United States       Prevention, Pesticides     EPA712-C-98-217
          Environmental Protection    and Toxic Substances     August 1998
          Agency        (7101)
&EPA   Health Effects Test
          OPPTS 870.5200
          Mouse Visible Specific
          Locus Test

     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

     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.).

     Final  Guideline Release: This guideline  is available from the U.S.
Government Printing Office,  Washington, DC 20402 on disks or paper
copies: call (202) 512-0132. This  guideline is also available electronically
in PDF (portable document format) from EPA's  World Wide Web  site
(http://www.epa.gov/epahome/research.htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelines/OPPTS  Harmonized Test

OPPTS 870.5200 Mouse visible 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 798.5200 Mouse visible
specific locus test  and OPP 84-2 Mutagenicity Testing (Pesticide Assess-
ment Guidelines, Subdivision F—Hazard Evaluation; Human and Domes-
tic Animals) EPA report 540/09-82-025, 1982.

     (b) Purpose.  The mouse visible specific locus  test  (MSLT) may be
used to detect and quantitate mutations in the germ  line  of a mammalian

     (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.

     Germ line is the cells in the gonads  of higher eukaryotes  which are
the carriers of the genetic information for the species.

     Visible specific locus mutation is a genetic change that alters factors
responsible for coat color and other visible characteristics  of certain mouse

     (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 var-
ious 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:

     (A) The visible  specific locus  test using either five  or seven 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 fe-
males 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 should be used
as  the  test  species.  Male  mice  should  be  either  (C3Hxl01)Fi  or
(101xC3H)Fi hybrids. Females should be T stock virgins.

    (ii) Age. Healthy sexually mature animals should be used.

    (iii) Number. A decision on the minimum number of treated animals
should take into account the spontaneous variation of the biological charac-
terization 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 should  be randomized  and  as-
signed 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. How-
ever,  any laboratory which has  had no prior  experience  with the test
should,  at its first attempt,  produce a negative control sample of 20,000
and a positive  control,  using 100 mg/kg 1-ethylnitrosourea, 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
should be dissolved or suspended in appropriate vehicles. The vehicle used
should 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 rec-
ommended 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

     (e)  Test  performance—(1) Treatment and mating.  Hybrid FI
(C3Hxl01) or (101xC3H) male mice should be treated with the test  sub-
stance and immediately mated to virgin T stock females. Each treated male
should be mated to a fresh group of two to four virgin females each week
for 7 weeks, after which he should 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  should
be recorded.

     (ii) Nonmutant progeny should be  discarded.  Mutant progeny  should
be subjected to  genetic tests for verification.

     (f) Data and  report—(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 re-
corded 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 and negative control
data, if they are available,  should be tabulated so that it is  possible to
differentiate between concurrent (when available)  and long-term accumu-
lated mutation frequencies.

     (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  result, one of  which is a statistically significant dose-
related increase in the number of specific locus mutations. Another cri-
terion may be based upon detection of a reproducible and statistically sig-
nificant positive response for at least one of the test points.

     (ii) A test  substance which does not produce either a statistically sig-
nificant 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,  the  following  specific information
should 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, rationale for dose selection,
and 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) Additional requirements. Testing facilities conducting the mouse
visible specific locus test in  accordance with this section should, in addi-
tion to adhering to the provisions  of 40 CFR 792.190 and 792.195 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.

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

     (1) Russell, L.B. et al. 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

     (2) [Reserved]