United States Prevention, Pesticides EPA712-C-96-232
Environmental Protection and Toxic Substances June 1996
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.5575
Mitotic Gene Conversion
in Saccharomyces
cerevisiae
'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 870.5575 Mitotic gene conversion in Saccharomyces
cere visiae.
(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 798.5575 Mitotic Gene
Gene Conversion in Saccharomyces cerevisiae amd OPP 84-2 Mutagenic-
ity Testing (Pesticide Assessment Guidelines, Subdivision F—Hazard
Evaluation; Human and Domestic Animals) EPA report 540/09-82-025,
1982.
(b) Purpose. The mitotic gene conversion assay in the yeast,
Saccharomyces cerevisiae, measures the conversion of differentially inac-
tive alleles to wild-type alleles by mutagenic agents. Heteroallelic diploid
yeast strains carry two different inactive alleles of the same gene locus.
The presence of these alleles causes a nutritional requirement, e.g. these
heteroallelic diploids grow only in medium supplemented with a specific
nutrient such as tryptophan. When gene conversion occurs, a fully active
wild-type phenotype is produced from these inactive alleles through
intragenic recombination. These wild-type colonies grow on a medium
lacking the specific nutritional requirement (selective medium).
(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.
Heteroallelic diploids are diploid strains of yeast carrying two dif-
ferent, inactive alleles of the same gene locus causing a nutritional require-
ment.
Mitotic gene conversion is detected by the change of inactive alleles
of the same gene to wild-type alleles through intragenic recombination
in mitotic cells.
(d) Reference substances. These may include, but need not be lim-
ited to, hydrazine sulfate or 2-acetylaminofluorene.
(e) Test method—(1) Principle. The method is based on the fact
that heteroallelic diploid yeast strains carry two inactive alleles of the same
gene locus making them dependent on a specific nutritional requirement
(e.g., tryptophan) for their survival. Treatment of such strains with muta-
genic agents can cause conversion of these alleles back to the wild-type
condition which allows growth on a medium lacking the required nutrient
(selective medium).
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(2) Description. Heteroallelic diploid strains such as D7, requiring
a specific nutrient in the medium, are treated with test chemical with and
without metabolic activation and plated on a selective medium lacking the
required nutrient. The wild-type colonies that grow on the selective me-
dium as a result of gene conversion are scored.
(3) Strain selection—(i) Designation. At the present time, S.
cerevisiae strain D7 is recommended for use in this assay. The use of
other strains may also be appropriate.
(ii) Preparation and storage. Stock culture preparation and storage,
growth requirements, method of strain identification and demonstration of
appropriate phenotypic requirements should be performed using good
microbiological techniques and should be documented.
(iii) Media. YEP glucose medium enriched with the appropriate
growth factors may be used for cell growth and maintenance. Other media
may also be appropriate.
(4) Selection of cultures. Cells should be grown with aeration in liq-
uid medium enriched with growth factors to early stationary phase. Cells
should then be seeded on selective medium to determine the rate of sponta-
neous conversion. Cultures with a high rate of spontaneous conversion
should be discarded.
(5) Metabolic activation. Cells should be exposed to test chemical
both in the presence and absence of an appropriate metabolic activation
system.
(6) Control groups. Concurrent positive and negative (untreated and/
or vehicle) controls both with and without metabolic activation should be
included in each experiment.
(7) Test chemicals—(i) Vehicle. Test chemicals and positive control
reference substances should be dissolved in an appropriate vehicle and then
further diluted in vehicle for use in the assay. Dimethylsulfoxide should
be avoided as a vehicle.
(ii) Exposure concentrations. (A) The test should initially be per-
formed over a broad range of concentrations. Among the criteria to be
taken into consideration for determining the upper limits of test chemical
concentration are cytotoxicity and solubility. Cytotoxicity of the test chem-
ical may be altered in the presence of metabolic activation systems. For
cytotoxic chemicals, the highest dose tested should not reduce survival
to less than 10 percent of that seen in the untreated control cultures. Rel-
atively insoluble chemicals should be tested up to the limits of solubility.
For freely soluble nontoxic chemicals, the upper test chemical concentra-
tion should be determined on a case by case basis.
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(B) When appropriate, a positive response should be confirmed by
using a narrow range of concentrations.
(f) Test performance—(1) Treatment. Cultures should be treated
in liquid suspension. Resting cells should be treated in buffer; growing
cells should be treated in a synthetic medium. Cultures with low sponta-
neous convertant frequencies should be centrifuged, washed and
resuspended in liquid at the appropriate density. Cells should be exposed
to test chemical both in the presence and absence of a metabolic activation
system. Independent tubes should be treated for each concentration. At
the end of the treatment period, cells should be centrifuged, washed and
resuspended in distilled water prior to plating on selective medium for
convertant selection and on complete medium to determine survival. At
the end of the incubation period, plates should be scored for survival and
the presence of convertant colonies.
(2) Number of cultures. At least six individual plates per treatment
concentration and control should be used.
(3) Incubation conditions. All plates in a given experiment should
be incubated for the same time period. This incubation period may be
from 4 to 6 days at 28 °C.
(g) Data and report—(1) Treatment of results. Individual plate
counts for test substance and control should be presented for both
convertants and survivors. The mean number of colonies per plate and
standard deviation should also be presented. Data should be presented in
tabular form indicating numbers of viable and convertant colonies scored,
survival frequency and convertant frequencies for each treatment and con-
trol culture. Conversion frequencies should be expressed as number of
convertants per number of survivors. Sufficient detail should be provided
for verification of survival and convertant frequencies.
(2) Statistical evaluation. Data should be evaluated by appropriate
statistical methods.
(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 gene convertants. Another criterion may
be based upon detection of a reproducible and statistically significant posi-
tive 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 gene conversions or a statis-
tically significant and reproducible positive response at any one of the
test points is considered nonmutagenic in this system.
(iii) Both biological and statistical significance should be considered
together in the evaluation.
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(4) Test evaluation, (i) Positive results in this assay indicate that
under the test conditions the test chemical causes mitotic gene conversion
in the yeast S. cerevisiae.
(ii) Negative results indicate that under the test conditions the test
chemical does not cause mitotic gene conversion in S. cerevisiae.
(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) Strain of organism used in the assay.
(ii) Test chemical vehicle, doses used, and rationale for dosage selec-
tion.
(iii) Method used to select cultures.
(iv) Treatment protocol including cell density at treatment and length
of exposure to test substance.
(v) Details of both the protocol used to prepare the metabolic activa-
tion system and its use in the assay.
(vi) Incubation times and temperatures.
(vii) Dose-response relationship, if applicable.
(h) References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1) Ames, B.N. et al. Methods for detecting carcinogens and mutagens
with the &z/m0«e//a/mammalian-microsome mutagenicity test. Mutation
Research 31:347-364 (1975).
(2) Callen, D.F. and Philpot, R.M. Cytochrome P-450 and the activa-
tion of promutagens in Saccharomyces cerevisiae. Mutation Research
45:309-324 (1975).
(3) Zimmermann, F.K. Procedures used in the induction of mitotic
recombination and mutation in the yeast Saccharomyces cerevisiae. Hand-
book of mutagenicity test procedures. Eds. Kilby, B.J., Legator, M., Nicols,
W., Ramel, C. Elsevier/North Holland Biomedical Press, Amsterdam
(1979). pp. 119-134.
(4) Zimmermann, F.K. et al. A yeast strain for simultaneous detection
of induced mitotic crossing over, mitotic gene conversion and reverse mu-
tation. Mutation Research 28:381-388 (1975).
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