[Federal Register Volume 61, Number 80 (Wednesday, April 24, 1996)]
[Notices]
[Pages 18198-18202]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 96-10021]
[[Page 18197]]
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Part II
Department of Health and Human Services
_______________________________________________________________________
Food and Drug Administration
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International Conference on Harmonisation; Guidance on Specific Aspects
of Regulatory Genotoxicity Tests for Pharmaceuticals; Availability;
Notice
Federal Register / Vol. 61, No. 80 / Wednesday, April 24, 1996 /
Notices
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DEPARTMENT OF HEALTH AND HUMAN SERVICES
Food and Drug Administration
[Docket No. 94D-0324]
International Conference on Harmonisation; Guidance on Specific
Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals;
Availability
AGENCY: Food and Drug Administration, HHS.
ACTION: Notice.
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SUMMARY: The Food and Drug Administration (FDA) is publishing a
guideline entitled ``Guidance on Specific Aspects of Regulatory
Genotoxicity Tests for Pharmaceuticals.'' This guideline was prepared
under the auspices of the International Conference on Harmonisation of
Technical Requirements for Registration of Pharmaceuticals for Human
Use (ICH). The guideline is intended to provide guidance on
genotoxicity testing for pharmaceuticals.
DATES: Effective April 24, 1996. Submit written comments at any time.
ADDRESSES: Submit written comments on the guidance to the Dockets
Management Branch (HFA-305), Food and Drug Administration, 12420
Parklawn Dr., rm. 1-23, Rockville, MD 20857. Copies of the guideline
entitled ``Guidance on Specific Aspects of Regulatory Genotoxicity
Tests for Pharmaceuticals'' are available from the Consumer Affairs
Branch (HFD-8) (previously the CDER Executive Secretariat Staff),
Center for Drug Evaluation and Research, Food and Drug Administration,
7500 Standish Pl., Rockville, MD 20855.
FOR FURTHER INFORMATION CONTACT:
Regarding the guideline: Robert E. Osterberg, Center for Drug
Evaluation and Research (HFD-520), Food and Drug Administration, 5600
Fishers Lane, Rockville, MD 20857, 301-443-4300.
Regarding 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 the Federal Register of September 22, 1994 (59 FR 48734), FDA
published a draft tripartite guideline entitled ``Notes for Guidance on
Specific Aspects of Regulatory Genotoxicity Tests.'' The notice gave
interested persons an opportunity to submit comments by December 6,
1994.
After consideration of the comments received and revisions to the
guideline, a final draft of the guideline was submitted to the ICH
Steering Committee and endorsed by the three participating regulatory
agencies at the ICH meeting held in July 1995.
The guideline recommends methods for testing and assessing the
genotoxic potential of pharmaceuticals. A companion document (S2B:
``Genotoxicity: Standard Battery Tests'') providing guidance on a
``core test battery'' is under development. These recommendations are
based on a retrospective review of relevant databases from the
international pharmaceutical industry and regulatory agencies in the
European Community, Japan, and the United States. Because these tests
have not been designed nor validated for biological products (e.g.,
large molecules), they should not be routinely applied to such
products. When there is cause for concern, specific endpoints should be
identified and relevant tests should be developed and applied.
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). Although this
guideline does not create or confer any rights for or on any person,
and does not operate to bind FDA, it does represent the agency's
current thinking on recommended methods for testing and assessing the
genotoxic potential of pharmaceuticals.
As with all of FDA's guidelines, the public is encouraged to submit
written comments with new data or other new information pertinent to
this guideline. The comments in the docket will be periodically
reviewed, and, where appropriate, the guideline will be amended. The
public will be notified of any such amendments through a notice in the
Federal Register.
Interested persons may, at any time, submit written comments on the
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 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 guideline follows:
Guidance on Specific Aspects of Regulatory Genotoxicity Tests for
Pharmaceuticals
1. Introduction
Guidelines for the testing of pharmaceuticals for genetic
toxicity have been established in the European Community (EEC, 1987)
and Japan (Japanese Ministry of Health and Welfare, 1989). FDA's
Centers for Drug Evaluation and Research 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 (58 FR 16536, March 29, 1993) to be applicable
to pharmaceuticals.
