
no qualitative differences have been described. Where marked 
quantitative differences exist, there is invariably a difference in 
toxicity between the sexes. If there is a clear qualitative 
difference in metabolites between male and female rodents, then both 
sexes should be used in the micronucleus test. Similar principles 
can be applied for other established in vivo tests (see note 10). 
Both rats and mice are deemed acceptable for use in the bone marrow 
mironucleus test (see note 11). In summary, unless there are obvious 
differences in toxicity or metabolism between male and female 
rodents, males alone are sufficient for use in bone marrow 
micronucleus tests. If gender- specific drugs are to be tested, the 
appropriate sex should be used.

Notes

    (1) Analysis of the database held by the Japanese Ministry of 
Labour on 5,526 compounds (and supported by smaller databases held 
by various pharmaceutical companies) has shown that approximately 
7.5 percent of the bacterial mutagens identified are detected by E. 
coli WP2 uvrA but not by the standard set of four Salmonella 
strains. Although animal carcinogenicity data are not available on 
these compounds, it is likely that such compounds would carry the 
same carcinogenic potential as mutagens-inducing changes in the 
standard set of Salmonella strains.
    (2) TA1537, TA97, and TA97a contain cytosine runs at the 
mutation-sensitive site within the relevant target histidine loci 
and show similar sensitivity to frameshift mutagens that induce 
deletion of bases in these frameshift hotspots. There was consensus 
agreement at the International Workshop on Standardization of 
Genotoxicity Procedures, Melbourne, 1993 (Gatehouse, et al., 1994) 
that all three strains could be used interchangeably, and that 
decision is endorsed here.
    (3) As the mechanisms of micronucleus formation are related to 
those inducing chromosomal aberrations (e.g., Hayashi, et al., 
1984), both micronuclei and chromosomal aberrations can be accepted 
as assay systems to screen for clastogenicity induced by test 
compounds. Comparisons of data where both the mouse micronucleus 
test and rat bone marrow metaphase analysis have been carried out on 
the same compounds has shown impressive correlation both 
qualitatively, i.e., detecting clastogenicity, and quantitatively, 
e.g., determination of the lowest clastogenic dose. Even closer 
correlations can be expected where the data are generated in the 
same species.
    (4) The peripheral blood micronucleus test in the mouse using 
acridine orange supravital staining was introduced by Hayashi, et 
al. (1990). The test has recently been the subject of a major 
collaborative study by the Japanese Collaborative Study Group for 
the Micronucleus Test (see Mutation Research (1992) 278, Nos. 2/3). 
The tests were carried out in CD-1 mice using 23 test substances of 
various modes of action. Peripheral blood sampled from the same 
animal was examined 0, 24, 48, and 72 hours (or longer) after 
treatment. As a rule one chemical was studied by 2 different 
laboratories (46 laboratories took part). All chemicals were 
detected as inducers of micronuclei. There were quantitative 
differences between laboratories, but no qualitative differences. 
Most chemicals gave the greatest response 48 hours after treatment. 
Thus, the results suggest that the peripheral blood assay using 
acridine orange supravital staining can generate reproducible and 
reliable data to evaluate the clastogenicity of chemicals. Based on 
these data, the Melbourne workshop concluded that this assay is 
equivalent in accuracy to the bone marrow micronucleus assay.
    (5) Apart from the cytogenetic assays in bone marrow cells, the 
largest database for in vivo assays exists for the liver unscheduled 
DNA synthesis (UDS) assay. A review of the literature shows that a 
combination of the liver UDS test and the bone marrow micronucleus 
test will detect most genotoxic carcinogens with few false positive 
results (Tweats, 1994). Unstable genotoxins and certain aromatic 
amines are problematical for all existing in vivo screens. The 
choice of a second test, however, should not be restricted to UDS 
tests because other assays may be more appropriate (e.g., 32P 
post-labelling, DNA strand-breakage assays, etc.), depending on the 
compound in question.
    (6) The bone marrow is a well-perfused tissue and it can be 
deduced therefore that levels of drug-related materials in blood or 
plasma will be similar to those observed in bone marrow. This is 
supported by direct comparisons of drug levels in the two 
compartments for a large series of different pharmaceuticals. 
Although drug levels are not always the same, there is sufficient 
correlation for measurements in blood or plasma to be adequate for 
validating bone marrow exposure.
    (7) Laboratories in Japan carrying out genotoxicity tests have 
much experience in testing precipitates, and have identified 
examples of substances that are clearly genotoxic only in the 
precipitating range of concentrations. These compounds include 
polymers and mixtures of compounds, some polycyclic hydrocarbons, 
some phenylene diamines, heptachlor, etc. Collaborative studies with 
some of these compounds have shown that they may be detectable in 
the soluble range; however, it does seem clear that genotoxic 
activity increases well into the insoluble range. A discussion of 
these factors is found in the report of the ``in vitro'' subgroup of 
the Melbourne conference (Kirkland, 1994).
    (8) Testing compounds in the precipitating range is 
problematical with respect to defining the exposure periods for 
assays where the cells grow in suspension. After the defined 
exposure period, the cells are normally pelleted by centrifugation 
and are then resuspended in fresh medium without the test compound. 
If a precipitate is present, the compound will be carried through to 
the later stages of the assay, making control of exposure 
impossible. If such cells are used (e.g., human peripheral 
lymphocytes or mouse lymphoma cells), it is reasonable to use the 
lowest precipitating concentration as the highest concentration 
tested.
    (9) As the induction of micronuclei and chromosomal aberrations 
are related, it is reasonable to assume that the same conditions can 
be applied for using male animals in bone marrow chromosomal 
aberration assays. The peripheral blood micronucleus test has been 
validated only in male rodents (The Collaborative Study Group for 
the Micronucleus Test, 1992), as has the ex vivo UDS test (Madle, et 
al., 1994).
    (10) A detailed collaborative study was carried out under the 
auspices of the Japanese Environmental Mutagen Society (The 
Collaborative Study Group for the Micronucleus Test, 1986). This 
study indicated that, in general, male mice were more sensitive than 
female mice for micronucleus induction; where differences were seen, 
they were only quantitative, not qualitative. This analysis has been 
extended by the group considering the micronucleus test at the 
Melbourne Harmonisation workshop and, having analyzed data on 53 in 
vivo clastogens (and 48 nonclastogens), the same conclusions were 
drawn (Hayashi, et al., 1994).
    (11) Both the rat and mouse are suitable species for use in the 
micronucleus test. However, data are accumulating to show that some 
species-specific carcinogens are species-specific genotoxins (e.g., 
Albanese, et al., 1988). When more data have accumulated, there may 
be a case for carrying out micronucleus tests in both the rat and 
the mouse.

