[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: 40CFR798.5955]

[Page 208-210]
 
                   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

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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.
    (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.

[[Page 210]]

    (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.
    (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]