The following notes for guidance should be applied in
conjunction with existing guidelines in the United States, the
European Community, and Japan. The recommendations below are derived
from considerations of historical information held within the
international pharmaceutical industry, the three regulatory bodies,
and the scientific literature. Where relevant, the
[[Page 18199]]
recommendations from the latest review of the Organization for
Economic Cooperation and Development (OECD) guidelines (OECD, 1994)
and the 1993 International Workshop on Standardisation of
Genotoxicity Test Procedures (Mutation Research, 312(3), 1994) have
been considered.
2. Specific guidance and recommendations
2.1 Specific guidance for in vitro tests
2.1.1 The base set of strains used in bacterial mutation assays
Current guidelines for the detection of bacterial mutagens
employ several strains to detect base substitution and frameshift
point mutations. The Salmonella typhimurium strains mentioned in
guidelines (normally TA1535, TA1537, TA98, and TA100) will detect
such changes at G-C (guanine-cytosine) sites within target histidine
genes. It is clear from the literature that some mutagenic
carcinogens also modify A-T (adenine-thymine) base pairs. 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 detects such mutations within
multiple copies of hisG genes, or Escherichia coli WP2 uvrA, which
detects these mutations in the trpE gene, or the same strain
possessing the plasmid (pKM101), which carries mucAB genes that
enhance error prone repair (see note 1). 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).
In order to detect cross-linking agents it may be preferable to
select S. typhimurium TA 102 or to add a repair proficient E. coli
strain, such as WP2 pKM101. It is noted that such compounds are
detected in assays that measure chromosome damage.
2.1.2 Definition of the top concentration for in vitro tests
2.1.2.1 High concentration for nontoxic compounds
For freely soluble, nontoxic compounds, the desired upper
treatment levels are 5 milligrams (mg)/plate for bacteria and 5 mg/
milliliter (mL) or 10 millimolar (mM) (whichever is the lower) for
mammalian cells.
2.1.2.2 Desired level of cytotoxicity
Some genotoxic carcinogens are not detectable in in vitro
genotoxicity assays unless the concentrations tested induce some
degree of cytotoxicity. It is also apparent that excessive toxicity
often does not allow a proper evaluation of the relevant genetic
endpoint. Indeed, at very low survival levels in mammalian cells,
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.). 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 considered acceptable for in
vitro bacterial and mammalian cell tests (concentrations should not
exceed the levels specified in 2.1.2.1):
(i) In the bacterial reverse mutation test, the highest
concentration of test compound is desired to show evidence of
significant toxicity. Toxicity may be detected by a reduction in the
number of revertants, a clearing or diminution of the background
lawn.
(ii) The desired level of toxicity for in vitro cytogenetic
tests using cell lines should be greater than 50 percent reduction
in cell number or culture confluency. For lymphocyte cultures, an
inhibition of mitotic index by greater than 50 percent is considered
sufficient.
(iii) In mammalian cell mutation tests, ideally the highest
concentration should produce at least 80 percent toxicity (no more
than 20 percent survival). Toxicity can be measured either by
assessment of cloning efficiency (e.g., immediately after
treatment), or by calculation of relative total growth, i.e., the
product of relative suspension growth during the expression period
and relative plating efficiency at the time of mutant selection.
Caution is due with positive results obtained at levels of survival
lower than 10 percent.
2.1.2.3 Testing of poorly soluble compounds
There is some evidence that dose-related genotoxic activity can
be detected when testing certain compounds in the insoluble range in
both bacterial and mammalian cell genotoxicity tests. This is
generally associated with dose-related toxicity (see note 3). 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 have endocytic activity (e.g., Chinese hamster V79,
CHO and CHL cells) and can ingest solid particles that may
subsequently disperse into the cytoplasm. An insoluble compound may
also contain soluble genotoxic impurities. It should also be noted
that a number of insoluble pharmaceuticals are administered to
humans as suspensions or as particulate materials.
