United States Prevention, Pesticides EPA712-C-96-221
Environmental Protection and Toxic Substances June 1996
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.5300
Detection of Gene
Mutations in Somatic
Cells in Culture
'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 870.5300 Detection of gene mutations in somatic cells in
culture.
(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 material used in developing this har-
monized OPPTS test guideline is OPPT 40 CFR 798.5300 Detection of
Gene Mutations in Somatic Cells in Culture and OECD guideline 476 Ge-
netic 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 measure
mutation at the thymidine kinase (TK), hypoxanthine-guanine-
phosphoribosyl 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 lim-
ited to, ethyl methanesulfonate, TV-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 time
and subcultured to determine cytotoxicity and to allow phenotypic expres-
sion 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
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the "salvage" enzyme thymidine kinase means that these antimetabolites
are not incorporated into cellular nucleotides and the nucleotides needed
for cellular metabolism are obtained solely from de novo synthesis. How-
ever, in the presence of thymidine kinase, BrdU, FdU or TFT are incor-
porated into the nucleotides, resulting in inhibition of cellular metabolism
and cytotoxicity. Thus mutant cells are able to proliferate in the presence
of BrdU, FdU or TFT whereas normal cells, which contain thymidine ki-
nase, 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 pe-
riod of time—characteristic of each selected locus—to allow near-optimal
phenotypic expression of induced mutations. Mutant frequency is deter-
mined by seeding known numbers of cells in media containing the selec-
tive 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, CHO
cells or V-79 cells. Cell types used in this assay should have a dem-
onstrated sensitivity to chemical mutagens, a high cloning efficiency and
a low spontaneous mutation frequency. Cells should be checked for Myco-
plasma contamination and may be periodically checked for karyotype sta-
bility.
(ii) Cell growth and maintenance. Appropriate culture media and
incubation conditions (culture vessels, CO2 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
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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 pa-
rameters. The number of cells per culture is based on the expected back-
ground 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 concentration
should approximate the negative control. Cytotoxicity should be deter-
mined 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 under
culture conditions. For freely-soluble nontoxic substances the highest con-
centration used should be determined on a case-by-case basis.
(f) Test performance. (1) Cells should be exposed to the test sub-
stance 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, cells
should be grown in media with and without selective agent(s) for deter-
mination 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 pre-
sented in tabular form. Individual colony counts for the treated and control
groups should be presented for both mutation induction and survival. Sur-
vival and cloning efficiencies should be given as a percentage of the con-
trols. 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.
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(3) Interpretation of results, (i) There are several criteria for deter-
mining a positive result, one of which is a statistically significant con-
centration-related increase in the mutant frequency. Another criterion may
be based upon detection of a reproducible and statistically significant posi-
tive response for at least one of the test substance concentrations.
(ii) A test substance which does not produce either a statistically sig-
nificant concentration-related increase in the mutant frequency 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.
(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 as
specified under 40 CFR part 792, subpart J, the following specific informa-
tion should be reported:
(i) Cell type used, number of cell cultures, methods used for mainte-
nance of cell cultures.
(ii) Rationale for selection of concentrations and number of cultures.
(iii) Test conditions: composition of media, CCh concentration, con-
centration of test substance, vehicle, incubation temperature, incubation
time, duration of treatment, cell density during treatment, type of metabolic
activation system, positive and negative controls, length of expression pe-
riod (including number of cells seeded and subculture and feeding sched-
ules, 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 ad-
ditional 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).
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(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.H/- 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 quan-
tify 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).
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