[Code of Federal Regulations]
[Title 40, Volume 28]
[Revised as of July 1, 2002]
From the U.S. Government Printing Office via GPO Access
[CITE: 40CFR798.5460]
[Page 203-205]
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. 798.5460 Rodent heritable translocation assays.
(a) Purpose. This test detects transmitted chromosomal damage which
manifests as balanced reciprocal translocations in progeny descended
from parental males treated with chemical mutagens.
(b) Definitions. (1) A heritable translocation is one in which
distal segments of nonhomologous chromosomes are involved in a
reciprocal exchange.
(2) Diakinesis and metaphase I are stages of meiotic prophase scored
cytologically for the presence of multivalent chromosome association
characteristic of translocation carriers.
(c) Reference substances. Not applicable.
(d) Test method--(1) Principle. When a balanced reciprocal
translocation is induced in a parental male germ cell, the resulting
progeny is translocation heterozygote.
(i) Basis for fertility screening. Male translocation heterozygotes
may be completely sterile. This class consists of two types of
translocations:
(A) Translocations between non-homologous chromosomes in which at
least one of the breaks occurs close to one end of a chromosome.
(B) Those that carry multiple translocations. The majority of male
translocation heterozygotes are semisterile--they carry one or (rarely)
two translocations. The degree of semisterility is dependent upon the
proportions of balanced and unbalanced (duplication-deficiency) gametes
produced in the ejaculate as a function of meiotic segregation. Balanced
and unbalanced sperm are equally capable of fertilizing an egg. Balanced
sperm lead to viable progeny. Unbalanced sperm result in early embryonic
lethality.
(ii) Basis for cytological screening. The great majority of male
translocation heterozygotes can be identified cytologically through
analysis of diakinesis metaphase I spermatocytes. Translocation
heterozygotes are characterized by the presence of multivalent
chromosome association such as a ring or chain of four chromosomes held
together by chiasmata in paired homologous regions. Some translocation
carriers can be identified by the presence of extra long and/or extra
short chromosomes in spermatogonial and somatic cell metaphase
preparations.
(2) Description. Essentially, two methods have been used to screen
for translocation heterozygosity; one method uses a mating sequence to
identify sterile and semisterile males followed by cytological
examination of suspect male individuals; the other method deletes the
mating sequence altogether and all F1 male progeny are
examined cytologically for presence of translocation. In the former
approach, the mating sequence serves as a screen which eliminates most
fully fertile animals for cytological confirmation as translocation
heterozygotes.
(3) Animal selection--(i) Species. The mouse is the species
generally used, and is recommended.
(ii) Age. Healthy sexually mature animals shall be used.
(iii) Number. (A) The number of male animals necessary is determined
by the following factors:
(1) The use of either historical or concurrent controls.
(2) The power of the test.
(3) The minimal rate of induction required.
(4) Whether positive controls are used.
(5) The level of significance desired.
(B) [Reserved]
(iv) Assignment to groups. Animals shall be randomized and assigned
to treatment and control groups.
(4) Control groups--(i) Concurrent controls. No concurrent positive
or negative (vehicle) controls are recommended as routine parts of the
heritable translocation assay. However, investigators not experienced in
performing translocation testing shall include a substance known to
produce translocations in the assay as a positive control reference
chemical.
(ii) Historical controls. At the present time, historical control
data must be used in tests for significance. When
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statistically reliable historical controls are not available, negative
(vehicle) controls shall be used.
(5) Test chemicals--(i) Vehicle. When appropriate for the route of
administration, solid and liquid test substances should be dissolved or
suspended in distilled water or isotonic saline. Water-insoluble
chemicals may be dissolved or suspended in appropriate vehicles. The
vehicle used shall neither interfere with the test chemical nor produce
toxic effects. Fresh preparations of the test chemical should be
employed.
(ii) Dose levels. At least two dose levels shall be used. The
highest dose level shall result in toxic effects (which shall not
produce an incidence of fatalities which would prevent a meaningful
evaluation) or shall be the highest dose attainable or 5g/kg body
weight.
(iii) Route of administration. Acceptable routes of administration
include oral, inhalation, admixture with food or water, and IP or IV
injection.
