United States Prevention, Pesticides EPA712-C-96-220
Environmental Protection and Toxic Substances June 1996
Agency (7101)
&EPA 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 pub-
lished 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. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7U.S.C. I36,etseq.).
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 "Environ-
mental 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 com-
ments. 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, Ar-
lington, 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 Bul-
letin 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 test-
ing 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 har-
monized 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 one-
fifth 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. Sex-
linked 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 lim-
ited to, ethyl methanesulfonate or jV-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 pres-
ence 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 ar-
ranged 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
PI 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
methane sulfonate and jV-nitrosodimethylamine.
(iii) Other positive controls. Other positive control reference sub-
stances may be used.
(iv) Negative controls. Negative (vehicle) controls should be in-
cluded. The size of the negative (vehicle) control group should be deter-
mined 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 Tween-
60 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 suffi-
cient 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 Muller-
5 stock or females from another appropriately marked (with multiply-in-
verted 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, spermato-
cytes and spermatogonia at the time of treatment.
(2) Fl matings. Heterozygous FI 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
FI 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 pre-
determined 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 num-
ber 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 tab-
ulated 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 deter-
mining a positive result, one of which is a statistically significant dose-
related increase in the number of sex-lined recessive lethals. Another cri-
terion may be based upon detection of a reproducible and statistically sig-
nificant positive response for at least one of the test points.
(ii) A test substance which does not produce either a statistically sig-
nificant 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 informa-
tion 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 ad-
ditional background material on this test guideline.
(1) Sobels, F.H. and Vogel, E. The capacity of Drosophila for detect-
ing relevant genetic damage. Mutation Research 41:95-106 (1976).
(2) Wurgler F.E. et al. Drosophila as assay system for detecting ge-
netic changes. Handbook of mutagenicity test procedures. Eds. Kilbey,
B.J., Legator, M., Nichols, W., Ramel, C. Elsevier/North Holland Bio-
medical, Amsterdam (1977) pp. 335-373.
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