[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]
_______________________________________________________________________
Part VII
Department of Health and Human Services
_______________________________________________________________________
Food and Drug Administration
_______________________________________________________________________
International Conference on Harmonisation, Draft Guideline on Specific
Aspects of Regulatory Genetoxicity Tests; Notice
=======================================================================
-----------------------------------------------------------------------
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.
-----------------------------------------------------------------------
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
