40 CFR § 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.
(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 F2generation. 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.
(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.