On the other hand, heavy precipitates can interfere with scoring
the desired parameter and render control of exposure very difficult
(e.g., where (a) centrifugation step(s) is (are) included in a
protocol to remove cells from exposure media) (see note 4), 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 below refers to the test
article in the culture medium.
If no cytotoxicity is observed, then the lowest precipitating
concentration should be used as the top concentration but not
exceeding 5 mg per plate for bacterial tests and 5 mg/mL or 10 mM
for mammalian cell tests. If dose-related cytotoxicity or
mutagenicity is noted, irrespective of solubility, then the top
concentration should be based on toxicity as described above. This
may require the testing of more than one precipitating concentration
(not to exceed the above stated levels). 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 at the beginning and at the
end of the treatment period using the naked eye.
2.2 Specific guidance for in vivo tests
2.2.1 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 or chromosomes 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 5). Micronuclei can also result from lagging of
one or more whole chromosome(s) at anaphase and thus micronucleus
tests have the potential to detect some aneuploidy inducers (see
note 6).
In conclusion, either the analysis of chromosomal aberrations in
bone marrow cells or the measurement of micronucleated polychromatic
erythrocytes in bone marrow cells in vivo is acceptable for the
detection of clastogens. The measurement of micronucleated immature
(e.g., polychromatic) erythrocytes in peripheral blood is an
acceptable alternative in the mouse, or in any other species in
which the inability of the spleen to remove micronucleated
erythrocytes has been demonstrated, or which has shown an adequate
sensitivity to detect clastogens/aneuploidy inducers in peripheral
blood (see note 7).
2.2.2 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 8). Quantitative differences in 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. Similar principles can be applied for other established in
vivo tests (see note 9). Both rats and mice are deemed acceptable
for use in the bone marrow micronucleus test (see note 10).
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, animals of the corresponding sex
should normally be used.
[[Page 18200]]
2.3 Guidance on the evaluation of test 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. Genotoxicity test
batteries (of in vitro and in vivo tests) detect carcinogens that
are thought to act primarily via a mechanism involving direct
genetic damage, such as the majority of known human carcinogens.
Therefore, these batteries may not detect nongenotoxic carcinogens.
Experimental conditions, such as the limited capability of the in
vitro metabolic activation systems, can also lead to false negative
results in in vitro tests. The test battery approach is designed to
reduce the risk of false negative results for compounds with
genotoxic potential, while a positive result in any assay for
genotoxicity does not necessarily mean that the test compound poses
a genotoxic/carcinogenic hazard to humans.
2.3.1 Guidance on the evaluation of in vitro test results
2.3.1.1 In vitro positive results
The scientific literature gives a number of conditions that may
lead to a positive in vitro result of questionable relevance.
Therefore, any in vitro positive test result should be evaluated for
its biological relevance taking into account the following
considerations (this list is not exhaustive, but is given as an aid
to decision-making):
(i) Is the increase in response over the negative or solvent
control background regarded as a meaningful genotoxic effect for the
cells?
(ii) Is the response concentration-related?
(iii) For weak/equivocal responses, is the effect reproducible?
(iv) Is the positive result a consequence of an in vitro
specific metabolic activation pathway/in vitro specific active
metabolite (see also note 12)?
(v) Can the effect be attributed to extreme culture conditions
that do not occur in in vivo situations, e.g., extremes of pH;
osmolality; heavy precipitates, especially in cell suspensions (see
note 4)?
(vi) For mammalian cells, is the effect only seen at extremely
low survival levels (see section 2.1.2.2 for acceptable levels of
toxicity)?
(vii) Is the positive result attributable to a contaminant (this
may be the case if the compound shows no structural alerts or is
weakly mutagenic or mutagenic only at very high concentrations)?
(viii) Do the results obtained for a given genotoxic endpoint
conform to that for other compounds of the same chemical class?
