[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: 40CFR799.9530]
[Page 423-429]
TITLE 40--PROTECTION OF ENVIRONMENT
CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
PART 799_IDENTIFICATION OF SPECIFIC CHEMICAL SUBSTANCE AND MIXTURE
TESTING REQUIREMENTS--Table of Contents
Subpart H_Health Effects Test Guidelines
Sec. 799.9530 TSCA in vitro mammalian cell gene mutation test.
(a) Scope. This section is intended to meet the testing requirements
under section 4 of TSCA. The in vitro mammalian cell gene mutation test
can be used to detect gene mutations induced by chemical substances.
Suitable cell lines include L5178Y mouse lymphoma cells, the CHO, AS52
and V79 lines of Chinese hamster cells, and TK6 human lymphoblastoid
cells under paragraph (g)(1) of this section. In these cell lines the
most commonly-used genetic endpoints measure mutation at thymidine
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 (For a discussion see the references in paragraphs (g)(2),
(g)(3), (g)(4),(g)(5), and (g)(6) of this section).
(b) Source. The source material used in developing this TSCA test
guideline is the OECD guideline 476 (February 1997). This source is
available at the address in paragraph (g) of this section.
(c) Definitions. The following definitions apply to this section:
Base pair substitution mutagens are substances which cause
substitution 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
deletion 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
compared to a control population of cells; calculated as the product of
suspension growth relative to the negative control times cloning
efficiency relative 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, osmolality or high levels of
cytotoxicity.
(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 mechanisms or mechanisms absent in bacterial cells.
(e) Test method--(1) Principle. (i) Cells deficient in thymidine
kinase (TK) due
[[Page 424]]
to the mutation TK=/- -<= TK-/
- 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 suspected selective
toxicity by the test substance for mutant and non-mutant cells should be
investigated. Thus, performance of the selection system/agent shall be
confirmed when testing chemicals structurally related to the selective
agent.
(ii) Cells in suspension or monolayer culture shall be exposed to
the 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. Cytotoxicity is
usually determined by measuring the relative cloning efficiency
(survival) or relative total growth of the cultures after the treatment
period. The treated cultures shall be 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 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 (viability). After a suitable incubation time, colonies shall
be counted. The mutant frequency is derived from the number of mutant
colonies in selective medium and the number of colonies in non-selective
medium.
(2) Description--(i) Preparations--(A) Cells. (1) 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
efficiency and a stable spontaneous mutant frequency. Cells should be
checked for mycoplasma contamination and should not be used if
contaminated.
(2) 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 parameters
discussed in the reference under paragraph (g)(13) of this section may
be used. The minimal number of viable cells surviving 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 number which is at least ten
times the inverse of the spontaneous mutation frequency. However, it is
recommended to utilize at least 10\6\ cells. Adequate 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, CO2
concentration and humidity) shall be used. Media should be chosen
according to the selective 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
cultures, seeded in culture medium and incubated at 37 [deg]C. Prior to
use in this test, cultures may need to be cleansed of pre-existing
mutant cells.
(D) Metabolic activation. Cells shall be exposed to the test
substance both in the presence and absence of an appropriate metabolic
activation 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 or a
combination of phenobarbitone and [beta]-naphthoflavone. The post-
mitochondrial fraction is usually used at concentrations in the range
from 1-10% v/v in the final test
[[Page 425]]
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 activating
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
dissolved or suspended in appropriate solvents or vehicles and diluted
if appropriate 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 acceptability of storage.
(ii) Test conditions--(A) Solvent/vehicle. The solvent/vehicle shall
not be suspected of chemical reaction with the test substance and shall
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 supported by data indicating their compatibility. It is recommended
that wherever 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. (1) Among the criteria to be considered
when determining the highest concentration are cytotoxicity and
solubility in the test system and changes in pH or osmolality.
