Guideline for Industry

Specific Aspects of Regulatory

Genotoxicity Tests for

Pharmaceuticals

 

April 1996

ICH S2A
i

Table of Contents

I. INTRODUCTION (1) .................................................. 1

II. SPECIFIC GUIDANCE AND RECOMMENDATIONS (2) ..................... 1

A. Specific guidance for in vitro tests (2.1) ............................... 1

1. The base set of strains used in bacterial mutation assays (2.1.1) ........ 1

2. Definition of the top concentration for in vitro tests (2.1.2) ........... 2

a. High concentration for nontoxic compounds (2.1.2.1) ......... 2

b. Desired level of cytotoxicity (2.1.2.2) ..................... 2

c. Testing of poorly soluble compounds (2.1.2.3) .............. 3

B. Specific guidance for in vivo tests (2.2) ............................... 4

1. Acceptable bone marrow tests for the detection of clastogens in vivo

(2.2.1) ................................................... 4

2. Use of male/female rodents in bone marrow micronucleus tests (2.2.2)

........................................................ 5

C. Guidance on the evaluation of test results (2.3) .......................... 5

1. Guidance on the evaluation of in vitro test results (2.3.1) ............ 5

a. In vitro positive results (2.3.1.1) ......................... 5

b. In vitro negative results (2.3.1.2) ......................... 6

2. Guidance on the evaluation of in vivo test results (2.3.2) ............. 6

a. Principles for demonstration of target tissue exposure for negative

in vivo test results (2.3.2.1) ............................. 7

b. Detection of germ cell mutagens (2.3.2.2) .................. 8
ii

GLOSSARY (4) .......................................................... A-1

REFERENCES (5) ........................................................ B-1

NOTES (3) .............................................................. C-1
This guideline was developed within the Expert Working Group (Safety) of the International Conference on †

Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) and has been

subject to consultation by the regulatory parties, in accordance with the ICH process. This document has been endorsed

by the ICH Steering Committee at Step 4 of the ICH process, July, 1995. At Step 4 of the process, the final draft is

recommended for adoption to the regulatory bodies of the European Union, Japan and the USA. This guideline was

published in the Federal Register on April 24, 1996 (61 FR 18199) and is applicable to drug and biological products. 

Although this guideline does not create or confer any rights for or on any person and does not operate to bind FDA or the

industry, it does represent the agency’s current thinking on recommended methods for testing and assessing the

genotoxic potential of pharmaceuticals. For additional copies of this guideline, contact the Drug Information Branch,

HFD-210, CDER, FDA, 5600 Fishers Lane, Rockville, MD 20857 (Phone: 301-827-4573) or the Manufacturers

Assistance and Communication Staff (HFM-42), CBER, FDA, 1401 Rockville Pike, Rockville, MD 20852-1448. Send

one self-addressed adhesive label to assist the offices in processing your request. An electronic version of this guidance

is also available via Internet using the World Wide Web (WWW) (connect to the CDER Home Page at

http://www.fda.gov/cder and go to the “Regulatory Guidance” section).

GUIDELINE FOR INDUSTRY†

SPECIFIC ASPECTS OF REGULATORY GENOTOXICITY

TESTS FOR PHARMACEUTICALS

I. INTRODUCTION (1)

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 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.

II. SPECIFIC GUIDANCE AND RECOMMENDATIONS (2)

A. Specific guidance for in vitro tests (2.1)

1. The base set of strains used in bacterial mutation assays (2.1.1)

Current guidelines for the detection of bacterial mutagens employ several strains to

detect base substitution and frameshift point mutations. The Salmonella
2

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.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 ; 5. TA102 or 2

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. Definition of the top concentration for in vitro tests (2.1.2)

a. High concentration for nontoxic compounds (2.1.2.1)

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.

b. Desired level of cytotoxicity (2.1.2.2)

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
3

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.

c. Testing of poorly soluble compounds (2.1.2.3)

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. It is possible that solubilization of a precipitate is 3

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)
4

centrifugation step(s) is (are) included in a protocol to remove cells from

exposure media), or render the test compound unavailable to enter cells 4

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.

B. Specific guidance for in vivo tests (2.2)

1. Acceptable bone marrow tests for the detection of clastogens in vivo

(2.2.1)

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.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.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.7
5

2. Use of male/female rodents in bone marrow micronucleus tests (2.2.2)

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. Quantitative differences in micronucleus 8

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. Both 9

rats and mice are deemed acceptable for use in the bone marrow micronucleus

test.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.

C. Guidance on the evaluation of test results (2.3)

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.

1. Guidance on the evaluation of in vitro test results (2.3.1)

a. In vitro positive results (2.3.1.1)

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
6

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?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?4

vi. For mammalian cells, is the effect only seen at extremely

low survival levels (see section II.A.2.b (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?

b. In vitro negative results (2.3.1.2)

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. Guidance on the evaluation of in vivo test results (2.3.2)

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
7

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 II.C.1.a (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.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 II.C.1.a (2.3.1.1), and

II.C.2.a (2.3.2.1)).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.

a. Principles for demonstration of target tissue exposure for negative

in vivo test results (2.3.2.1)

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.
8

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.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.

b. Detection of germ cell mutagens (2.3.2.2)

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.14
A-1

ATTACHMENT A

GLOSSARY (4)

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.
A-2

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.
B-1

ATTACHMENT B

REFERENCES (5)

Albanese, R., E. Mirkova, D. Gatehouse, and J. Ashby, “Species-specific response to the rodent

carcinogens, 1,2-dimethylhydrazine and 1,2-dibromochloropropane in rodent bone marrow

micronucleus assays,” Mutagenesis, 3:35-38, 1988.

