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’’
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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 the

National 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 procedures

that 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 the

EPA 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.’’
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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 all

X-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 mutant

gene. 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 SLRL

test 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
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a sex-linked recessive lethal mutation has occurred in a germ cell of the

P1 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 or

suspended 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 mated

individually 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 effects

on mature sperm, mid or late stage spermatids, early spermatids, spermatocytes and spermatogonia at the time of treatment.

(2) F1 matings. Heterozygous F1 females from the above crosses

should be allowed to mate individually (i.e., one female per vial) with
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their brothers. In the F2 generation, each culture should be scored for the

absence of wild-type males. If a culture appears to have arisen from an

F1 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 frequency

observed 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 as

specified under 40 CFR part 792, subpart J the following specific information should be reported.
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(i) Drosophila stock used in the assay, age of insects, number of

males treated, number of sterile males, number of F2 cultures established,

number of F2 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.