United States

Environmental Protection

Agency

Prevention, Pesticides

and Toxic Substances

(7101)

EPA712–C–96–232

June 1996

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 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 870.5575 Mitotic gene conversion in Saccharomyces

cerevisiae.

(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 798.5575 Mitotic Gene

Gene Conversion in Saccharomyces cerevisiae amd OPP 84–2 Mutagenicity 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 inactive 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 different, inactive alleles of the same gene locus causing a nutritional requirement.

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 limited 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 mutagenic 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 medium 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 liquid medium enriched with growth factors to early stationary phase. Cells

should then be seeded on selective medium to determine the rate of spontaneous 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 performed 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 chemical 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. Relatively insoluble chemicals should be tested up to the limits of solubility.

For freely soluble nontoxic chemicals, the upper test chemical concentration 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 spontaneous 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 control 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 determining a positive result, one of which is a statistically significant doserelated increase in the number of gene convertants. 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 gene conversions or a statistically 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 information should be reported:

(i) Strain of organism used in the assay.

(ii) Test chemical vehicle, doses used, and rationale for dosage selection.

(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 activation 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 additional background material on this test guideline.

(1) Ames, B.N. et al. Methods for detecting carcinogens and mutagens

with the Salmonella/mammalian-microsome mutagenicity test. Mutation

Research 31:347–364 (1975).

(2) Callen, D.F. and Philpot, R.M. Cytochrome P–450 and the activation 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. Handbook 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 mutation. Mutation Research 28:381–388 (1975).