ASTM E1280-97(2008)
(Guide)Standard Guide for Performing the Mouse Lymphoma Assay for Mammalian Cell Mutagenicity (Withdrawn 2015)
Standard Guide for Performing the Mouse Lymphoma Assay for Mammalian Cell Mutagenicity (Withdrawn 2015)
SIGNIFICANCE AND USE
This guide is limited to procedures used solely for the testing of substances to determine their mutagenicity and does not apply to other methods and uses such as exploring mechanisms of mutation.
Recent evidence suggests that this assay measures a dual genetic end point; therefore, some discussion of the relationships between mammalian cell mutagenicity testing results and the results observed both in pure gene mutational assays and in cytogenetic assays is necessary. However, it is not the intent of this guide to discuss other relationships between this mammalian cell mutagenicity testing results and the results observed in other tests for mutagenicity and carcinogenicity.
SCOPE
1.1 The purpose and scope of this guide is to present background material and to establish criteria by which protocols and procedures for conducting the L5178Y/TK+/−-3.7.2C mouse lymphoma mutagenicity assay (commonly referred to as the mouse lymphoma assay, (MLA)) can be properly understood and evaluated. This guide is also intended to aid researchers and others to gain a better understanding of the critical elements involved with mammalian cell mutagenicity testing. More specifically, this guide is intended to provide for researchers the accomplishment of the following goals:
1.1.1 Provide an understanding of the critical procedures (steps) in the performance of this mammalian cell mutagenicity test.
1.1.2 Provide generalized criteria by which researchers can evaluate if they are properly performing, utilizing, and interpreting this assay.
1.1.3 Provide criteria by which individuals responsible for evaluating MLA data can determine if the experiments have been properly performed and interpreted.
1.1.4 Provide a basis from which new procedures and developments in testing procedures can be evaluated.
1.1.5 Provide an understanding of the types of genetic damage (that is, gene and chromosome mutation) that may be detected in this mammalian cell mutagenicity test.
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
WITHDRAWN RATIONALE
The purpose and scope of this guide was to present background material and to establish criteria by which protocols and procedures for conducting the L5178Y/TK+/−-3.7.2C mouse lymphoma mutagenicity assay (commonly referred to as the mouse lymphoma assay, (MLA)) could be properly understood and evaluated.
Formerly under the jurisdiction of Committee F04 on Medical and Surgical Materials and Devices, this guide was withdrawn in November 2014. This standard is being withdrawn without replacement due to its limited use by industry.
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NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation: E1280 − 97(Reapproved 2008)
Standard Guide for
Performing the Mouse Lymphoma Assay for Mammalian
Cell Mutagenicity
This standard is issued under the fixed designation E1280; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
INTRODUCTION
This guide was developed at the request of ASTM Subcommittee E47.09 on Biomarkers in order
to aid toxicologists, geneticists, biochemists, other researchers, and interested persons in the
understanding, performance, and analysis of the mammalian cell mutagenicity test that uses the
+/−
TK -3.7.2C strain of L5178Ymouse lymphoma cells. In this rapidly changing area of toxicology, it
is not intended for this guide to replace, alter, or diminish the usefulness of presently available
protocols and procedures.
1. Scope 1.2 The values stated in SI units are to be regarded as
standard. No other units of measurement are included in this
1.1 The purpose and scope of this guide is to present
standard.
background material and to establish criteria by which proto-
+/−
cols and procedures for conducting the L5178Y/TK -3.7.2C
2. Terminology
mouselymphomamutagenicityassay(commonlyreferredtoas
2.1 Definitions:
the mouse lymphoma assay, (MLA)) can be properly under-
2.1.1 clastogen—any agent that is capable of inducing
stood and evaluated. This guide is also intended to aid
chromosome breaks.
researchers and others to gain a better understanding of the
2.1.2 gene mutation—any heritable change whose physical
critical elements involved with mammalian cell mutagenicity
extent is restricted to the limits of a single gene.
testing. More specifically, this guide is intended to provide for
researchers the accomplishment of the following goals:
2.1.3 mutagen—any physical or chemical agent capable of
1.1.1 Provide an understanding of the critical procedures inducing a mutation.
(steps)intheperformanceofthismammaliancellmutagenicity
2.1.4 mutation—any heritable change in the genetic
test.
material, not caused by genetic segregation or genetic
1.1.2 Provide generalized criteria by which researchers can recombination, and that is transmitted to daughter cells.
evaluate if they are properly performing, utilizing, and inter-
2.2 Definitions of Terms Specific to This Standard:
preting this assay.
2.2.1 chromosome mutation—a mutation resulting from a
1.1.3 Provide criteria by which individuals responsible for
structural change to a chromosome involving the gain, loss, or
evaluating MLA data can determine if the experiments have
relocation of chromosome segments. Chromosome mutations
been properly performed and interpreted.
can be either intrachromosomal or interchromosomal.
