[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: 40CFR798.5955]
[Page 210-212]
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. 798.5955 Heritable translocation test in drosophila melanogaster.
(a) Purpose. The heritable translocation test in Drosophila measures
the induction of chromosomal translocations in germ cells of insects.
Stocks carrying genetic markers on two or more chromosomes are used to
follow the assortment of chromosomes in meiosis. The F1 male
progeny of treated parents are individually mated to females and the
F2 progeny phenotypes are scored. The observed spectrum of
phenotypes is used to determine the presence or absence of a
translocation. This is usually indicated by a lack of independent
assortment of genes on different chromosomes.
(b) Definitions--(1) Chromosome mutations are chromosomal changes
resulting from breakage and reunion of chromosomes. Chromosomal
mutations are also produced through nondisjunction of chromosomes during
cell division.
(2) Reciprocal translocations are chromosomal translocations
resulting from reciprocal exchanges between two or more chromosomes.
(3) Heritable translocations are reciprocal translocations
transmitted from parent to the succeeding progeny.
(c) Reference substances. These may include, but need not be limited
to, ethyl methanesulfonate or N-dimethyl-nitrosamine.
(d) Test method--(1) Principle. The method is based on the principle
that balanced reciprocal chromosomal translocations can be induced by
chemicals in the germ cells of treated flies and that these
translocations are detected in the F2 progeny using genetic
markers (mutations). Different mutations may be used as genetic markers
and two or more of the four chromosomes may be genetically marked for
inclusion in this test.
(2) Description. Wild-type males are treated with chemical and bred
with females of known genetic markers. The F1 males are
collected and individually bred with virgin females of the female
parental stock. The resulting F2 progeny are scored. Putative
translocation carriers are confirmed with an F3 cross.
(i) Illustrative example. The following example serves to illustrate
the method. Males carrying genes for red eye color on chromosomes II and
III are bred with females of white eye color carrying alleles for brown
(bw) on the second chromosome and scarlet (st) and pink (pp) on the
third chromosome. The F1 male progeny are bred with virgin
females of the female parental stock and the resulting F2
progeny are examined for eye color phenotypes. If there is no
translocation in the F1 male, then the resulting
F2 progeny will have four eye color phenotypes: red, white,
orange, and brown. If the F1 male carries a translocation
between chromosomes II and III, only red and white eye phenotypes are
obtained in the F2 generation. This happens because the
F1 translocation heterozygote produces two balanced (carrying
either the parental or the translocated configuration of markers) and
two unbalanced gametes. The unbalanced gametes (carrying one normal and
one translocated chromosome) are unable to develop into normal
individuals in the F2 generation.
(ii) [Reserved]
(3) Drosophila stocks. Wild-type males and females of the genotype
bw:st:pp (white eyes) may be used in the heritable translocation test.
Other appropriately marked Drosophila stocks may also be used.
(4) Control groups. (i) Concurrent positive and negative (vehicle)
controls should be included in each experiment.
(ii) Negative (vehicle) controls should be included. The size of the
negative (vehicle) control group should be determined by the
availability of appropriate laboratory historical control data.
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(iii) If the historical control data are of sufficient numbers,
concurrent controls may not be necessary.
(5) Test chemicals--(i) Vehicle. Test chemicals should be dissolved
in water. Compounds which are insoluble in water may be dissolved or
suspended in appropriate vehicles (e.g., a mixture of ethanol and Tween-
60 or 80), and then diluted in water or saline prior to administration.
Dimethylsulfoxide should be avoided as a vehicle.
(ii) Dose levels. For the initial assessment of mutagenicity, it may
be sufficient to test a single dose of the test substance. This dose
should be the maximum tolerated dose or that which produces some
indication of toxicity. If the test is being used to verify mutagenic
activity, at least two additional exposure levels should be used.
(iii) Route of administration. Exposure may be oral, by injection or
by exposure to gases or vapours. Feeding of the test compound may be
done in sugar solution. When necessary, substances may be dissolved in
0.7 percent NaCl solution and injected into the thorax or abdomen.
(e) Test performance--(1) P1 mating. (i) In the primary screen of a
chemical, it is enough to sample one germ cell stage, either mature
sperm or spermatids (for indirect acting mutagens). Other stages may be
sampled if needed, i.e., when mature germ cells give a positive result
and data from earlier germ cells are needed for the purpose of risk
assessment. Thus, the treated males may be mated only once for a period
of 3 days to sample sperm or transferred every 2 to 3 days to cover the
entire germ cell cycle.
(ii) Mass matings may be performed because the control rate for
translocations in the available literature is very low (near 0) and
clustered events are extremely rare. Mated females may be aged for 2
weeks in order to recover an enhanced incidence of translocation due to
the storage effect. The females are then allowed to lay eggs and
F1 males are collected for test mating.
(2) F1 mating. F1 males should be bred with
virgin females of the parental female stock. Since each F1
male represents one treated gamete of the male parent, the F1
males have to be mated individually to virgin females. Each
F1 male should be mated to three females to ensure sufficient
progeny.
(3) Scoring the F2 generation. F2 cultures
(each representing 1 F1 male tested) should be scored for the
presence or absence of phenotype variations (linkage of markers) from
the expected types. The test should be designed with a predetermined
sensitivity and power. The number of flies in each group should reflect
these defined parameters. The spontaneous mutant frequency observed in
the appropriate control group will strongly influence the number of
treated chromosomes that must be analyzed to detect substances which
show mutation rates close to those of the controls. A positive test
should be confirmed by F3 mating trials.
(4) Number of replicate experiments. Replicate experiments are
usually performed for each dose of the compound tested. If a chemical is
a potent inducer of translocations, one experiment may be sufficient.
Otherwise two or three replicate experiments should be done.
(f) Data and report--(1) Treatment of results. Data should be
tabulated to show the number of translocations and the number of fertile
F1 males at each exposure for each germ cell stage sampled.
(2) Statistical evaluation. Data should 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.
[[Page 212]]
(4) Test evaluation. (i) Positive results in the heritable
translocation test in Drosophila indicate that under the test conditions
the test substance causes chromosome damage in germ cells of this
insect.
(ii) Negative results indicate that under the test conditions the
test substance does not cause chromosomal damage in D. melanogaster.
(5) Test report. In addition to the reporting recommendations as
specified under 40 CFR part 792, subpart J, the following specific
information should be reported:
(i) Drosophila stock used in the assay, age of insects, number of
males treated, number of F2 cultures established, number of
replicate experiments.
(ii) Test chemical vehicle, treatment and mating schedule, exposure
levels, toxicity data, dose and route of exposure.
(iii) Positive and negative (vehicle) controls.
(iv) Historical control data, if available.
(v) Number of chromosomes scored.
(vi) Criteria for scoring mutant chromosomes.
(vii) Dose-response relationship, if applicable.
(g) References. For additional background information on this test
guideline the following references should be consulted:
(1) Wurgler, F.E., Sobels, F.H., Vogel, E. ``Drosophila as assay
system for detecting genetic changes,'' Handbook of mutagenicity test
procedures. Eds. Kilby, B.J., Legator, M., Nichols, W., Ramel, C.
(Amsterdam: Elsevier/North Holland Biomedical Press, 1979) pp. 335-374.
(2) [Reserved]