Ashby, J., “The unique role of rodents in the detection of possible human carcinogens and

mutagens,” Mutation Research, 115:177-213, 1983.

EEC, “Notes for Guidance for the Testing of Medicinal Products for their Mutagenic Potential,”

Official Journal European Community, L73, 1987.

Gatehouse, D., S. Haworth, T. Cebula, E. Gocke, L. Kier, T. Matsushima, C. Melcion, T. Nohmi,

T. Ohta, S. Venitt, and E. Zeiger, “Report from the working group on bacterial mutation assays:

International workshop on standardisation of genotoxicity test procedures,” Mutation Research,

312:217-233, 1994.

Hayashi, M., T. Sofuni, and M. Ishidate, Jr., “Kinetics of micronucleus formation in relation to

chromosomal aberration in mouse bone marrow,” Mutation Research, 127:129-137, 1984.

Hayashi, M., T. Morita, Y. Kodama, T. Sofuni, and M. Ishidate, Jr., “The micronucleus assay

with mouse peripheral blood reticulocytes using acridine orange-coated slides,” Mutation

Research, 245:245-249, 1990.

Hayashi, M., R. R. Tice, J. T. MacGregor, D. Anderson, D. H. Blakey, M. Kirsch-Volders, F. B.

Oleson, Jr., F. Pacchierotti, F. Romagna, H. Shimada, S. Sutou, and B. Vannier, “In vivo rodent

erythrocyte micronucleus assay,” Mutation Research, 312(3):293-304, 1994.

Hayashi, M., “Acceptability of in vivo MN and CA tests,” in Proceedings of the Second

International Conference on Harmonisation (ICH 2), edited by P. F. D'Arcy and D. W. G. Harron,

Greystoke Books Ltd., N. Ireland, pp. 232-237, 1994.

Japanese Ministry of Health and Welfare, Guidelines for toxicity studies of drugs, 1989.

Kenelly, J. C., R. Waters, J. Ashby, P. A. Lefevre, B. Burlinson, D. J. Benford, S. W. Dean, and I.

deG. Mitchell, “In vivo rat liver UDS assay,” in Supplementary Mutagenicity Tests, UKEMS

Recommended Procedures, edited by D. J. Kirkland and M. Fox, Cambridge University Press, pp.

52-77, 1993.

Kirkland, D., “Report of the in vitro sub-group. International workshop on standardisation of

Genotoxicity Test Procedures,” Mutation Research, 312:211-215, 1994.
B-2

Levin, D. E., M. Hollstein, M. F. Christman, E. A. Schwiers, and B. N. Ames, “A new Salmonella

tester strain (TA102) with A-T base pairs at the site of mutation detects oxidative mutagens,”

Proceedings of the National Academy of Sciences of the USA, 79:7445-7449, 1982.

Madle, S., S. W. Dean, U. Andrae, G. Brambilla, B. Burlinson, D. J. Doolittle, C. Furihata, T.

Hertner, C. A. McQueen, and H. Mori, “Recommendations for the performance of UDS tests in

vitro and in vivo,” Mutation Research, 312:263-285, 1994.

OECD Guidelines for Genetic Toxicology (1983, 1984, 1986) Published by OECD, Paris; latest

revisions discussed at ad hoc expert committee meeting, Rome, September 1994.

Probst, G., “Validation of target tissue exposure for in vivo tests,” in Proceedings of the Second

International Conference on Harmonisation (ICH 2), edited by P. F. D'Arcy and D. W. G. Harron,

Greystoke Books Ltd., N. Ireland, pp. 249-252, 1994.

The Collaborative Study Group for the Micronucleus Test, CSGMT, “Sex difference in the

micronucleus test,” Mutation Research, 172:151-163, 1986.

The Collaborative Study Group for the Micronucleus Test, CSGMT, “Micronucleus test with

mouse peripheral blood erythrocytes by acridine orange supravital staining: The summary report

of the 5th collaborative study by CSGMT/JEMS: MMS,” Mutation Research, 278:83-98, 1992.

Tweats, D. J., “Follow-up of in vitro positive results,” in Proceedings of the Second International

Conference on Harmonisation (ICH 2), edited by P. F. D'Arcy and D. W. G. Harron, Greystoke

Books Ltd., N. Ireland, pp. 240-244, 1994.

Wilcox, P., A. Naidoo, D. J. Wedd, and D. G. Gatehouse, “Comparison of Salmonella

typhimurium TA102 with Escherichia coli WP2 tester strains,” Mutagenesis, 5:285-291, 1990.
C-1

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 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

ATTACHMENT C

NOTES (3)
C-2

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.
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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., <SUP>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.