.... United States Environmental Protection Agency Prevention, Pesticides and Toxic Substances (7101) EPA712–C–96–220 June 1996 

# Health Effects Test Guidelines OPPTS 870.5275 Sex-Linked Recessive Lethal Test in Drosophila melanogaster

‘‘Public Draft’’

### i INTRODUCTION This guideline is one of a series of test guidelines that have been developed by the Office of Prevention, Pesticides and Toxic Substances, United States Environmental Protection Agency for use in the testing of pesticides and toxic substances, and the development of test data that must be submitted to the Agency for review under Federal regulations. The Office of Prevention, Pesticides and Toxic Substances (OPPTS) has developed this guideline through a process of harmonization that blended the testing guidance and requirements that existed in the Office of Pollution Prevention and Toxics (OPPT) and appeared in Title 40, Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the Office of Pesticide Programs (OPP) which appeared in publications of theNational Technical Information Service (NTIS) and the guidelines published by the Organization for Economic Cooperation and Development (OECD). The purpose of harmonizing these guidelines into a single set of OPPTS guidelines is to minimize variations among the testing proceduresthat must be performed to meet the data requirements of the U. S. Environmental Protection Agency under the Toxic Substances Control Act (15 U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act (7 U.S.C. 136, et seq. ).

Public Draft Access Information: This draft guideline is part of a series of related harmonized guidelines that need to be considered as a unit. For copies: These guidelines are available electronically from theEPA Public Access Gopher (gopher.epa.gov) under the heading ‘‘Environmental Test Methods and Guidelines’’ or in paper by contacting the OPP Public Docket at (703) 305–5805 or by e-mail: guidelines@epamail.epa.gov.

To Submit Comments: Interested persons are invited to submit comments. By mail: Public Docket and Freedom of Information Section, Office of Pesticide Programs, Field Operations Division (7506C), Environmental Protection Agency, 401 M St. SW., Washington, DC 20460. In person:bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Arlington, VA. Comments may also be submitted electronically by sending electronic mail (e-mail) to: guidelines@epamail.epa.gov.

Final Guideline Release: This guideline is available from the U.S. Government Printing Office, Washington, DC 20402 on The Federal Bulletin Board. By modem dial 202–512–1387, telnet and ftp: fedbbs.access.gpo.gov (IP 162.140.64.19), or call 202–512–0132 for disks or paper copies. This guideline is also available electronically in ASCII and PDF (portable document format) from the EPA Public Access Gopher (gopher.epa.gov) under the heading ‘‘Environmental Test Methods and Guidelines.’’

### 1 OPPTS 870.5275 Sex-linked recessive lethal test in Drosophila melanogaster. (a) Scope—(1) Applicability. This guideline is intended to meet testing requirements of both the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq. ) and the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601). (2) Background. The source materials used in developing this harmonized OPPTS test guideline are OPPT 40 CFR 798.5275 Sex-Linked Recessive Lethal Test in Drosophila melanogaster and OECD 477 Genetic Toxicology: Sex-Linked Recessive Lethal Test in Drosophila melanogaster

. (b) Purpose. The sex-linked recessive lethal (SLRL) test using Drosophila melanogaster detects the occurrence of mutations, both point mutations and small deletions, in the germ line of the insect. This test is a forward mutation assay capable of screening for mutations at about 800 loci on the X-chromosome. This represents about 80 percent of allX-chromosome loci. The X-chromosome represents approximately onefifth of the entire haploid genome. (c) Definitions. The definitions in section 3 of TSCA and in 40 CFR Part 792—Good Laboratory Practice Standards (GLP) apply to this test guideline. The following definitions also apply to this test guideline.

Lethal mutation is a change in the genome which, when expressed, causes death to the carrier.

Recessive mutation is a change in the genome which is expressed in the homozygous or hemizygous condition. Sex-linked genes are present on the sex (X or Y) chromosomes. Sexlinked genes in the context of this guideline refer only to those located on the X-chromosome. (d) Reference substances. These may include, but need not be limited to, ethyl methanesulfonate or N -nitrosodimethylamine. (e) Test method—(1) Principle. Mutations in the X-chromosome of D. melanogaster are phenotypically expressed in males carrying the mutantgene. When the mutation is lethal in the hemizygous condition, its presence is inferred from the absence of one class of male offspring out of the two that are normally produced by a heterozygous female. The SLRLtest takes advantage of these facts by means of specially marked and arranged chromosomes. (2) Description. Wild-type males are treated and mated to appropriate females. Female offspring are mated individually to their brothers, and in the next generation the progeny from each separate dose are scored for phenotypically wild-type males. Absence of these males indicates that