(2) Cytotoxicity should be determined with and without metabolic
activation in the main experiment using an appropriate indicator of cell
integrity 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 analyzable concentrations shall be used. Where
there is cytotoxicity, these concentrations shall cover a range from the
maximum to little or no toxicity; this will usually mean that the
concentration levels should be separated by no more than a factor
between 2 and [radic]10. If the maximum concentration is based on
cytotoxicity then it shall result in approximately 10-20% but not less
than 10% relative survival (relative cloning efficiency) or relative
total growth. For relatively non-cytotoxic compounds the maximum
concentration should be 5 mg/ml, 5 [micro]l/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 interfere with the scoring.
(C) Controls. (1) Concurrent positive and negative (solvent or
vehicle) controls both with and without metabolic activation shall be
included in each experiment. When metabolic activation is used the
positive control chemical shall be one that requires activation to give
a mutagenic response.
(2) Examples of positive control substances include:
----------------------------------------------------------------------------------------------------------------
Metabolic Activation condition Locus Chemical CAS No.
----------------------------------------------------------------------------------------------------------------
Absence of exogenous metabolic HPRT................... Ethylmethanesulfonate.. [CAS no. 62-50-0]
activation
....................... Ethylnitrosourea....... [CAS no. 759-73-9]
TK (small and large Methylmethanesulfonate. [CAS no. 66-27-3]
colonies).
XPRT................... Ethylmethanesulfonate.. [CAS no. 62-50-0]
Ethylnitrosourea....... [CAS no. 759-73-9]
[[Page 426]]
Presence of exogenous metabolic HPRT................... 3-Methylcholanthrene... [CAS no. 56-49-5]
activation.
N-Nitrosodimethylamine. [CAS no. 62-75-9]
7,12- [CAS no. 57-97-6]
Dimethylbenzanthracene.
TK (small and large Cyclophosphamide [CAS no. 50-18-0]
colonies). (monohydrate). [CAS no. 6055-19-2]
Benzo(a)pyrene......... [CAS no. 50-32-8]
3-Methylcholanthrene... [CAS no. 56-49-5]
XPRT................... N-Nitrosodimethylamine [CAS no. 62-75-9]
(for high levels of S-
9).
Benzo(a)pyrene......... [CAS no. 50-32-8]
----------------------------------------------------------------------------------------------------------------
(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
shall 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.
(3) Procedure--(i) Treatment with test substance. (A) Proliferating
cells shall be exposed to the test substance both with and without
metabolic activation. Exposure shall be for a suitable period of time
(usually 3 to 6 hrs 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
concentrations should be increased to ensure an adequate number of
cultures for analysis (e.g. at least eight analyzsable concentrations).
Duplicate negative (solvent) control cultures should be used.
(C) Gaseous or volatile substances should be tested by appropriate
methods, such as in sealed culture vessels. Methods described in the
references under paragraphs (g)(20) and (g)(21) of this section may be
used.
(ii) Measurement of survival, viability, and mutant frequency. (A)
At the end of the exposure period, cells shall 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 numbers
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=/
- test, 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=/- test, colony sizing should be
performed on the negative and positive controls. In studies using
TK6TK=/-, colony sizing may also be performed.
(f) Data and reporting--(1) Treatment of results. (i) Data shall
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=/- test,
colonies are scored using the criteria of small and large colonies on at
least one concentration of the test substance (highest positive
concentration) and on the negative and positive control. The molecular
and cytogenetic nature of both large and small colony mutants
[[Page 427]]
has been explored in detail and is discussed in the references under
paragraphs (g)(22) and (g)(23) of this section. In the TK=/
- test, colonies are scored using the criteria of normal
growth (large) and slow growth (small) colonies (a scoring system
similar to the one described in the reference under paragraph (g)(24) of
this section may be used). Mutant cells that have suffered the most
extensive genetic damage have prolonged doubling times and thus form
small colonies. This damage typically ranges in scale from the losses of
the entire gene to karyotypically visible chromosome aberrations. The
induction of small colony mutants has been associated with chemicals
that induce gross chromosome aberrations. Less seriously affected mutant
cells grow at rates similar to the parental cells and form large
colonies.
