United States Prevention, Pesticides EPA712-C-98-221
Environmental Protection and Toxic Substances August 1998
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.5300
In Vitro Mammalian Cell
Gene Mutation Test
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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.).
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on disks or paper
copies: call (202) 512-0132. This guideline is also available electronically
in PDF (portable document format) from EPA's World Wide Web site
(http://www.epa.gov/epahome/research.htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelines/OPPTS Harmonized Test
Guidelines."
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OPPTS 870.5300 In vitro mammalian cell gene mutation test.
(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 798.5300 Detection of
gene mutations in somatic cells in culture and OECD 476, In Vitro Mam-
malian Cell Gene Mutation Test.
(b) Introduction. The in vitro mammalian cell gene mutation test
can be used to detect gene mutations induced by chemical substances. Suit-
able cell lines include L5178Y mouse lymphoma cells, the CHO, AS52
and V79 lines of Chinese hamster cells, and TK6 human lymphoblastoid
cells (see reference in paragraph (g)(l) of this guideline). In these cell
lines the most commonly-used genetic endpoints measure mutation at thy-
midine kinase (TK) and hypoxanthine-guanine phosphoribosyl transferase
(HPRT), and a transgene of xanthine-guanine phosphoribosyl transferase
(XPRT). The TK, HPRT and XPRT mutation tests detect different spectra
of genetic events. The autosomal location of TK and XPRT may allow
the detection of genetic events (e.g., large deletions) not detected at the
HPRT locus on X-chromosomes (see references in paragraphs (g)(2),
(g)(3), (g)(4),(g)(5), and (g)(6) of this guideline).
(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 substitution mutagens are substances which cause substi-
tution of one or several base pairs in the DNA.
Forward mutation is a gene mutation from the parental type to the
mutant form which gives rise to an alteration or a loss of the enzymatic
activity or the function of the encoded protein.
Frameshift mutagens are substances which cause the addition or dele-
tion of single or multiple base pairs in the DNA molecule.
Mutant frequency is the number of mutant cells observed divided by
the number of viable cells.
Phenotypic expression time is a period during which unaltered gene
products are depleted from newly mutated cells.
Relative suspension growth is an increase in cell number over the
expression period relative to the negative control.
Relative total growth is an increase in cell number over time com-
pared to a control population of cells; calculated as the product of suspen-
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sion growth relative to the negative control times cloning efficiency rel-
ative to negative control.
Survival is the cloning efficiency of the treated cells when plated at
the end of the treatment period; survival is usually expressed in relation
to the survival of the control cell population.
Viability is the cloning efficiency of the treated cells at the time of
plating in selective conditions after the expression period.
(d) Initial considerations. (1) In the in vitro mammalian cell gene
mutation test, cultures of established cell lines or cell strains can be used.
The cells used are selected on the basis of growth ability in culture and
stability of the spontaneous mutation frequency. Tests conducted in vitro
generally require the use of an exogenous source of metabolic activation.
This metabolic activation system cannot mimic entirely the mammalian
in vivo conditions. Care should be taken to avoid conditions which would
lead to results not reflecting intrinsic mutagenicity. Positive results which
do not reflect intrinsic mutagenicity may arise from changes in pH, osmo-
lality or high levels of cytotoxicity (see reference in paragraph (g)(7) of
this guideline).
(2) This test is used to screen for possible mammalian mutagens and
carcinogens. Many compounds that are positive in this test are mammalian
carcinogens; however, there is not a perfect correlation between this test
and carcinogenicity. Correlation is dependent on chemical class and there
is increasing evidence that there are carcinogens that are not detected by
this test because they appear to act through other, non-genotoxic mecha-
nisms or mechanisms absent in bacterial cells (see reference in paragraph
(g)(6) of this guideline).
(e) Test method—(1) Principle, (i) Cells deficient in thymidine ki-
nase (TK) due to the mutation TK+/- -> TK-A are resistant to the cytotoxic
effects of the pyrimidine analogue trifluorothymidine (TFT). Thymidine
kinase proficient cells are sensitive to TFT, which causes the inhibition
of cellular metabolism and halts further cell division. Thus mutant cells
are able to proliferate in the presence of TFT, whereas normal cells, which
contain thymidine kinase, are not. Similarly, cells deficient in HPRT or
XPRT are selected by resistance to 6-thioguanine (TG) or 8-azaguanine
(AG). The properties of the test substance should be considered carefully
if a base analogue or a compound related to the selective agent is tested
in any of the mammalian cell gene mutation tests. For example, any sus-
pected selective toxicity by the test substance for mutant and non-mutant
cells should be investigated. Thus, performance of the selection system/
agent must be confirmed when testing chemicals structurally related to
the selective agent (see reference in paragraph (g)(8) of this guideline).