### 2 a sex-linked recessive lethal mutation has occurred in a germ cell of the P 1 male. (3) Drosophila stocks. Males of a well-defined wild type stock and females of the Muller-5 stock may be used. Other appropriately marked female stocks with multiple inverted X-chromosomes may also be used. (4) Control groups—(i) Concurrent controls. Concurrent positive and negative (vehicle) controls should be included in each experiment. (ii) Positive controls. Examples of positive controls include ethyl methanesulfonate and N -nitrosodimethylamine. (iii) Other positive controls. Other positive control reference substances may be used. (iv) Negative controls. 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. (5) Test chemicals—(i) Vehicle. Test chemicals should be dissolved in water. Compounds which are insoluble in water may be dissolved orsuspended in appropriate vehicles (e.g., a mixture of ethanol and Tween60 or 80) and then diluted in water or saline prior to administration. The use of dimethylsulfoxide as a vehicle should be avoided. (ii) Dose levels. For the initial assessment of mutagenicity, it is sufficient to test a single dose of the test substance for screening purposes. This dose should be the maximum tolerated dose, or that which produces some indication of toxicity, or should be the highest dose attainable. For dose-response purposes, at least three additional dose levels should be used.(iii) Route of administration. Exposure may be oral, by injection or by exposure to gases or vapors. 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. (f) Test performance—(1) Treatment and mating. Wild-type males (3 to 5 days old) should be treated with the test substance and matedindividually to an appropriate number of virgin females from the Muller5 stock or females from another appropriately marked (with multiply-inverted X-chromosomes) stock. The females should be replaced with fresh virgins every 2 to 3 days to cover the entire germ cell cycle. The offspring of these females are scored for lethal effects corresponding to the effectson mature sperm, mid or late stage spermatids, early spermatids, spermatocytes and spermatogonia at the time of treatment. (2) F1 matings. Heterozygous F 1 females from the above crosses should be allowed to mate individually (i.e., one female per vial) with

### 3 their brothers. In the F 2 generation, each culture should be scored for the absence of wild-type males. If a culture appears to have arisen from an F 1 female carrying a lethal in the parental X-chromosome (i.e., no males with the treated chromosome are observed), daughters of that female with the same genotype should be tested to ascertain if the lethality is repeated in the next generation. (3) Number of matings. (i) 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 frequencyobserved in the appropriate control group will strongly influence the number of treated chromosomes that must be analysed to detect substances which show mutation rates close to those of the controls. (ii) Test results should be confirmed in a separate experiment. (g) Data and report—(1) Treatment of results. Data should be tabulated to show the number of chromosomes tested, the number of nonfertile males and the number of lethal chromosomes at each exposure concentration and for each mating period for each male treated. Numbers of clusters of different size per male should be reported. (2) Statistical evaluation. Data should be evaluated by appropriate statistical techniques. (3) Interpretation of results. (i) There are several criteria for determining a positive result, one of which is a statistically significant doserelated increase in the number of sex-lined recessive lethals. 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 sex-linked recessive lethals or a statistically significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system. (iii) Both biological and statistical significance should be considered together in the evaluation. (4) Test evaluation. (i) Positive results in the SLRL test in D. melanogaster indicate that under the test conditions the test agent causes mutations in germ cells of this insect. (ii) Negative results indicate that under the test conditions the test substance is not mutagenic in D. melanogaster

. (5) Test report. In addition to the reporting recommendations asspecified under 40 CFR part 792, subpart J the following specific information should be reported.

### 4 (i) Drosophila stock used in the assay, age of insects, number of males treated, number of sterile males, number of F 2 cultures established, number of F 2 cultures without progeny. (ii) Test chemical vehicle, treatment and sampling schedule, exposure levels, toxicity data, negative (vehicle) and positive controls, if appropriate. (iii) Criteria for scoring lethals. (iv) Number of chromosomes tested, number of chromosomes scored, number of chromosomes carrying a lethal mutation. (v) Historical control data, if available. (vi) Dose-response relationship, if applicable. (h) References. The following references should be consulted for additional background material on this test guideline. (1) Sobels, F.H. and Vogel, E. The capacity of Drosophila for detecting relevant genetic damage. Mutation Research 41:95–106 (1976). (2) Wurgler F.E. et al. Drosophila as assay system for detecting genetic changes. Handbook of mutagenicity test procedures. Eds. Kilbey,B.J., Legator, M., Nichols, W., Ramel, C. Elsevier/North Holland Biomedical, Amsterdam (1977) pp. 335–373.