United States

Environmental Protection

Agency

Prevention, Pesticides

and Toxic Substances

(7101)

EPA712–C–96–216

June 1996

Health Effects Test

Guidelines

OPPTS 870.5195

Mouse Biochemical

Specific Locus Test

‘‘Public Draft’’
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INTRODUCTION

This guideline is one of a series of test guidelines that have been

developed by the Office of Prevention, Pesticides and Toxic Substances,

United States Environmental Protection Agency for use in the testing of

pesticides and toxic substances, and the development of test data that must

be submitted to the Agency for review under Federal regulations.

The Office of Prevention, Pesticides and Toxic Substances (OPPTS)

has developed this guideline through a process of harmonization that

blended the testing guidance and requirements that existed in the Office

of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,

Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the

Office of Pesticide Programs (OPP) which appeared in publications of the

National Technical Information Service (NTIS) and the guidelines published by the Organization for Economic Cooperation and Development

(OECD).

The purpose of harmonizing these guidelines into a single set of

OPPTS guidelines is to minimize variations among the testing procedures

that must be performed to meet the data requirements of the U. S. Environmental Protection Agency under the Toxic Substances Control Act (15

U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act

(7 U.S.C. 136, et seq.).

Public Draft Access Information: This draft guideline is part of a

series of related harmonized guidelines that need to be considered as a

unit. For copies: These guidelines are available electronically from the

EPA Public Access Gopher (gopher.epa.gov) under the heading ‘‘Environmental Test Methods and Guidelines’’ or in paper by contacting the OPP

Public Docket at (703) 305–5805 or by e-mail:

guidelines@epamail.epa.gov.

To Submit Comments: Interested persons are invited to submit comments. By mail: Public Docket and Freedom of Information Section, Office

of Pesticide Programs, Field Operations Division (7506C), Environmental

Protection Agency, 401 M St. SW., Washington, DC 20460. In person:

bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Arlington, VA. Comments may also be submitted electronically by sending

electronic mail (e-mail) to: guidelines@epamail.epa.gov.

Final Guideline Release: This guideline is available from the U.S.

Government Printing Office, Washington, DC 20402 on The Federal Bulletin Board. By modem dial 202–512–1387, telnet and ftp:

fedbbs.access.gpo.gov (IP 162.140.64.19), or call 202–512–0132 for disks

or paper copies. This guideline is also available electronically in ASCII

and PDF (portable document format) from the EPA Public Access Gopher

(gopher.epa.gov) under the heading ‘‘Environmental Test Methods and

Guidelines.’’
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OPPTS 870.5195 Mouse biochemical specific locus test.

(a) Scope—(1) Applicability. This guideline is intended to meet testing requirements of both the Federal Insecticide, Fungicide, and

Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.) and the Toxic Substances

Control Act (TSCA) (15 U.S.C. 2601).

(2) Background. The source materials used in developing this harmonized OPPTS test guideline are OPPT 40 CFR 798.5195 Mouse Biochemical Specific Locus Test and OPP 84–2 Mutagenicity Testing (Pesticide Assessment Guidelines, Subdivision F—Hazard Evaluation; Human

and Domestic Animals) EPA report 540/09–82–025, 1982.

(b) Purpose. The mouse biochemical specific locus test (MBSL) may

be used to detect and quantitate mutations originating in the germ line

of a mammalian species.

(c) Definitions. The definitions in section 3 of TSCA and in 40 CFR

Part 792—Good Laboratory Practice Standards (GLP) apply to this test

guideline. The following definitions also apply to this test guideline.

A biochemical specific locus mutation is a genetic change resulting

from a DNA lesion causing alterations in proteins that can be detected

by electrophoretic methods.

The germ line comprises the cells in the gonads of higher eukaryotes,

which are the carriers of the genetic information for the species.

(d) Test method—(1) Principle. The principle of the MBSL is that

heritable damage to the genome can be detected by electrophoretic analysis

of proteins in the tissues of the progeny of mice treated with germ cell

mutagens.