(ii) Cells in suspension or monolayer culture are exposed to the test
substance, both with and without metabolic activation, for a suitable period
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of time and subcultured to determine cytotoxicity and to allow phenotypic
expression prior to mutant selection (see references in paragraphs (g)(9),
(g)(10), (g)(ll), (g)(12), and (g)(13) of this guideline). Cytotoxicity is usu-
ally determined by measuring the relative cloning efficiency (survival) or
relative total growth 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 and cell type, to allow near-optimal
phenotypic expression of induced mutations. Mutant frequency is deter-
mined by seeding known numbers of cells in medium containing the selec-
tive agent to detect mutant cells, and in medium without selective agent
to determine the cloning efficiency (viability). After a suitable incubation
time, colonies are counted. The mutant frequency is derived from the num-
ber of mutant colonies in selective medium and the number of colonies
in non-selective medium.
(2) Description—(i) Preparations—(A) Cells. (7) A variety of cell
types are available for use in this test including subclones of L5178Y,
CHO, CHO-AS52, V79, or TK6 cells. Cell types used in this test should
have a demonstrated sensitivity to chemical mutagens, a high cloning effi-
ciency and a stable spontaneous mutant frequency. Cells should be
checked for Mycoplasma contamination and should not be used if contami-
nated.
(2) The test should be designed to have a predetermined sensitivity
and power. The number of cells, cultures and concentrations of test sub-
stance used should reflect these defined parameters (see reference in para-
graph (g)(14) of this guideline). The minimal number of viable cells sur-
viving treatment and used at each stage in the test should be based on
the spontaneous mutation frequency. A general guide is to use a cell num-
ber which is at least ten times the inverse of the spontaneous mutation
frequency. However, it is recommended to utilise at least 106 cells. Ade-
quate historical data on the cell system used should be available to indicate
consistent performance of the test.
(B) Media and culture conditions. Appropriate culture media and
incubation conditions (culture vessels, temperature, CC>2 concentration and
humidity) should be used. Media should be chosen according to the selec-
tive systems and cell type used in the test. It is particularly important that
culture conditions should be chosen that ensure optimal growth of cells
during the expression period and colony forming ability of both mutant
and non-mutant cells.
(C) Preparation of cultures. Cells are propagated from stock cul-
tures, seeded in culture medium and incubated at 37 °C. Prior to use in
this test, cultures may need to be cleansed of pre-existing mutant cells.
(D) Metabolic activation. Cells should be exposed to the test sub-
stance both in the presence and absence of an appropriate metabolic activa-
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tion system. The most commonly used system is a co-factor-supplemented
post-mitochondrial fraction (S9) prepared from the livers of rodents treated
with enzyme-inducing agents such as Aroclor 1254 (see references in para-
graphs (g)(15), (g)(16), (g)(17), and (g)(18) of this guideline) or a com-
bination of phenobarbitone and p-naphthoflavone (see references in para-
graphs (g)(19) and (g)(20) of this guideline). The post-mitochondrial frac-
tion is usually used at concentrations in the range from 1-10 percent
v/v in the final test medium. The choice and condition of a metabolic
activation system may depend upon the class of chemical being tested.
In some cases it may be appropriate to utilize more than one concentration
of post-mitochondrial fraction. A number of developments, including the
construction of genetically engineered cell lines expressing specific activat-
ing enzymes, may provide the potential for endogenous activation. The
choice of the cell lines used should be scientifically justified (e.g., by the
relevance of the cytochrome P450 isoenzyme to the metabolism of the
test substance).
(E) Test substance/preparations. Solid test substances should be dis-
solved or suspended in appropriate solvents or vehicles and diluted if ap-
propriate prior to treatment of the cells. Liquid test substances may be
added directly to the test systems and/or diluted prior to treatment. Fresh
preparations should be employed unless stability data demonstrate the ac-
ceptability of storage.
