United States      Prevention, Pesticides    EPA712-C-98-220
          Environmental Protection   and Toxic Substances    August 1998
          Agency        (7101)
&EPA    Health Effects Test
          OPPTS 870.5275
          Sex-Linked Recessive
          Lethal Test in Drosophila

     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

     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.).

     Final  Guideline Release: This guideline  is available from the U.S.
Government Printing Office,  Washington, DC 20402 on disks or paper
copies: call (202) 512-0132. This  guideline is also available electronically
in PDF (portable document format) from EPA's  World Wide Web  site
(http://www.epa.gov/epahome/research.htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelines/OPPTS  Harmonized Test

OPPTS  870.5275  Sex-linked recessive lethal  test in  Drosophila
     (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

     (b)  Purpose.  The sex-linked  recessive lethal  (SLRL) test  using
Drosophila melanogaster  (D.  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 ap-
proximately 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

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 TV-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

     (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) FI matings. Heterozygous  FI females from the above crosses
should be allowed  to mate individually (i.e., one female per  vial) with

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 nonfer-
tile males and the number of lethal chromosomes at  each exposure con-
centration 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.

     (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.