References

    Albanese, R., Mirkova, E., Gatehouse, D., and Ashby, J. (1988). 
Species-specific response to the rodent carcinogens 1,2-
dimethylhydrazine and 1,2-dibromochloropropane in rodent bone marrow 
micronucleus assays. Mutagenesis, 3, 35-38.
    Ashby, J. (1983). The unique role of rodents in the detection of 
possible human carcinogens and mutagens. Mutat. Res., 115, 177-213.
    EEC (1987). Notes for Guidance for the Testing of Medicinal 
Products for their Mutagenic Potential, Official Journal Eur. Comm. 
L73
    Gatehouse, D., Haworth, S., Cebula, T., Goch, E., Kier, L., 
Matsushima, T., Melcion, C., Nohmi, T., Ohta, Venitt, S., and 
Zeiger, E. (1994). Report from the working group on bacterial 
mutation assays: International workshop on standardisation of 
genotoxicity test procedures. Mutat. Res. (in press).
    Haseman, J. K., and Clark, A. M. (1990). Carcinogenicity results 
for 114 laboratory animal studies used to assess the predictivity of 
four in vitro genetic toxicity assays for rodent carcinogenicity. 
Environ. Mol. Mutagen, 16, Suppl. 18, 15-31.
    Hayashi, M., Morita, T., Kodarna, Y., Sofuni, T., and Ishidate, 
M., Jr. (1990). The micronucleus assay with mouse peripheral blood 
reticulocytes using acridine orange-coated slides, Mutat. Res., 245, 
245-249.
    Hayashi, M., Tice, R. R., MacGregor, J. T., Anderson, D., 
Blakey, D. H., Kirsch-Volders, M., Oleson, F. B., Jr., Pacchierotti, 
F., Romagna, F., Shimada, H., Sutou, S., and Vannier, B. (1994). in 
vivo rodent erythrocyte micronucleus assay. Mutat. Res. (in press).
    Hayashi, M., Sofuni, T., and Ishidate, M., Jr. (1984).Kinetics 
of micronucleus formation in relation to chromosomal aberration in 
mouse bone marrow. Mutat. Res., 127, 129-137.
    Japanese Ministry of Health and Welfare (1989). Guidelines for 
toxicity studies of drugs.
    Kirkland, D. (1992). Chromosomal aberrations test in vitro: 
Problems with protocol design and interpretation of results. 
Mutagenesis, 7, 95-106.
    Kirkland, D. (1994). Report of the in-vitro sub-group. 
International workshop on standardization of Genotoxicity Test 
Procedures. Mutat. Res. (in press).
    Levin, D. E., Hollstein, M., Christman, M. F., Schwiers, E. A., 
and Ames, B. N. (1982). A new Salmonella tester strain (TA102) with 
A-T base pairs at the site of mutation detects oxidative mutagens. 
Proc. Nat. Acad. Sci. USA, 79, 7445-7449.
    Madle, S., Dean, S. W., Andrae, U., Brambilla, G., Burlinson, 
B., Doolittle, D. J., Furihata, C., Hertner, T., McQueen, C.A., and 
Mori, H. (1994). Recommendations for the performance of UDS tests in 
vitro and in vivo. Mutat. Res. (in press).
    Purves, D., Harvey, C., Tweats, D. J., and Lumley, C. (1994). 
Genotoxicity Testing: Current practices and strategies used by the 
pharmaceutical industry (submitted for publication).
    Shimada, H. (1993). Mutagenicity studies of Japanese regulatory 
guideline: the status quo and the point at issue. Environ. Mut. Res. 
Com., 15, 109-121.
    The Collaborative Study Group for the Micronucleus Test (1992). 
Micronucleus test with mouse peripheral blood erythrocytes by 
acridine orange supravital staining: The summary report of the 5th 
collaborative study by CSGMT/JEMS: MMS. Mutat. Res., 278, 83-98.
    Tweats, D. J. (1994). Follow-up of in vitro positive results. 
Proceedings of ICH 2 (in press).
    Wilcox, P., Naidoo, A., Wedd, D. J., and Gatehouse, D. G. 
(1990). Comparison of Salmonella typhimurium TA102 with Escherichia 
coli WP2 tester strains. Mutagenesis, 5, 285-291.

    Dated: September 15, 1994.
William K. Hubbard,
Interim Deputy Commissioner for Policy.
[FR Doc. 94-23377 Filed 9-21-94; 8:45 am]
BILLING CODE 4160-01-F