(ii) Test conditions—(A) Solvent/vehicle. The solvent/vehicle should
not be suspected of chemical reaction with the test substance and should
be compatible with the survival of the cells and the S9 activity. If other
than well-known solvent/vehicles are used, their inclusion should be sup-
ported by data indicating their compatibility. It is recommended that wher-
ever possible, the use of an aqueous solvent/vehicle be considered first.
When testing water-unstable substances, the organic solvents used should
be free of water. Water can be removed by adding a molecular sieve.
(B) Exposure concentrations. (7) Among the criteria to be consid-
ered when determining the highest concentration are cytotoxicity and solu-
bility in the test system and changes in pH or osmolality.
(2) Cytotoxicity should be determined with and without metabolic ac-
tivation in the main experiment using an appropriate indicator of cell integ-
rity and growth, such as relative cloning efficiency (survival) or relative
total growth. It may be useful to determine cytotoxicity and solubility in
a preliminary experiment.
(3) At least four analysable concentrations should be used. Where
there is cytotoxicity, these concentrations should cover a range from the
maximum to little or no toxicity; this will usually mean that the concentra-
tion levels should be separated by no more than a factor between 2 and
VlO. If the maximum concentration is based on cytotoxicity then it should
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result in approximately 10-20 percent but not less than 10 percent relative
survival (relative cloning efficiency) or relative total growth. For relatively
non-cytotoxic compounds the maximum concentration should be
5 mg/ml, 5(il/ml, or 0.01 M, whichever is the lowest.
(4) Relatively insoluble substances should be tested up to or beyond
their limit of solubility under culture conditions. Evidence of insolubility
should be determined in the final treatment medium to which cells are
exposed. It may be useful to assess solubility at the beginning and end
of the treatment, as solubility can change during the course of exposure
in the test system due to presence of cells, S9, serum etc. Insolubility
can be detected by using the unaided eye. The precipitate should not inter-
fere with the scoring.
(C) Controls. (7) Concurrent positive and negative (solvent or vehi-
cle) controls both with and without metabolic activation should be included
in each experiment. When metabolic activation is used the positive control
chemical should be one that requires activation to give a mutagenic re-
sponse.
(2) Examples of positive control substances include:
Metabolic Activation condition
Absence of exogenous meta-
bolic activation
Presence of exogenous meta-
bolic activation.
Locus
HPRT
TK (small and
large colonies).
XPRT
HPRT
TK (small and
large colonies).
XPRT
Chemical
Ethylmethanesulfonate
Ethylnitrosourea
Methylmethanesulfonate
Ethylmethanesulfonate
Ethylnitrosourea
3-Methylcholanthrene
N-Nitrosodimethylamine
7,12-Dimethylbenzanthracene
Cyclophosphamide
(monohydrate).
Benzo(a)pyrene
3-Methylcholanthrene
N-Nitrosodimethylamine
(for high levels of S-9).
Benzo(a)ovrene
CAS number
[62-50-0]
[759-73-9]
[66_27-3]
[62-50-0]
[759-73-9]
[56-49-5]
[62-75-9]
[57-97-6]
[50-18-0]
([6055-19-2])
[50-32-8]
[56-49-5]
[62-75-9]
[50-32-81
(3) Other appropriate positive control reference substances may be
used, e.g., if a laboratory has a historical data base on 5-Bromo 2'-
deoxyuridine [CAS no. 59-14-3], this reference substance could be used
as well. The use of chemical class-related positive control chemicals may
be considered, when available.
(4) Negative controls, consisting of solvent or vehicle alone in the
treatment medium, and treated in the same way as the treatment groups
should be included. In addition, untreated controls should also be used
unless there are historical control data demonstrating that no deleterious
or mutagenic effects are induced by the chosen solvent.
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(3) Procedure—(i) Treatment with test substance. (A) Proliferating
cells should be exposed to the test substance both with and without meta-
bolic activation. Exposure should be for a suitable period of time (usually
3 to 6 hours is effective). Exposure time may be extended over one or
more cell cycles.
(B) Either duplicate or single treated cultures may be used at each
concentration tested. When single cultures are used, the number of con-
centrations should be increased to ensure an adequate number of cultures
for analysis (e.g., at least eight analysable concentrations). Duplicate nega-
tive (solvent) control cultures should be used.
(C) Gaseous or volatile substances should be tested by appropriate
methods, such as in sealed culture vessels (see references in paragraphs
(g)(21) and (g)(22) of this guideline).
