18198 Federal Register / Vol. 61, No. 80 / Wednesday, April 24, 1996 / Notices

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 § 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 § 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
Federal Register / Vol. 61, No. 80 / Wednesday, April 24, 1996 / Notices 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 (guaninecytosine) 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.
18200 Federal Register / Vol. 61, No. 80 / Wednesday, April 24, 1996 / Notices

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 doselimiting 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 drugrelated 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
Federal Register / Vol. 61, No. 80 / Wednesday, April 24, 1996 / Notices 18201

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 speciesspecific 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.
18202 Federal Register / Vol. 61, No. 80 / Wednesday, April 24, 1996 / Notices

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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dimethylhydrazine and 1,2-

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Ashby, J., ‘‘The unique role of rodents in

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EEC, ‘‘Notes for Guidance for the Testing

of Medicinal Products for their Mutagenic

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Gatehouse, D., S. Haworth, T. Cebula, E.

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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 orangecoated slides,’’ Mutation Research, 245:245–

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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. Romagna,

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Hayashi, M., ‘‘Acceptability of in vivo MN

and CA tests,’’ in Proceedings of the Second

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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,

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Kirkland, D., ‘‘Report of the in vitro subgroup. International workshop on

standardisation of Genotoxicity Test

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215, 1994.

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.

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Mori, ‘‘Recommendations for the

performance of UDS tests in vitro and in

vivo,’’ Mutation Research, 312:263–285,

1994.

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(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

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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

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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.

Dated: April 15, 1996.

William K. Hubbard,

Associate Commissioner for Policy

Coordination.

[FR Doc. 96–10021 Filed 4–23–96; 8:45 am]

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