(ii) Survival (relative cloning efficiencies) or relative total
growth shall be given. Mutant frequency shall be expressed as number of
mutant cells per number of surviving cells.
(iii) Individual culture data shall be provided. Additionally, all
data shall be summarized in tabular form.
(iv) There is no requirement for verification of a clear positive
response. Equivocal results shall 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 confirmation of negative results is not considered necessary,
justification should be provided. Modification of study parameters to
extend the range of conditions assessed should be considered in follow-
up experiments for either equivocal 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
criteria described in paragraph (f)(2)(i) of this section is considered
non-mutagenic in this system.
(iii) Although most studies will give clearly positive or negative
results, 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
reproducible is most meaningful. Negative results indicate that, under
the test conditions, the test substance does not induce gene mutations
in the cultured mammalian cells used.
(3) Test report. The test report shall include the following
information:
(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 of mycoplasma.
(iv) Test conditions:
(A) Rationale for selection of concentrations and number of cell
cultures including e.g., cytotoxicity data and solubility limitations,
if available.
(B) Composition of media, CO2 concentration.
(C) Concentration of test substance.
(D) Volume of vehicle and test substance added.
[[Page 428]]
(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.
(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
(including 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
substance, 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=/- 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. For additional background information on this test
guideline, the following references should be consulted. These
references are available for inspection at the TSCA Nonconfidential
Information Center, Rm. NE-B607, Environmental Protection Agency, 401 M
St., SW., Washington, DC, 12 noon to 4 p.m., Monday through Friday,
except legal holidays.
(1) Chu, E.H.Y. and Malling, H.V. Mammalian Cell Genetics. II.
Chemical Induction of Specific Locus Mutations in Chinese Hamster Cells
In Vitro, Proc. National Academy Science (USA, 1968) 61, 1306-1312.
(2) Liber, H.L. and Thilly, W.G. Mutation Assay at the Thymidine
Kinase Locus in Diploid Human Lymphoblasts. Mutation Research. 94, 467-
485 (1982).
(3) Moore, M.M., Harrington-Brock, K., Doerr, C.L., and Dearfield,
K.L. Differential Mutant Quantitation at the Mouse Lymphoma TK and CHO
HGPRT Loci. Mutagenesis. 4, 394-403 (1989).
(4) 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).
(5) Aaron, C.S., Bolcsfoldi, G., Glatt, H.R., Moore, M., Nishi, Y.,
Stankowski, L., Theiss, J., and Thompson, E. Mammalian Cell Gene
Mutation Assays Working Group Report. Report of the International
Workshop on Standardization of Genotoxicity Test Procedures. Mutation
Research. 312, 235-239 (1994).
(6) Scott, D., Galloway, S.M., Marshall, R.R., Ishidate, M.,
Brusick, D., Ashby, J., and Myhr, B.C. Genotoxicity Under Extreme
Culture Conditions. A report from ICPEMC Task Group 9. Mutation
Research. 257, 147-204 (1991).
(7) Clive, D., McCuen, R., Spector, J.F.S., Piper, C., and
Mavournin, K.H. Specific Gene Mutations in L5178Y Cells in Culture. A
Report of the U.S. Environmental Protection Agency Gene-Tox Program.
Mutation Research. 115, 225-251 (1983).
(8) Li, A.P., Gupta, R.S., Heflich, R.H., and Wasson, J. S. A Review
and Analysis of the Chinese Hamster Ovary/Hypoxanthine Guanine
Phosphoribosyl Transferase System to Determine 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).
(9) Li, A.P., Carver, J.H., Choy, W.N., Hsie, A.W., Gupta, R.S.,
Loveday, K.S., O'Neill, J.P., Riddle, J.C., Stankowski, Jr., L.F., and
Yang, L.L. A Guide for the Performance of the Chinese Hamster Ovary
Cell/Hypoxanthine-Guanine Phosphoribosyl Transferase Gene Mutation
Assay. Mutation Research. 189, 135-141 (1987).