2.3.1.2 In vitro negative results
For in vitro negative results, special attention should be paid
to the following considerations (the examples given are not
exhaustive, but are given as an aid to decision-making): Does the
structure or known metabolism of the compound indicate that standard
techniques for in vitro metabolic activation (e.g., rodent liver S9)
may be inadequate? Does the structure or known reactivity of the
compound indicate that the use of other test methods/systems may be
appropriate?
2.3.2 Guidance on the evaluation of in vivo test results
In vivo tests, by their nature, have the advantage of taking
into account absorption, distribution, and excretion, which are not
factors in in vitro tests, but are relevant to human use. In
addition, metabolism is likely to be more relevant in vivo compared
to the systems normally used in vitro. There are a few validated in
vivo models accepted for assessment of genotoxicity. These include
the bone marrow or peripheral blood cytogenetic assays. If a
compound has been tested in vitro with negative results, it is
usually sufficient to carry out a single in vivo cytogenetics assay.
For a compound that induces a biologically relevant positive
result in one or more in vitro tests (see section 2.3.1.1), a
further in vivo test in addition to the in vivo cytogenetic assay,
using a tissue other than the bone marrow/peripheral blood, can
provide further useful information. The target cells exposed in vivo
and possibly the genetic end point measured in vitro guide the
choice of this additional in vivo test. However, there is no
validated, widely used in vivo system that measures gene mutation.
In vivo gene mutation assays using endogenous genes or transgenes in
several tissues of the rat and mouse are at various stages of
development. Until such tests for mutation become accepted, results
from other in vivo tests for genotoxicity in tissues other than the
bone marrow can provide valuable additional data but the assay of
choice should be scientifically justified (see note 11).
If in vivo and in vitro test results do not agree, then the
differences should be considered/explained on a case-by-case basis
(see sections 2.3.1.1, 2.3.2.1, and note 12).
In conclusion, the assessment of the genotoxic potential of a
compound should take into account the totality of the findings and
acknowledge the intrinsic values and limitations of both in vitro
and in vivo tests.
2.3.2.1 Principles for demonstration of target tissue exposure for
negative in vivo test results
In vivo tests have an important role in genotoxicity test
strategies. The significance of in vivo results in genotoxicity test
strategies is directly related to the demonstration of adequate
exposure of the target tissue to the test compound. This is
especially true for negative in vivo test results and when in vitro
test(s) have shown convincing evidence of genotoxicity. Although a
dose sufficient to elicit a biological response (e.g., toxicity) in
the tissue in question is preferable, such a dose could prove to be
unattainable since dose-limiting toxicity can occur in a tissue
other than the target tissue of interest. In such cases,
toxicokinetic data can be used to provide evidence of
bioavailability. If adequate exposure cannot be achieved, e.g., with
compounds showing very poor target tissue availability, extensive
protein binding, etc., conventional in vivo genotoxicity tests may
have little value.
The following recommendations apply to bone marrow cytogenetic
assays; as examples, if other target tissues are used, similar
principles should be applied.
For compounds showing positive results in any of the in vitro
tests employed, demonstration of in vivo exposure should be made by
any of the following measurements:
(i) By obtaining 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 13).
(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 or other relevant doses using the
same species/strain and dosing route used in the bone marrow assay.
If in vitro tests do not show genotoxic potential, in vivo
(systemic) exposure should be demonstrated 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
studies in rodents.
2.3.2.2 Detection of germ cell mutagens
With respect to the detection of germ cell mutagens, results of
comparative studies have shown that, in a qualitative sense, most
germ cell mutagens are likely to be detected as such in somatic cell
tests and negative results of in vivo somatic cell genotoxicity
tests generally indicate the absence of germ cell effects (see note
14).
3. Notes
(1) Relevant examples of genotoxic carcinogens that are detected
if bacterial strains with A-T target mutations are included in the
base set can be found in the literature (e.g., Levin et al., 1983;
Wilcox et al., 1990). Analysis of the data base held by the Japanese
Ministry of Labour on 5,526 compounds (and supported by smaller data
bases 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 all 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 Standardisation of
Genotoxicity Procedures, Melbourne, 1993 (Gatehouse et al., 1994)
that all three strains could be used interchangeably.