(e) Test performance--(1) Treatment and mating. The animals shall be
dosed with the test substances 7 days per week over a period of 35 days.
After treatment, each male shall be caged with 2 untreated females for a
period of 1 week. At the end of 1 week, females shall be separated from
males and caged individually. When females give birth, the day of birth,
litter size, and sex of progeny shall be recorded. All male progeny
should be weaned, and all female progeny should be discarded.
(2) Testing for translocation heterozygosity. When males are
sexually mature, testing for translocation heterozygosity shall begin.
One of two methods shall be used; the first method involves mating,
determining those F1 progeny which are sterile or semisterile
and subsequent cytological analysis of suspect progeny; the other method
does not involve mating and determining sterility or semisterility; all
progeny are examined cytologically.
(i) Determination of sterility or semisterility--(A) Conventional
method. Females are mated, usually three females for each male, and each
female is killed at midpregnancy. Living and dead implantations are
counted. Criteria for determining normal and semisterile males are
usually established for each new strain because the number of dead
implantations varies considerably among strains.
(B) Sequential method. Males to be tested are caged individually
with females and the majority of the presumably normal males are
identified on the basis of a predetermined size of 1 or 2 litters.
Breeding pens are examined daily on weekdays beginning 18 days after
pairing. Young are discarded immediately after they are scored. Males
that sire a litter whose size is the same as or greater than the minimum
set for a translocation-free condition are discarded with their litter.
If the litter size is smaller than the predetermined number, a second
litter is produced with the same rule applying. Males that cannot be
classified as normal after production of a second litter are tested
further by the conventional method or by cytological confirmation of
translocation.
(ii) Cytological analysis. For cytological analysis of suspected
semisteriles, the air-drying technique is used. Observation of at least
2 diakinesis-metaphase 1 cells with mutivalent association constitutes
the required evidence for the presence of a translocation. Sterile males
are examined by one of two methods, those with testes of normal size and
sperm in the epididymis are examined by the same techniques used for
semisteriles. Animals with small testes are examined by squash
preparations or, alternatively, by examination of mitotic metaphase
preparations. If squash preparations do not yield diakinesis-metaphase 1
cells, analysis of spermatogonia or bone marrow for the presence of
unusually long or short chromosomes should be performed.
(f) Data and report--(1) Treatment of results. (i) Data shall be
presented in tabular form and shall include the number of animals at
risk, the germ cell stage treated, the number of partial steriles and
semisteriles (if the fertility test is used), the number of
cytogenetically confirmed translocation heterozygotes (if the fertility
test is used, report the number of confirmed steriles and confirmed
partial steriles), the translocation rate, and either the
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standard error of the rate or the upper 95 percent confidence limit on
the rate.
(ii) These data shall be presented for both treated and control
groups. Historical or concurrent controls shall be specified, as well as
the randomization procedure used for concurrent controls.
(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 heritable
translocations. 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 heritable
translocations 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 heritable
translocation assay indicate that under the test conditions the test
substance causes heritable chromosomal damage in the test species.
(ii) Negative results indicate that under the test conditions the
test substance does not cause heritable chromosomal damage in the test
species.
(5) Test report. In addition to the reporting recommendations as
specified under 40 CFR part 792, subpart J, the following specific
information shall be reported:
(i) Species, strain, age, weight and number of animals of each sex
in each group.
(ii) Test chemical vehicle, route and schedule of administration,
toxicity data.
(iii) Dosing regimen, doses tested and rationale for dosage
selection.
(iv) Mating schedule, number of females mated to each male.
(v) The use of historical or concurrent controls.
(vi) Screening procedure including the decision criteria used and
the method by which they were determined.
(vii) Dose-response relationship, if applicable.
(g) References. For additional background information on this test
guideline the following references should be consulted:
(1) Generoso, W.M., Bishop, J.B., Goslee, D.G., Newell, G.W., Sheu,
G-J, von Halle, E. ``Heritable translocation test in mice,'' Mutation
Research, 76:191-215 (1980).
(2) [Reserved]
[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19081, May 20, 1987]