United States Prevention, Pesticides EPA712-C-96-216
Environmental Protection and Toxic Substances June 1996
Agency (7101)
&EPA 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 pub-
lished 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. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7U.S.C. I36,etseq.).
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 "Environ-
mental 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 com-
ments. 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, Ar-
lington, 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 Bul-
letin 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 test-
ing 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 har-
monized OPPTS test guideline are OPPT 40 CFR 798.5195 Mouse Bio-
chemical Specific Locus Test and OPP 84-2 Mutagenicity Testing (Pes-
ticide 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 fo-
cussing. 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 x DBA/2) Fl 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 fertil-
ity 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 num-
ber 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 un-
treated 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 sam-
ple (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 x 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 ap-
propriate 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 deter-
mined 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 lev-
els 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, vari-
ations 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. Scru-
tiny 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 ge-
netic nature of the change.
(f) Data and reports—(1) Treatment of results. Data should be pre-
sented 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 thor-
oughly described. In addition, concurrent positive control data (if em-
ployed) and spontaneous control data should also be tabulated. These con-
current controls should be added to, as well as compared with, the histori-
cal control data.
(2) Statistical evaluation. Data should be evaluated by appropriate
statistical methods.
(3) Interpretation of results, (i) There are several criteria for deter-
mining a positive response, one of which is a statistically significant dose-
related increase in the frequency of electrophoretic mutations. Another cri-
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terion may be based upon detection of a reproducible and statistically sig-
nificant 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 sys-
tem, provided that the sample size is sufficient to exclude a biologically
significant increase in mutation frequency.
(iii) Biological and statistical significance should be considered to-
gether 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, accept-
able 35-mm photographs (and their negatives) of the stained isoelectric-
focusing 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 ad-
ditional background material on this test guideline.
(1) Johnson, P.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, P.M. and S.E. Lewis. Mutation rate determinations based
on electrophoretic analysis of laboratory mice. Mutation Research 82:125-
135(1981).
(3) Johnson, P.M. and S.E. Lewis. Electrophoretically detected ger-
minal 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 ex-
pressed mutations induced in male mice exposed to ethylene oxide by in-
halation. EnvironmentalMutagenesis 8:867-872 (1986).
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