(ii) Measurement of survival, viability, and mutant frequency. (A)
At the end of the exposure period, cells should be washed and cultured
to determine survival and to allow for expression of the mutant phenotype.
Measurement of cytotoxicity by determining the relative cloning efficiency
(survival) or relative total growth of the cultures is usually initiated after
the treatment period.
(B) Each locus has a defined minimum time requirement to allow
near optimal phenotypic expression of newly induced mutants (HPRT and
XPRT require at least 6-8 days, and TK at least 2 days). Cells are grown
in medium with and without selective agent(s) for determination of num-
bers of mutants and cloning efficiency, respectively. The measurement of
viability (used to calculate mutant frequency) is initiated at the end of
the expression time by plating in non-selective medium.
(C) If the test substance is positive in the L5178Y TK+Atest, colony
sizing should be performed on at least one of the test cultures (the highest
positive concentration) and on the negative and positive controls. If the
test substance is negative in the L5178Y TK+Atest, colony sizing should
be performed on the negative and positive controls. In studies using
TK6TK+A, colony sizing may also beperformed.
(f) Data and reporting—(1) Treatment of results, (i) Data should
include cytotoxicity and viability determination, colony counts and mutant
frequencies for the treated and control cultures. In the case of a positive
response in the L5178Y TK+Atest, colonies are scored using the criteria
of small and large colonies on at least one concentration of the test sub-
stance (highest positive concentration) and on the negative and positive
control. The molecular and cytogenetic nature of both large and small col-
ony mutants has been explored in detail (see references in paragraphs
(g)(23) and (g)(24) of this guideline). In the TK+Atest, colonies are scored
using the criteria of normal growth (large) and slow growth (small) colo-
nies (see reference in paragraph (g)(25) of this guideline). Mutant cells
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that have suffered the most extensive genetic damage have prolonged dou-
bling times and thus form small colonies. This damage typically ranges
in scale from the losses of the entire gene to karyotypically visible chro-
mosome aberrations. The induction of small colony mutants has been asso-
ciated with chemicals that induce gross chromosome aberrations (see ref-
erence in paragraph (g)(26) of this guideline). Less seriously affected mu-
tant cells grow at rates similar to the parental cells and form large colonies.
(ii) Survival (relative cloning efficiencies) or relative total growth
should be given. Mutant frequency should be expressed as number of mu-
tant cells per number of surviving cells.
(iii) Individual culture data should be provided. Additionally, all data
should be summarized in tabular form.
(iv) There is no requirement for verification of a clear positive re-
sponse. Equivocal results should be clarified by further testing preferably
using a modification of experimental conditions. Negative results need to
be confirmed on a case-by-case basis. In those cases where confimation
of negative results is not considered necessary, justification should be pro-
vided. Modification of study parameters to extend the range of conditions
assessed should be considered in follow-up experiments for either equivo-
cal or negative results. Study parameters that might be modified include
the concentration spacing, and the metabolic activation conditions.
(2) Evaluation and interpretation of results, (i) There are several
criteria for determining a positive result, such as a concentration-related,
or a reproducible increase in mutant frequency. Biological relevance of
the results should be considered first. Statistical methods may be used as
an aid in evaluating the test results. Statistical significance should not be
the only determining factor for a positive response.
(ii) A test substance, for which the results do not meet the above
criteria is considered non-mutagenic in this system.
(iii) Although most studies will give clearly positive or negative re-
sults, in rare cases the data set will preclude making a definite judgement
about the activity of the test substance. Results may remain equivocal or
questionable regardless of the number of times the experiment is repeated.
(iv) Positive results for an in vitro mammalian cell gene mutation
test indicate that the test substance induces gene mutations in the cultured
mammalian cells used. A positive concentration-response that is reproduc-
ible is most meaningful. Negative results indicate that, under the test con-
ditions, the test substance does not induce gene mutations in the cultured
mammalian cells used.
(3) Test report. The test report should include the following informa-
tion:
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(i) Test substance:
(A) Identification data and CAS no., if known.
(B) Physical nature and purity.
(C) Physicochemical properties relevant to the conduct of the study.
(D) Stability of the test substance.
(ii) Solvent/vehicle:
(A) Justification for choice of vehicle/solvent.
(B) Solubility and stability of the test substance in solvent/vehicle,
if known.