(3) 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
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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 given in
the report of the in vitro subgroup of the International Workshop on
Standardisation of Genotoxicity Procedures, Melbourne, 1993
(Kirkland, 1994).
(4) 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 tested.
(5) As the mechanisms of micronucleus formation are related to
those inducing chromosomal aberrations (e.g., Hayashi et al., 1984
and 1994; Hayashi, 1994), 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 have shown
impressive correlation both qualitatively, i.e., detecting
clastogenicity, and quantitatively, i.e., determination of the
lowest clastogenic dose. Even closer correlations can be expected
where the data are generated in the same species.
(6) Although micronuclei can arise from lagging whole
chromosomes following interaction of a compound with the spindle
apparatus, the micronucleus test may not detect all aneuploidy
inducers. Specific aneuploidy assays may become available in the
near future. One approach is the evolving rapid and sensitive
technique for identifying individual (rodent) chromosomes in
interphase nuclei, e.g., via fluorescence in situ hybridization
(FISH).
(7) The peripheral blood micronucleus test in the mouse using
acridine orange supravital staining was originally introduced by
Hayashi et al. (1990). The test has been the subject of a major
collaborative study by the Japanese Collaborative Study Group for
the Micronucleus Test (Mutation Research, 278, 1992, Nos. 2/3). The
tests were carried out in CD-l 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 micronucleus
assay using acridine orange supravital staining can generate
reproducible and reliable data to evaluate the clastogenicity of
chemicals. Based on these data, the International Workshop on
Standardisation of Genotoxicity Procedures, Melbourne, 1993,
concluded that this assay is equivalent in accuracy to the bone
marrow micronucleus assay (Hayashi et al., 1994). The application of
the peripheral blood micronucleus assay to rats is under validation
by the Japanese Collaborative Study Group for the Micronucleus Test.
(8) A detailed collaborative study was carried out indicating
that, in general, male mice were more sensitive than female mice for
micronucleus induction; where differences were observed, they were
only quantitative and not qualitative (The Collaborative Study Group
for the Micronucleus Test, 1986). This analysis has been extended by
a group considering the micronucleus test at the International
Workshop on Standardisation of Genotoxicity Procedures, Melbourne,
1993. Having analyzed data on 53 in vivo clastogens (and 48
nonclastogens), the same conclusions were drawn (Hayashi et al.,
1994).
(9) As the induction of micronuclei and chromosomal aberrations
are related, it is reasonable to assume that the same conditions can
be applied when 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 unscheduled DNA
synthesis (UDS) test (Kennely et al., 1993; Madle et al., 1994).
(10) Both the rat and mouse are suitable species for use in the
micronucleus test with bone marrow. 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.
(11) Apart from the cytogenetic assays in bone marrow cells, a
large data base for in vivo assays exists for the liver UDS assay
(Madle et al., 1994). 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). False negative results with this combination
of assays have been generated for some unstable genotoxic compounds
and certain aromatic amines that are problematical for most existing
in vivo screens (Tweats, 1994). Therefore, further in vivo testing
should not be restricted to liver UDS tests as other assays may be
more appropriate (e.g., 32P postlabeling; DNA strand-breakage
assays, etc.), depending on the compound in question. It is
important to recognize that for these in vivo endpoints, their
relationship to mutation is not precisely known.
(12) Examples to consider regarding the differences between in
vitro and in vivo test results have been described in the literature
(e.g., Ashby, 1983). They include: (i) An active metabolite produced
in vitro may not be produced in vivo, (ii) an active metabolite may
be rapidly detoxified in vivo but not in vitro, and (iii) rapid and
efficient excretion of a compound may occur in vivo.
(13) 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
borne out by direct comparisons of drug levels in the two
compartments for a large series of different pharmaceuticals
(Probst, 1994). 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.
