.... United States Environmental Protection Agency Prevention, Pesticides and Toxic Substances (7101) EPA712–C–96–221 June 1996


# Health Effects Test Guidelines
OPPTS 870.5300 Detection of Gene Mutations in Somatic Cells in Culture

‘‘Public Draft’’

### i 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 theNational 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 proceduresthat 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 theEPA 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.’’

### 1 OPPTS 870.5300 Detection of gene mutations in somatic cells in culture. (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 material used in developing this harmonized OPPTS test guideline is OPPT 40 CFR 798.5300 Detection ofGene Mutations in Somatic Cells in Culture and OECD guideline 476 Genetic Toxicology: In Vivo Mammalian Cell Gene Mutation Tests.
(b) Purpose. Mammalian cell culture systems may be used to detect mutations induced by chemical substances. Widely used cell lines include L5178Y mouse lymphoma cells and the CHO and V–79 lines of Chinese hamster cells. In these cell lines the most commonly used systems measuremutation at the thymidine kinase (TK), hypoxanthine-guaninephosphoribosyl transferase (HPRT) and Na+/K+ ATPase loci. The TK and HPRT mutational systems detect base pair mutations, frameshift mutations, and small deletions; the Na+/K+. ATPase system detects base pair mutations only.
(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.

Base pair mutagens are agents which cause a base change in the DNA.

A forward mutation assay detects a gene mutation from the parental type to the mutant form which gives rise to a change in an enzymatic or functional protein.
Frameshift mutagens are agents which cause the addition or deletion of single or multiple base pairs in the DNA molecule.
Phenotypic expression time is a period during which unaltered gene products are depleted from newly mutated cells.
(d) Reference substances. These may include, but need not be limited to, ethyl methanesulfonate, N -nitroso-dimethylamine, 2-acetylamino fluorene, 7,12-dimethylbenzanthracene, or hycanthone.
(e) Test method—(1) Principle. Cells are exposed to test substance, both with and without metabolic activation, for a suitable period of timeand subcultured to determine cytotoxicity and to allow phenotypic expression prior to mutant selection. Cells deficient in thymidine kinase (TK) due to the forward mutation TK+ to TK

– are resistant to the cytotoxic effects of pyrimidine analogues such as bromodeoxyuridine (BrdU), fluorodeoxyuridine (FdU) or trifluorothymidine (TFT). The deficiency of

### 2 the ‘‘salvage’’ enzyme thymidine kinase means that these antimetabolites are not incorporated into cellular nucleotides and the nucleotides neededfor cellular metabolism are obtained solely from de novo synthesis. However, in the presence of thymidine kinase, BrdU, FdU or TFT are incorporated into the nucleotides, resulting in inhibition of cellular metabolism and cytotoxicity. Thus mutant cells are able to proliferate in the presenceof BrdU, FdU or TFT whereas normal cells, which contain thymidine kinase, are not. Similarly cells deficient in HPRT are selected by resistance to 8-azaguanine (AG) or 6-thioguanine (TG) and cells with altered Na+/ K+ ATPase are selected by resistance to ouabain.
(2) Description. Cells in suspension or monolayer culture are exposed to the test substance, both with and without metabolic activation, for a defined period of time. Cytotoxicity is determined by measuring the colony forming ability or growth rate of the cultures after the treatment period.The treated cultures are maintained in growth medium for a sufficient period of time—characteristic of each selected locus—to allow near-optimalphenotypic expression of induced mutations. Mutant frequency is determined by seeding known numbers of cells in media containing the selective agent to detect mutant cells, and in media without selective agent to determine the cloning efficiency. After a suitable incubation time, cell colonies are counted. The number of mutant colonies in selective media is adjusted by the number of colonies in nonselective media to derive the mutant frequency.
(3) Cells—(i) Type of cells used in the assay. A variety of cell lines are available for use in this assay including subclones of L5178Y, CHOcells or V–79 cells. Cell types used in this assay should have a demonstrated sensitivity to chemical mutagens, a high cloning efficiency and a low spontaneous mutation frequency. Cells should be checked for Mycoplasma contamination and may be periodically checked for karyotype stability.
(ii) Cell growth and maintenance. Appropriate culture media and incubation conditions (culture vessels, CO 2 concentrations, temperature, and humidity) should be used.
(4) Metabolic activation. Cells should be exposed to test substance both in the presence and absence of an appropriate metabolic activation system.
(5) Control groups. Positive and negative (untreated and/or vehicle) controls should be included in each experiment. When metabolic activation is used, the positive control substance should be known to require such activation.
(6) Test chemicals—(i) Vehicle. Test substances may be prepared in culture media or dissolved or suspended in appropriate vehicles prior to treatment of the cells. The final concentration of the vehicle should

### 3 not interfere with cell viability or growth rate. Treatment vessels should be chosen to ensure that there is no visible interaction, such as etching, between the solvent, the test chemical, and the vessel.

