[Code of Federal Regulations]
[Title 40, Volume 31]
[Revised as of July 1, 2007]
From the U.S. Government Printing Office via GPO Access
[CITE: 40CFR798.5195]

[Page 183-185]
 
                   TITLE 40--PROTECTION OF ENVIRONMENT
 
         CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
 
PART 798_HEALTH EFFECTS TESTING GUIDELINES--Table of Contents
 
                       Subpart F_Genetic Toxicity
 
Sec.  798.5195  Mouse biochemical specific locus test.


    (a) 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.
    (b) Definitions. (1) 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.
    (2) The germ line is comprised of the cells in the gonads of higher 
eukaryotes, which are the carriers of the genetic information for the 
species.
    (c) Reference substances. Not applicable.
    (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 shall be used as 
the test species. Although the biochemical specific locus test could be 
performed in a number of in bred strains, in the most frequently used 
cross, C57BL/6 females are mated to DBA/2 males to produce (C57BL/6xDBA/
2) F1 progeny for screening.
    (ii) Age. Healthy, sexually-mature (at least 8 weeks old) animals 
shall be used for treatment and breeding.
    (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 shall be analyzed in each experiment. These 
should be partly derived from matings of untreated animals (from 5 to 20 
percent ofthe 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 shall produce a spontaneous control 
sample of about 5,000 progeny animals and a positive control (using 100 
mg/kg ethylnitrosourea) sample of at least 1,200 offspring.
    (ii) Historical controls. Long-term, accumulated spontaneous control 
data (currently, 1 mutation in 1,200,000 control loci screened) are 
available for comparative purposes.
    (5) Test chemicals--(i) Vehicle. When possible, test chemicals shall 
be dissolved or suspended in distilled water or buffered isotonic 
saline. Water-insoluble chemicals shall be dissolved or suspended in 
appropriate vehicles. The vehicle used shall 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 are turn of males to fertility within 80 days after

[[Page 184]]

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 
shall be treated with the test chemical and mated to virgin C57BL/6 
females immediately after cessation of treatment. Each treated male 
shall 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. 
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 shall 
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 shall also be sought. Scrutiny of 
such visible characteristics of all animals shall be made during all 
subsequent manipulations of the animals.
    (ii) Tissue sampling. Blood (about 0.1 mL) and one kidney shall be 
removed from progeny mice under anesthesia. Both tissues are then 
prepared for analysis by electrophoresis.
    (iii) Electrophoresis. The gene products of 6 loci shall be analyzed 
in the blood sample by broad-range isoelectric focussing and of 27 loci 
in the kidney sample by starch-gel electrophoresis and enzyme-specific 
staining. Details on these procedures are included in paragraphs (g)(1) 
through (g)(3) of this section.
    (iv) Mutant identification. Presumptive electrophoretic mutants 
shall be identified by variation from the normal electrophoretic banding 
patterns. Reruns of all variant samples shall be performed to confirm 
the presence of altered banding patterns. Samples from parents of 
progeny exhibiting banding pattern variations shall 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 shall be 
presented in tabular form and shall permit independent analysis of cell 
stage-specific effects, and dose-dependent phenomena. The data shall be 
recorded and analyzed in such a way that clusters of identical mutations 
are clearly identified. The individual mutants detected shall be 
thoroughly described. In addition, concurrent positive control data (if 
employed) and spontaneous control data shall also be tabulated. These 
concurrent controls shall be added to, as well as compared with, the 
historical control data.
    (2) Statistical evaluation. Data shall 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 dose-related increase in the frequency of electrophoretic 
mutations. Another criterion 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) Both biological and statistical significance should be 
considered together in the evaluation.

[[Page 185]]

    (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, and paragraph (h) of this 
section, the following specific information shall 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) References. For additional background information on this test 
guideline, the following references should be consulted:
    (1) Personal communication from Susan E. Lewis, Ph.D. to Dr. Michael 
Cimino, U.S. EPA, OPPT, October 5, 1989.
    (2) Johnson, F.M., G.T. Roberts, R.K. Sharma, F.Chasalow, R. 
Zweidinger, A. Morgan, R.W. Hendren, and S.E.Lewis. ``The detection of 
mutants in mice by electrophoresis: Results of a model induction 
experiment with procarbazine.'' Genetics 97:113-124 (1981).
    (3) Johnson, F.M. and S.E. Lewis. ``Mutation rate determinations 
based on electrophoretic analysis of laboratory mice.'' Mutation 
Research 82:125-135 (1981a).
    (4) 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 (1981b).
    (5) Lewis, S.E., C. Felton, L.B. Barnett, W. Generoso, N. Cacheiro, 
and M.D. Shelby. ``Dominant visible and electrophoretically expressed 
mutations induced in male mice exposed to ethylene oxide by 
inhalation.'' Environmental Mutagenesis 8:867-872 (1986).
    (h) Additional requirements. Testing facilities conducting the mouse 
biochemical specific locus test in accordance with this section shall, 
in addition to adhering to the provisions of Sec.  Sec.  792.190 and 
792.195 of this chapter, obtain, adequately identify, and retain for at 
least 10 years, acceptable 35-mm photographs (and their negatives) of 
the stained isoelectric-focussing columns and the stained starch-gels 
obtained following analyses of blood and kidney preparations, 
respectively, from mutant mice, their siblings, and their parents.

[55 FR 12641, Apr. 5, 1990]