(10) Liber, H.L., Yandell, D.W., and Little, J.B. A Comparison of
Mutation
[[Page 429]]
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).
(11) 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 Mutation in AS52 Cells. Mutation Reseach. 160, 133-147 (1986).
(12) Turner, N.T., Batson, A.G., and Clive, D. Eds. Kilbey, B.J. et
al. Procedures for the L5178Y/TK=/-
TK=/- Mouse Lymphoma Cell Mutagenicity Assay.
Handbook of Mutagenicity Test Procedures (Elsevier Science Publishers,
New York, 1984) pp. 239-268.
(13) Arlett, C.F., Smith, D.M., Clarke, G.M., Green, M.H.L., Cole,
J., McGregor, D.B., and Asquith, J.C. Ed. Kirkland, D.J. Mammalian Cell
Gene Mutation Assays Based Upon Colony Formation. Statistical Evaluation
of Mutagenicity Test Data (Cambridge University Press, 1989) pp. 66-101.
(14) Abbondandolo, A., Bonatti, S., Corti, G., Fiorio, R., Loprieno,
N., and Mazzaccaro, A. Induction of 6-Thioguanine-Resistant Mutants in
V79 Chinese Hamster Cells by Mouse-Liver Microsome-Activated
Dimethylnitrosamine. Mutation Research. 46, 365-373 (1977).
(15) Ames, B.N., McCann, J., and Yamasaki, E. Methods for Detecting
Carcinogens and Mutagens with the Salmonella/Mammalian-Microsome
Mutagenicity Test. Mutation Reseach. 31, 347-364 (1975).
(16) Clive, D., Johnson, K.O., Spector, J.F.S., Batson, A.G., and
Brown M.M.M. Validation and Characterization of the L5178Y/
TK=/- Mouse Lymphoma Mutagen Assay System.
Mutation Reseach. 59, 61-108 (1979).
(17) Maron, D.M. and Ames, B.N. Revised Methods for the Salmonella
Mutagenicity Test. Mutation Reseach. 113, 173, 215 (1983).
(18) Elliott, B.M., Combes, R.D., Elcombe, C.R., Gatehouse, D.G.,
Gibson, G.G., Mackay, J.M., and Wolf, R.C. Alternatives to Aroclor 1254-
Induced S9 in In Vitro Genotoxicity Assays. Mutagenesis. 7, 175-177
(1992).
(19) Matsushima, T., Sawamura, M., Hara, K., and Sugimura, T. A Safe
Substitute for Polychlorinated 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 Activation in Mutagenesis Testing
(Elsevier, North-Holland, 1976) pp. 85-88.
(20) Krahn, D.F., Barsky, F.C., and McCooey, K.T. Eds. Tice, R.R.,
Costa, D.L., and Schaich, K.M. CHO/HGPRT Mutation Assay: Evaluation of
Gases and Volatile Liquids. Genotoxic Effects of Airborne Agents (New
York, Plenum, 1982) pp. 91-103.
(21) Zamora, P.O., Benson, J.M., Li, A.P., and Brooks, A.L.
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).
(22) Applegate, M.L., Moore, M.M., Broder, C.B., Burrell, A., and
Hozier, J.C. Molecular Dissection of Mutations at the Heterozygous
Thymidine Kinase Locus in Mouse Lymphoma Cells. Proc. National Academy
Science (USA, 1990) 87, 51-55.
(23) Moore, M.M., Clive, D., Hozier, J.C., Howard, B.E., Batson,
A.G., Turner, N.T., and Sawyer, J. Analysis of Trifluorothymidine-
Resistant (TFT\r\) Mutants of L5178Y/TK=/- Mouse
Lymphoma Cells. Mutation Research. 151, 161-174 (1985).
(24) 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).
(25) Moore, M.M. and Doerr, C.L. Comparison of Chromosome Aberration
Frequency and Small-Colony TK-Deficient Mutant Frequency in L5178Y/
TK=/- 3.7.2C Mouse Lymphoma Cells. Mutagenesis. 5,
609-614 (1990).