[Code of Federal Regulations]
[Title 40, Volume 31]
[Revised as of July 1, 2006]
From the U.S. Government Printing Office via GPO Access
[CITE: 40CFR798.5200]
[Page 185-187]
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. 798.5200 Mouse visible specific locus test.
(a) Purpose. The mouse visible specific locus test (MSLT) may be
used to detect and quantitate mutations in the germ line of a mammalian
species.
(b) Definitions. (1) A visible specific locus mutation is a genetic
change that alters factors responsible for coat color and other visible
characteristics of certain mouse strains.
(2) The germ line is 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. (i) The principle of the MSLT is to
cross individuals who differ with respect to the genes present at
certain specific loci, so that a genetic alteration involving the
standard gene at any one of these loci will produce an offspring
detectably different from the standard heterozygote. The genetic change
may be detectable by various means, depending on the loci chosen to be
marked.
(ii) Three variations of the method currently exist for detecting
newly arising point mutations in mouse germ cells:
[[Page 186]]
(A) The visible specific locus test using either 5 or 7 loci.
(B) The biochemical specific locus test using up to 20 enzymes.
(C) The test for mutations at histocompatibility loci.
(iii) Of the three tests, the visible specific locus test has been
most widely used in assessing genetic hazard due to environmental
agents. It is the method described in this guideline.
(2) Description. For technical reasons, males rather than females
are generally treated with the test agent. Treated males are then mated
to females which are genetically homozygous for certain specific visible
marker loci. Offspring are examined in the next generation for evidence
that a new mutation has arisen.
(3) Animal selection--(i) Species and strain. Mice shall be used as
the test species. Male mice shall be either
(C3Hx101)F1 or (101xC3H)F1
hybrids. Females shall be T stock virgins.
(ii) Age. Healthy sexually mature animals shall be used.
(iii) Number. A decision on the minimum number of treated animals
should take into account the spontaneous variation of the biological
characterization being evaluated. Other considerations should include:
(A) The use of either historical or concurrent controls.
(B) The power of the test.
(C) The minimal rate of induction required.
(D) The use of positive controls.
(E) The level of significance desired.
(iv) Assignment to groups. Animals shall be randomized and assigned
to treatment and control groups.
(4) Control groups--(i) Concurrent controls. The use of positive or
spontaneous controls is left to the discretion of the investigator.
However, any laboratory which has had no prior experience with the test
shall, at its first attempt, produce a negative control sample of 20,000
and a positive control, using 100 mg/kg 1-ethyl-nitrosourea, in a sample
of 5,000 offspring.
(ii) Historical controls. Long term, accumulated spontaneous control
data of 43/801,406 are available for comparative purposes.
(5) Test chemicals--(i) Vehicle. When possible, test chemicals
should be dissolved or suspended in distilled water or isotonic saline
buffered appropriately, if needed, for stability. Water-insoluble
chemicals shall be dissolved or suspended in appropriate vehicles. The
vehicle used shall neither interfere with the test compound nor produce
major toxic effects. Fresh preparations of the test chemical should be
employed.
(ii) Dose levels. Usually, only one dose level need be tested. This
should be the highest dose tolerated without toxic effects, provided
that any temporary sterility induced due to elimination of spermatagonia
is 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 gavage, inhalation, admixture with food or water, and IP or IV
injections.
(e) Test performance--(1) Treatment and mating. Hybrid F1
(C3 Hx101 or 101xC3 H) male mice shall be treated
with the test substance and immediately mated to virgin T stock females.
Each treated male shall be mated to a fresh group of 2 to 4 virgin
females each week for 7 weeks, after which he shall be returned to the
first group of females and rotated through the seven sets of females
repeatedly. This mating schedule generally permits sampling of all
postspermatagonial stages of germ cell development during the first 7
weeks and rapid accumulation of data for exposed spermatagonial stem
cells thereafter. Repeated mating cycles should be conducted until the
entire spermatogonial cycle has been evaluated and enough offspring have
been obtained to meet the power criterion of the assay.
(2) Examination of offspring. (i) Offspring may be examined at (or
soon after) birth but must be examined at about 3 weeks of age at which
time the numbers of mutant and nonmutant offspring in each litter shall
be recorded.
(ii) Nonmutant progeny should be discarded. Mutant progeny shall be
subjected to genetic tests for verification.
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(f) Data and report--(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 and negative
control data, if they are available, shall be tabulated so that it is
possible to differentiate between concurrent (when available) and long-
term accumulated mutation frequencies.
(2) Statistical evaluation. Data shall be evaluated by appropriate
statistical methods.
(3) Interpretation of results. (i) There are several criteria for
determining a positive result, one of which is a statistically
significant dose-related increase in the number of specific locus
mutations. Another criterion may be based upon detection of a
reproducible and statistically significant positive response for at
least one of the test points.
(ii) A test substance which does not produce either a statistically
significant dose-related increase in the number of specific locus
mutations or a statistically significant and reproducible positive
response at any one of the test points is considered nonmutagenic in
this system.
(iii) Both biological and statistical significance should be
considered together in the evaluation.
(4) Test evaluation. (i) Positive results in the MSLT indicate that
under the test conditions the test substance induces heritable gene
mutations in the test species.
(ii) Negative results indicate that under the test conditions the
test substance does not induce heritable gene mutations in the test
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, number of animals of
each sex in experimental and control groups.
(ii) Test chemical vehicle, doses used and rationale for dose
selection, toxicity data.
(iii) Route and duration of exposure.
(iv) Mating schedule.
(v) Time of examination for mutant progeny.
(vi) Criteria for scoring mutants.
(vii) Use of concurrent or negative controls.
(viii) Dose response relationship, if applicable.
(g) References. For additional background information on this test
guideline the following references should be consulted:
(1) Russell, L.B., Shelby, P.B., von Halle, E., Sheridan, W.,
Valcovic, L. The mouse specific locus test with agents other than
radiations: interpretation of data and recommendations for future work:
A report of the U.S. EPA's Gene-Tox Program,'' Mutation Research,
86:329-354 (1981).
(2) [Reserved]
(h) Additional requirements. Testing facilities conducting the mouse
visible 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, and retain for at least 10 years, acceptable
35-mm color photographs (and their negatives) demonstrating the visible
mutations observed in mutant animals and the lack of such mutations in
their siblings and parents.
[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19078, May 20, 1987;
55 FR 12643, Apr. 5, 1990]