[Code of Federal Regulations]
[Title 40, Volume 31]
[Revised as of July 1, 2006]
From the U.S. Government Printing Office via GPO Access
[CITE: 40CFR798.5300]
[Page 192-195]
TITLE 40--PROTECTION OF ENVIRONMENT
CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
PART 798_HEALTH EFFECTS TESTING GUIDELINES--Table of Contents
Subpart F_Genetic Toxicity
Sec. 798.5300 Detection of gene mutations in somatic cells in culture.
(a) 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.
(b) Definitions. (1) 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.
(2) Base pair mutagens are agents which cause a base change in the
DNA.
(3) Frameshift mutagens are agents which cause the addition or
deletion of single or multiple base pairs in the DNA molecule.
(4) Phenotypic expression time is a period during which unaltered
gene products are depleted from newly mutated cells.
(c) Reference substances. These may include, but need not be limited
to, ethyl methanesulfonate, N-nitroso-dimethylamine, 2-
acetylaminofluorene, 7,12-dimethylbenzanthracene or hycanthone.
(d) 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
expression prior to mutant selection. Cells deficient in thymidine
kinase (TK) due to the forward mutation TK=[rarr]
TK- are resistant to the cytotoxic effects of pyrimidine
analogues such as bromodeoxyuridine (BrdU), fluorodeoxyuridine (FdU) or
[[Page 193]]
trifluorothymidine (TFT). The deficiency of 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. 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 presence
of 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-optimal phenotypic expression of induced
mutations. Mutant frequency is determined by seeding known numbers of
cells in medium containing the selective agent to detect mutant cells,
and in medium without selective agent to determine the cloning
efficiency. After a suitable incubation time, cell colonies are counted.
The number of mutant colonies in selective medium is adjusted by the
number of colonies in nonselective medium 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
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, CO2 concentrations,
temperature and humidity) shall be used.
(4) Metabolic activation. Cells shall 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 shall be included in each experiment. When metabolic activation
is used, the positive control substance shall 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 shall 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 4) of the test
substance shall be used. Generally, these shall yield a concentration-
related toxic effect. The highest concentration shall produce a low
level of survival (approximately 10 percent), and the survival in the
lowest concentration shall approximate the negative control.
Cytotoxicity shall 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 under culture conditions. For freely-
soluble nontoxic substances the highest concentration used should be
determined on a case-by-case basis.
(e) Test performance. (1) Cells shall be exposed to the test
substance both with
[[Page 194]]
and without exogenous metabolic activation. Exposure shall be for a
suitable period of time, in most cases 1 to 5 hours is effective;
exposure time may be extended over one or more cell cycles.
(2) At the end of the exposure period, cells shall 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 shall be sufficient
to allow near optimal phenotypic expression of induced mutants, cells
should be grown in medium with and without selective agent(s) for
determination of number of mutants and cloning efficiency, respectively.
(4) Results shall be confirmed in an independent experiment. When
appropriate, a single positive response should be confirmed by testing
over a narrow range of concentrations.
(f) Data and report--(1) Treatment of results. Data shall be
presented in tabular form. Individual colony counts for the treated and
control groups shall be presented for both mutation induction and
survival. Survival and cloning efficiencies shall be given as a
percentage of the controls. Mutant frequency shall be expressed as
number of mutants per number of surviving cells.
(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 concentration-related increase in the mutant frequency.
Another criterion may be 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 as
specified under 40 CFR part 792, subpart J the following specific
information shall 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, CO2
concentration, concentration 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 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.
(g) References. For additional background information on this test
guideline the following references should be consulted:
(1) Amacher, D.E., Paillet, S.C., Ray, V. ``Point mutations at the
thymidine kinase locus in L5178Y mouse lymphoma cells. I. Application to
genetic toxicology testing,'' Mutation Research, 64:391-406 (1979).
(2) Amacher, D.E., Paillet, S.C., Turner, G.N., Ray, V.A. Salsburg,
V.A. ``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., Bhuyan B., Francis, M.C., Langenback, R.,
Peterson, A., Huberman, E. ``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).
[[Page 195]]
(4) Clive, D., Johnson, K.O., Spector, J.F.S., Batson, A.G., Brown,
M.M. ``Validation and characterization of the L5178Y TK=/
- mouse lymphoma mutagen assay system,'' Mutation Research,
59:61-108 (1979).
(5) Clive, D., 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., Casciano, D.A., Couch, D.B., Krahn, D.F., O'Neill,
J.P., Whitfield, B.L. ``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).
[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19079, May 20, 1987]