(iii) Cells:
(A) Type and source of cells.
(B) Number of cell cultures.
(C) Number of cell passages, if applicable.
(D) Methods for maintenance of cell cultures, if applicable.
(E) Absence ofMycoplasma.
(iv) Test conditions:
(A) Rationale for selection of concentrations and number of cell cul-
tures including e.g., cytotoxicity data and solubility limitations, if avail-
able.
(B) Composition of media, CCh concentration.
(C) Concentration of test substance.
(D) Volume of vehicle and test substance added.
(E) Incubation temperature.
(F) Incubation time.
(G) Duration of treatment.
(H) Cell density during treatment.
(I) Type and composition of metabolic activation system including
acceptability criteria.
(J) Positive and negative controls.
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(K) Length of expression period (including number of cells seeded,
and subcultures and feeding schedules, if appropriate).
(L) Selective agent(s).
(M) Criteria for considering tests as positive, negative or equivocal.
(N) Methods used to enumerate numbers of viable and mutant cells.
(O) Definition of colonies of which size and type are considered (in-
cluding criteria for "small" and "large" colonies, as appropriate).
(v) Results:
(A) Signs of toxicity.
(B) Signs of precipitation.
(C) Data on pH and osmolality during the exposure to the test sub-
stance, if determined.
(D) Colony size if scored for at least negative and positive controls.
(E) Laboratory's adequacy to detect small colony mutants with the
L5178Y TK+A system, where appropriate.
(F) Dose-response relationship, where possible.
(G) Statistical analyses, if any.
(H) Concurrent negative (solvent/vehicle) and positive control data.
(I) Historical negative (solvent/vehicle) and positive control data with
ranges, means, and standard deviations.
(J) Mutant frequency.
(vi) Discussion of the results.
(vii) Conclusion.
(g) References. The following references should be consulted for ad-
ditional background information on this test guideline.
(1) Moore, M.M., DeMarini, D.M., DeSerres, F.J., and Tindall, K.R.
(Eds.) Banbury Report 28: Mammalian Cell Mutagenesis, Cold Spring
Harbor Laboratory (New York, New York, 1987).
(2) Chu, E.H.Y. and Mailing, H.V. Mammalian Cell Genetics. II.
Chemical Induction of Specific Locus Mutations in Chinese Hamster Cells
In Vitro, Proceedings of the National Academy of Science USA, 61, 1306-
1312(1968).
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(3) Liber, H.L. and Thilly, W.G. Mutation Assay at the Thymidine
Kinase Locus in Diploid Human Lymphoblasts. Mutation Research 94,
467-485 (1982).
(4) Moore, M.M. et al. Differential Mutant Quantitation at the Mouse
Lymphoma TK and CHO HGPRT Loci. Mutagenesis 4, 394-403 (1989).
(5) Aaron, C.S. and Stankowski, Jr., L.F. Comparison of the AS52/
XPRT and the CHO/HPRT Assays: Evaluation of Six Drug Candidates.
Mutation Research 223, 121-128 (1989).
(6) Aaron, C.S. et al. Mammalian Cell Gene Mutation Assays Work-
ing Group Report. Report of the International Workshop on Standardiza-
tion of Genotoxicity Test Procedures. Mutation Research 312, 235-239
(1994).
(7) Scott, D. et al. Genotoxicity Under Extreme Culture Conditions.
A report from ICPEMC Task Group 9. Mutation Research 257, 147-204
(1991).
(8) Clive, D. et al. Specific Gene Mutations in L5178Y Cells in Cul-
ture. A Report of the U.S. Environmental Protection Agency Gene-Tox
Program. Mutation Research 115, 225-251 (1983).
(9) Li, A.P. et al. A Review and Analysis of the Chinese Hamster
Ovary/Hypoxanthine Guanine Phosphoribosyl Transferase System to De-
termine the Mutagenicity of Chemical Agents: A Report of Phase III of
the U.S. Environmental Protection Agency Gene-Tox Program. Mutation
Research 196, 17-36 (1988).
(10) Li, A.P. et al. A Guide for the Performance of the Chinese Ham-
ster Ovary Cell/Hypoxanthine-Guanine Phosphoribosyl Transferase Gene
Mutation Assay. Mutation Research 189, 135-141 (1987).