(ii) Exposure concentrations. (A) The test should be designed to have a predetermined sensitivity and power. The number of cells, cultures,and concentrations of test substance used should reflect these defined parameters. The number of cells per culture is based on the expected background mutant frequency; a general guide is to use a number which is 10 times the inverse of this frequency.
(B) Several concentrations (usually at least four) of the test substance should be used. Generally, these should yield a concentration-related toxic effect. The highest concentration should produce a low level of survival (approximately 10 percent), and the survival in the lowest concentrationshould approximate the negative control. Cytotoxicity should be determined after treatment with the test substance both in the presence and in the absence of an exogenous metabolic activation system. Relatively insoluble substances should be tested up to their limit of solubility underculture conditions. For freely-soluble nontoxic substances the highest concentration used should be determined on a case-by-case basis.

(f) Test performance. (1) Cells should be exposed to the test substance both with and without exogenous metabolic activation. Exposure should be for a suitable period of time, in most cases 1 to 5 h is effective; exposure time may be extended over one or more cell cycles.
(2) At the end of the exposure period, cells should be washed and cultured to determine viability and to allow for expression of the mutant phenotype.
(3) At the end of the expression period, which should be sufficient to allow near optimal phenotypic expression of induced mutants, cellsshould be grown in media with and without selective agent(s) for determination of number of mutants and cloning efficiency, respectively.
(4) Results should be confirmed in an independent experiment. When appropriate, a single positive response should be confirmed by testing over a narrow range of concentrations.
(g) Data and report—(1) Treatment of results. Data should be presented in tabular form. Individual colony counts for the treated and controlgroups should be presented for both mutation induction and survival. Survival and cloning efficiencies should be given as a percentage of the controls. Mutant frequency should be expressed as number of mutants per number of surviving cells.

(2) Statistical evaluation. Data should be evaluated by appropriate statistical methods.

### 4 (3) Interpretation of results. (i) There are several criteria for determining a positive result, one of which is a statistically significant concentration-related increase in the mutant frequency. Another criterion maybe based upon detection of a reproducible and statistically significant positive response for at least one of the test substance concentrations.

(ii) A test substance which does not produce either a statistically significant concentration-related increase in the mutant frequency 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.

(4) Test evaluation. (i) Positive results for an in vitro mammalian cell gene mutation test indicate that, under the test conditions, a substance induces gene mutations in the cultured mammalian cells used.

(ii) Negative results indicate that, under the test conditions, the test substance does not induce gene mutations in the cultured mammalian cells used.

(5) Test report. In addition to the reporting recommendations asspecified under 40 CFR part 792, subpart J, the following specific information should be reported:

(i) Cell type used, number of cell cultures, methods used for maintenance of cell cultures.

(ii) Rationale for selection of concentrations and number of cultures.

(iii) Test conditions: composition of media, CO 2 concentration, concentration of test substance, vehicle, incubation temperature, incubation time, duration of treatment, cell density during treatment, type of metabolicactivation system, positive and negative controls, length of expression period (including number of cells seeded and subculture and feeding schedules, if appropriate), selective agent(s).

(iv) Methods used to enumerate numbers of viable and mutant cells.

(v) Dose-response relationship, where possible.

(h) References. The following references should be consulted for additional background material on this test guideline.

(1) Amacher, D.E. et al. Point mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells. I. Application to genetic toxicology testing. Mutation Research 64:391–406 (1979).

### 5 (2) Amacher, D.E. et al. Point mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells. II. Test validation and interpretation. Mutation Research 72:447–474 (1980).
(3) Bradley, M.O. et al. Mutagenesis by chemical agents in V–79 Chinese hamster cells: a review and analysis of the literature: a report of the Gene-Tox Program. Mutation Research 87:81–142 (1981).

(4) Clive, D. et al. Validation and characterization of the L5178Y TK ¶/

· mouse lymphoma mutagen assay system. Mutation Research 59:61–108 (1979).
(5) Clive, D. and Spector, J.F.S. Laboratory procedures for assessing specific locus mutations at the TK locus in cultured L5178Y mouse lymphoma cells. Mutation Research 31:17–29 (1975).
(6) Hsie, A.W. et al. The use of Chinese hamster ovary cells to quantify specific locus mutation and to determine mutagenicity of chemicals: a report of the U.S. EPA’s Gene-Tox Program. Mutation Research 86:193– 214 (1981).