(14) There may be specific types of mutagens, e.g., aneuploidy
inducers, that act preferentially during meiotic gametogenesis
stages. There is no conclusive experimental evidence to date for the
existence of such substances.
4. Glossary
Aneuploidy: Numerical deviation of the modal number of
chromosomes in a cell or organism.
Base substitution: The substitution of one or more base(s) for
another in the nucleotide sequence. This may lead to an altered
protein.
Cell proliferation: The ability of cells to divide and to form
daughter cells.
Clastogen: An agent that produces structural changes of
chromosomes, usually detectable by light microscopy.
Cloning efficiency: The efficiency of single cells to form
clones. Usually measured after seeding low numbers of cells in a
suitable environment.
Culture confluency: A quantification of the cell density in a
culture (cell proliferation is usually inhibited at high degrees of
confluency).
Frameshift mutation: A mutation (change in the genetic code) in
which one base or two adjacent bases are added (inserted) or deleted
to the nucleotide sequence of a gene. This may lead to an altered or
truncated protein.
Gene mutation: A detectable permanent change within a single
gene or its regulating sequences. The changes may be point
mutations, insertions, or deletions.
Genetic endpoint: The precise type or type class of genetic
change investigated (e.g., gene mutations, chromosomal aberrations,
DNA-repair, DNA-adduct formation, etc.).
Genetic toxicity, genotoxicity: A broad term that refers to any
deleterious change in the genetic material regardless of the
mechanism by which the change is induced.
Micronucleus: Particle in a cell that contains microscopically
detectable nuclear DNA; it might contain a whole chromosome(s) or a
broken centric or acentric part(s) of chromosome(s). The size of a
micronucleus is usually defined as being less than 1/5 but more than
1/20 of the main nucleus.
Mitotic index: Percentage of cells in the different stages of
mitosis among the cells not in mitosis (interphase) in a preparation
(slide).
Plasmid: Genetic element additional to the normal bacterial
genome. A plasmid might be inserted into the host chromosome or form
an extrachromosomal element.
[[Page 18202]]
Point mutations: Changes in the genetic code, usually confined
to a single DNA base pair.
Polychromatic erythrocyte: An immature erythrocyte in an
intermediate stage of development that still contains ribosomes and,
as such, can be distinguished from mature normochromatic
erythrocytes (lacking ribosomes) by stains selective for ribosomes.
Survival (in the context of mutagenicity testing): Proportion of
cells in a living stage among dead cells, usually determined by
staining and colony counting methods after a certain treatment
interval.
Unscheduled DNA synthesis (UDS): DNA synthesis that occurs at
some stage in the cell cycle (other than S-phase) in response to DNA
damage. It is usually associated with DNA excision repair.
5. References
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Ashby, J., ``The unique role of rodents in the detection of
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EEC, ``Notes for Guidance for the Testing of Medicinal Products
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Hayashi, M., T. Morita, Y. Kodama, T. Sofuni, and M. Ishidate,
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reticulocytes using acridine orange-coated slides,'' Mutation
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Hayashi, M., R. R. Tice, J. T. MacGregor, D. Anderson, D. H.
Blakey, M. Kirsch-Volders, F. B. Oleson, Jr., F. Pacchierotti, F.
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Madle, S., S. W. Dean, U. Andrae, G. Brambilla, B. Burlinson, D.
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expert committee meeting, Rome, September 1994.
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tests,'' in Proceedings of the Second International Conference on
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The Collaborative Study Group for the Micronucleus Test, CSGMT,
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The Collaborative Study Group for the Micronucleus Test, CSGMT,
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acridine orange supravital staining: The summary report of the 5th
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WP2 tester strains,'' Mutagenesis, 5:285-291, 1990.
Dated: April 15, 1996.
William K. Hubbard,
Associate Commissioner for Policy Coordination.
[FR Doc. 96-10021 Filed 4-23-96; 8:45 am]
BILLING CODE 4160-01-F