(11) Liber, H.L., Yandell, D.W., and Little, J.B. A Comparison of
Mutation Induction at the tk and hprt Loci in Human Lymphoblastoid
Cells; Quantitative Differences are Due to an Additional Class of
Mutations at the Autosomal TK Locus. Mutation Research 216, 9-17
(1989).
(12) Stankowski, L.F. Jr., Tindall, K.R., and Hsie, A.W. Quantitative
and Molecular Analyses of Ethyl Methanesulfonate- and ICR 191-Induced
Molecular Analyses of Ethyl Methanesulfonate- and ICR 191-Induced Mu-
tation in AS52 Cells. Mutation Research 160, 133-147 (1986).
(13) Turner, N.T., Batson, A.G., and Clive, D. Procedures for the
L5178/TK+/-> TK+/-Mouse Lymphoma Cell Mutagenicity Assay. (Eds.)
Kilbey, B.J. et al. Handbook of Mutagenicity Test Procedures (Elsevier
Science Publishers, New York, 1984) pp. 239-268.
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(14) Arlett, C.F. et al. Mammalian Cell Gene Mutation Assays Based
Upon Colony Formation. (Ed.) Kirkland, D.J. Statistical Evaluation of Mu-
tagenicity Test Data (Cambridge University Press, 1989) pp. 66-101.
(15) Abbondandolo, A. et al. Induction of 6-Thioguanine-Resistant
Mutants in V79 Chinese Hamster Cells by Mouse-Liver Microsome-Acti-
vated Dimethylnitrosamine. Mutation Research 46, 365-373 (1977).
(16) Ames, B.N., McCann, J., and Yamasaki, E. Methods for Detect-
ing Carcinogens and Mutagens with the Salmonella/Mammalian-
Microsome Mutagenicity Test. Mutation Research 31, 347-364 (1975).
(17) Clive, D. et al. Validation and Characterization of the
L5178Y/TK+AMouse Lymphoma Mutagen Assay System. Mutation Re-
search 59, 61-108 (1979).
(18) Maron, D.M. and Ames, B.N. Revised Methods for the Sal-
monella Mutagenicity Test. Mutation Research 113, 173, 215 (1983).
(19) Elliott, B.M. et al. Alternatives to Aroclor 1254-Induced S9 in
In Vitro Genotoxicity Assays. Mutagenesis 7, 175-177 (1992).
(20) Matsushima, T. et al. A Safe Substitute for Fob/chlorinated
Biphenyls as an Inducer of Metabolic Activation Systems. (Eds.) de Serres,
F.J., Fouts, J.R., Bend, J.R., and Philpot, R.M. In Vitro Metabolic Activa-
tion in Mutagenesis Testing (Elsevier, North-Holland, 1976) pp. 85-88.
(21) Krahn, D.F., Barsky, F.C., and McCooey, K.T. CHO/HGPRT
Mutation Assay: Evaluation of Gases and Volatile Liquids. (Eds.) Tice,
R.R., Costa, D.L., and Schaich, K.M. Genotoxic Effects of Airborne
Agents. (New York, Plenum, 1982) pp. 91-103.
(22) Zamora, P.O. et al. Evaluation of an Exposure System Using
Cells Grown on Collagen Gels for Detecting Highly Volatile Mutagens
in the CHO/HGPRT Mutation Assay. Environmental Mutagenesis 5, 795-
801 (1983).
(23) Applegate, M.L. et al. Molecular Dissection of Mutations at the
Heterozygous Thymidine Kinase Locus in Mouse Lymphoma Cells. Pro-
ceedings of the National Academy of Science USA, 87, 51-55 (1990).
(24) Moore, M.M. et al. Analysis of Trifluorothymidine-Resistant
(TFTr) Mutants of L5178Y/TK+/-Mouse Lymphoma Cells. Mutation Re-
search 151, 161-174(1985).
(25) Yandell, D.W., Dryja, T.P., and Little J.B. Molecular Genetic
Analysis of Recessive Mutations at a Heterozygous Autosomal Locus in
Human Cells. Mutation Research 229, 89-102 (1990).
(26) Moore, M.M. and Doerr, C.L. Comparison of Chromosome Ab-
erration Frequency and Small-Colony TK-Deficient Mutant Frequency in
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L5178Y/TK+/-3.7.2C Mouse Lymphoma Cells. Mutagenesis 5, 609-614
(1990).
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