[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]




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Part II





Department of Health and Human Services





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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.

-----------------------------------------------------------------------

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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assays,'' Mutagenesis, 3:35-38, 1988.
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for their Mutagenic Potential,'' Official Journal European 
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    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