National Toxicology Program

The assays for induction sex-linked recessive lethal (SLRL) mutations and chromosomal reciprocal translocations (RT) in germ cells of male Drosophila melanogaster were performed with adult flies as described by Zimmering et al. (1985) and with larvae as described by Zimmering et al. (1989). All test chemicals were supplied as coded aliquots from Radian Corporation (Austin, TX). The SLRL mutation test was conducted first. If the chemical produced a positive response in this assay, it was subsequently tested for induction of reciprocal translocations using the same route that produced the response in the SLRL test.

Sex-Linked Recessive Lethal Mutation Test

The test chemical was assayed in the SLRL test by feeding for 3 days to adult Canton-S wild-type males no more than 24 hours old at the beginning of treatment. If no response was obtained, the chemical was retested by injection into adult males. Although feeding was the preferred route of administration, sometimes, due to the physical nature of the test chemical, feeding experiments were not possible, and injection was therefore selected as the method of administration.

To administer a chemical by injection, a glass Pasteur pipette is drawn out in a flame to a microfine filament and the tip is broken off to allow delivery of the test solution. Injection is performed either manually, by attaching a rubber bulb to the other end of the pipette and forcing through sufficient solution (0.2-0.3 uL) to slightly distend the abdomen of the fly, or by attaching the pipette to a microinjector that automatically delivers a calibrated volume. Flies are anaesthetized with ether and immobilized on a strip of tape. Injection into the thorax, under the wing, is performed with the aid of a dissecting microscope.

Toxicity tests were performed to attempt to set concentrations of test chemical at a level that ideally would induce 30% mortality after 72 hours of feeding or 24 hours after injection, while keeping induced sterility at an acceptable level. Canton-S males were allowed to feed for 72 hours on a 5% sucrose solution into which the test chemical was dissolved (solvents included water, ethyl alcohol, or DMSO). In the injection experiments, 24- to 72-hr. old Canton-S males were treated with a solution of test chemical dissolved in saline or suspended in peanut oil and allowed to recover for 24 hrs. A concurrent solvent control group was also included. In the adult exposures, treated males were mated individually to three Basc females for 3 days and given fresh females at 2-day intervals to produce three matings of 3, 2, and 2 days (sperm from successive matings had been treated at successively earlier post-meiotic stages).

For the larval feeding experiment, Canton-S females and males were mated and eggs were exposed in vials with standard cornmeal food containing the test chemical in solvent (5% ethanol) or the solvent alone (Valencia et al., 1989). Adult emergent males were mated individually at approximately 24 hours of age with two successive harems of 3 to 5 Basc females to establish two single-day broods.

For both the adult and larval exposure experiments, F1 heterozygous females were mated with their siblings and then placed in individual vials. F1 daughters from the same parental male were kept together to identify clusters. (A cluster occurs when a number of mutants from a given male result from a single spontaneous premeiotic mutation event, and is identified when the number of mutants from that male significantly exceeds the number predicted by a Poisson distribution). If a cluster was identified, all data from the male in question were discarded. Presumptive lethal mutations were identified as vials containing fewer than 5% of the expected number of wild-type males after 17 days; these were retested to confirm the response.

SLRL data were analyzed by simultaneous comparison with the concurrent and historical controls (Mason et al., 1992) using a normal approximation to the binomial test (Margolin et al., 1983). A test result was considered positive if the P value was less than 0.01 and the mutation frequency in the tested group was greater that 0.10%, or if the P value was less than 0.05 and the frequency in the treatment group was greater than 0.15%. A test was considered to be inconclusive if (a) the P value was between 0.05 and 0.01 but the frequency in the treatment group was between 0.10% and 0.15%, or (b) the P value was between 0.10 and 0.05 but the frequency in the treatment group was greater than 0.10%. A test was considered negative if the P value was greater than 0.10 or if the frequency in the treatment group was less than 0.10%.

Reciprocal Translocation Test

Using the route that produced a positive result in the SLRL test, the test chemical was assayed for induction of reciprocal translocations. The treatment regimen was essentially the same as for the SLRL test, except that Canton-S males were mated en masse to marker (bw;st or bw;e) females. The females were transferred to fresh medium every 3 to 4 days for a period of about 3 weeks to produce a total of 6 broods. The results of the SLRL test were used to determine the germ cell stages most likely to be affected by the test chemical. F1 heterozygous males were backcrossed individually to bw;st females and the F2 progeny were screened for pseudolinkage, which results from the induction of a translocation in a germ cell of the parental male. Flies suspected of carrying reciprocal translocations were retested to confirm the findings. The translocation data were compared to the historical control and significance was analyzed according to the conditional binomial response test of Kastenbaum and Bowman (1970).

Zimmering S, Mason JM, Valencia R, Woodruff RC. (1985) Chemical mutagenesis testing in Drosophila. II. Results of 20 coded compounds tested for the National Toxicology Program. Environ. Mol. Mutagen. 7(1): 87-100.

Zimmering S, Mason JM, Valencia R. (1989) Chemical mutagenesis testing in Drosophila. VII. Results of 22 coded compounds tested in larval feeding experiments. Environ. Mol. Mutagen. 14(4): 245-251.