(2) Description. For technical reasons, males rather than females are

generally treated with the test chemical. Treated males are then mated to

untreated females to produce F1 progeny. Both blood and kidney samples

are taken from progeny for electrophoretic analysis. Up to 33 loci can

be examined by starch-gel electrophoresis and broad-range isoelectric focussing. Mutants are identified by variations from the normal

electrophoretic pattern. Presumed mutants are bred to confirm the genetic

nature of the change.

(3) Animal selection—(i) Species and strain. Mice should be used

as the test species. Although the biochemical specific locus test could be

performed in a number of inbred strains, in the most frequently used cross,

(C57BL/6 × DBA/2) F1 progeny are produced by mating C57BL/6 females

to DBA/2 males.

(ii) Age. Healthy, sexually mature (at least 8 weeks old) animals

should be used for treatment and breeding.
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(iii) Number. A decision on the minimum number of treated animals

should take into account possible effects of the test chemical on the fertility of the treated animals. Other considerations should include:

(A) The production of concurrent spontaneous controls.

(B) The use of positive controls.

(C) The power of the test.

(4) Control groups—(i) Concurrent controls. An appropriate number of concurrent control loci should be analyzed in each experiment.

These should be partly derived from matings of untreated animals (from

5 to 20 percent of the treated matings), although some data on control

loci can be taken from the study of the alleles transmitted from the untreated parent in the experimental cross. However, any laboratory which

has had no prior experience with the test should produce a spontaneous

control sample of about 5,000 progeny animals and a positive control sample (using 100 mg/kg ethylnitrosourea) of at least 1,200 offspring.

(ii) Historical controls. Long-term, accumulated spontaneous control

data (currently, 1 mutation in 1.2 × 106 control loci screened) are available

for comparative purposes.

(5) Test chemicals—(i) Vehicle. When possible, test chemicals

should be dissolved or suspended in distilled water or buffered isotonic

saline. Water-insoluble chemicals should be dissolved or suspended in appropriate vehicles. The vehicle used should neither interfere with the test

chemical nor produce major toxic effects. Fresh preparations of the test

chemical should be employed.

(ii) Dose levels. Usually, only one dose need be tested. This should

be the maximum tolerated dose (MTD), the highest dose tolerated without

toxic effects. Any temporary sterility induced due to elimination of

spermatogonia at this dose must be of only moderate duration, as determined by a return of males to fertility within 80 days after treatment. For

evaluation of dose-response, it is recommended that at least two dose levels be tested.

(iii) Route of administration. Acceptable routes of administration

include, but are not limited to, gavage,inhalation, and mixture with food

or water, and intraperitoneal or intravenous injections.

(e) Test performance—(1) Treatment and mating. Male DBA/2

mice should be treated with the test chemical and mated to virgin

C57BL/6 females immediately after cessation of treatment. Each treated

male should be mated to new virgin C57BL/6 females each week. Each

pairing will continue for a week until the next week’s mating is to begin.

This mating schedule permits sampling of all post-spermatogonial stages

of germ-cell development during the first 7 weeks after exposure.
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Spermatogonial stem cells are studied thereafter. Repeated mating cycles

should be conducted until sufficient offspring have been obtained to meet

the power criterion of the assay for spermatogonial stem cells.

(2) Examination of offspring—(i) Birth and weaning. Offspring

should be examined at birth and at weaning for externally detectable

changes in morphology and behavior; these could be due to dominant

mutations. Such characteristics may include, but are not limited to, variations in coat color, appearance of eyes, size (in which case weighing

of variant animals and littermates should be carried out), fur texture, etc.

Gross changes in external form and behavior should also be sought. Scrutiny of such visible characteristics of all animals should be made during

all subsequent manipulations of the animals.

(ii) Tissue sampling. Blood (about 0.1 mL) and one kidney should

be removed from progeny mice under anesthesia. Both tissues are then

prepared for analysis by electrophoresis.

(iii) Electrophoresis. The gene products of 6 loci should be analyzed

in the blood sample by broad-range isoelectric focusing and of 27 loci

in the kidney sample by starch-gel electrophoresis and enzyme-specific

staining. Details on these procedures are included in paragraphs (h)(2) and

(h)(3) of this guideline.

(iv) Mutant identification. Presumptive electrophoretic mutants

should be identified by variation from the normal electrophoretic banding

patterns. Reruns of all variant samples should be performed to confirm

the presence of altered banding patterns. Samples from parents of progeny

exhibiting banding pattern variations should be assayed to determine

whether the variant was induced by the experimental treatment or was

pre-existing. All treatment-induced variants are bred to determine the genetic nature of the change.

(f) Data and reports—(1) Treatment of results. Data should be presented in tabular form and should permit independent analysis of cell

stage-specific effects, and dose-dependent phenomena. The data should be

recorded and analyzed in such a way that clusters of identical mutations

are clearly identified. The individual mutants detected should be thoroughly described. In addition, concurrent positive control data (if employed) and spontaneous control data should also be tabulated. These concurrent controls should be added to, as well as compared with, the historical control data.

(2) Statistical evaluation. Data should be evaluated by appropriate

statistical methods.

(3) Interpretation of results. (i) There are several criteria for determining a positive response, one of which is a statistically significant doserelated increase in the frequency of electrophoretic mutations. Another cri-
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terion may be based upon detection of a reproducible and statistically significant positive response for at least one of these test points.

(ii) A test chemical which does not produce a statistically significant

increase in the frequency of electrophoretic mutations over the spontaneous

frequency, or a statistically significant and reproducible positive response

for at least one of the test points, is considered nonmutagenic in this system, provided that the sample size is sufficient to exclude a biologically

significant increase in mutation frequency.

(iii) Biological and statistical significance should be considered together in the evaluation.

(4) Test evaluation. (i) Positive results in the MBSL indicate that,

under the test conditions, the test chemical induces heritable gene

mutations in a mammalian species.

(ii) Negative results indicate that, under the test conditions, the test

chemical does not induce heritable genemutations in a mammalian species.

(5) Test report. In addition to the reporting requirements as specified

under 40 CFR part 792, subpart J, the following specific information

should be reported:

(i) Strain, age and weight of animals used; numbers of animals of

each sex in experimental and control groups.

(ii) Test chemical vehicle, doses used, rationale for dose selection,

and toxicity data, if available.

(iii) Route and duration of exposure.

(iv) Mating schedule.

(v) Number of loci screened for both treated and spontaneous data.

(vi) Criteria for scoring mutants.

(vii) Number of mutants found/locus.

(viii) Loci at which mutations were found.

(ix) Use of concurrent negative and positive controls.

(x) Dose-response relationship, if applicable.

(g) Additional Requirements. Testing facilities conducting the

mouse biochemical specific locus test in accordance with this section

should, in addition to adhering to the provisions of 40 CFR 792.190 and

792.195 obtain, adequately identify, and retain for at least 10 years, acceptable 35–mm photographs (and their negatives) of the stained isoelectricfocusing columns and the stained starch-gels obtained following analyses
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of blood and kidney preparations, respectively, from mutant mice, their

siblings, and their parents.

(h) References. The following references should be consulted for additional background material on this test guideline.

(1) Johnson, F.M. et al. The detection of mutants in mice by

electrophoresis: Results of a model induction experiment with

procarbazine. Genetics 97:113–124 (1981).

(2) Johnson, F.M. and S.E. Lewis. Mutation rate determinations based

on electrophoretic analysis of laboratory mice. Mutation Research 82:125–

135 (1981).

(3) Johnson, F.M. and S.E. Lewis. Electrophoretically detected germinal mutations induced by ethylnitrosourea in the mouse. Proceedings

of the National Academy of Sciences 78:3138–93141 (1981).

(4) Lewis, S.E. et al. Dominant visible and electrophoretically expressed mutations induced in male mice exposed to ethylene oxide by inhalation. Environmental Mutagenesis 8:867–872 (1986).