1.1.4 Provide a basis from which new procedures and
2.2.2 relative suspension growth (RSG)—used to measure
developments in testing procedures can be evaluated.
the cytotoxicity of a given treatment based on the growth of
1.1.5 Provide an understanding of the types of genetic
cells in suspension culture relative to the untreated or solvent
damage (that is, gene and chromosome mutation) that may be control(s). RSG is calculated according to the method of Clive
detected in this mammalian cell mutagenicity test.
and Spector (1).
2.2.3 relative total growth (RTG)—used as a means to
measure the relative toxicity to cells (survival) following
treatment in the mouse lymphoma assay. RTG is calculated
This guide is under the jurisdiction of Committee F04on Medical and Surgical
Materials and Devices and is the direct responsibility of Subcommittee F04.16 on
Biocompatibility Test Methods.
Current edition approved Aug. 1, 2008. Published August 2008. Originally
approved in 1989. Last previous edition approved in 2003 as E1280–97 (2003). Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
DOI: 10.1520/E1280-97R08. this guide.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1280 − 97 (2008)
according to the method of Clive and Spector (1) and includes usedforavarietyofmediapreparations.PluronicF68 mustbe
RSG as well as the ability to form colonies in the clonal phase added to the base medium to facilitate growth in suspension
culture. Other supplements usually include antibiotics, sodium
of the assay.
pyruvate, and occasionally, glutamine. Refer to references in
2.3 Symbols:
4.1 for suggested concentrations.
2.3.1 BrUdR—5-bromo-2`-deoxyuridine.
4.1.2 Growth Medium—Growth medium is prepared by
supplementing the base medium with horse serum, usually
2.3.2 BUdR—bromouracil deoxyriboside.
10% by volume.
2.3.3 CAS—chemical abstract service.
4.1.3 Cloning Medium—Cloning medium is growth me-
2.3.4 DMSO—dimethylsulfoxide. dium further supplemented with agar (Noble, purified, or
Baltimore Biological Laboratories (BBL); see Ref. (8)) and
2.3.5 MLA—mouse lymphoma assay.
often with additional serum. Each investigator should deter-
2.3.6 NADP—nicotinamide-adenine dinucleotide phos-
mine serum and agar concentrations that yield the best cloning
phate.
conditionsintheirlaboratory.Seereferencesin4.1foragarand
serumconcentrationsastheyvarybetweenlaboratories.Serum
2.3.7 TFT—trifluorothymidine.
concentration is often adjusted to 20% in the cloning medium
2.3.8 THMG—thymidine + hypoxanthine + methotrexate +
since this concentration has been reported to provide the
glycine.
highest cloning efficiency for L5178Y cells (9); however, this
optimum may vary among lots of horse serum and among
2.3.9 VC—viable count(s).
laboratories.
4.1.4 Selective Media—There are two types of selective
3. Significance and Use
media routinely used in the MLA: cloning medium supple-
3.1 This guide is limited to procedures used solely for the
mentedwithTFTtopermitquantitationandcharacterizationof
−/−
testing of substances to determine their mutagenicity and does TK mutants;andTHMGcleansingmediumwhichkeepsthe
−/−
not apply to other methods and uses such as exploring spontaneous TK mutant frequency at a minimum, thereby
optimizing the assay sensitivity.
mechanisms of mutation.
4.1.4.1 TFTSelection—Cloningmediumsupplementedwith
3.2 Recentevidencesuggeststhatthisassaymeasuresadual
+/−
TFTis used to arrest growth ofTK cells and to allow clonal
genetic end point; therefore, some discussion of the relation- −/−
growth of TK cells. The optimal concentration of TFT may
shipsbetweenmammaliancellmutagenicitytestingresultsand
vary among laboratories, but is usually in the range of 1 to 5
theresultsobservedbothinpuregenemutationalassaysandin
µg/ml. Those laboratories utilizing RPMI 1640 medium may
cytogeneticassaysisnecessary.However,itisnottheintentof
find it necessary to use a TFT concentration at the higher end
this guide to discuss other relationships between this mamma-
of this range. Each laboratory should establish the efficacy of
liancellmutagenicitytestingresultsandtheresultsobservedin
their TFT selection by appropriate means. Differential lots of
other tests for mutagenicity and carcinogenicity.
horse serum vary in their ability to inactivate TFT, possibly
resulting from varying amounts of the enzyme thymidine
4. Test Materials
phosphorylase. This enzyme, in the presence of inorganic
phosphate, converts TFT to an inactive form. The approxi-
4.1 Media—Fischer—(2) successfully adapted L5178Y
mately sixfold higher level of inorganic phosphate present in
mouse leukemic cells to growth in suspension culture using
RPMI 1640 medium (relative to Fischer’s medium) drives this
F10 (Gibco H-11) medium. In developing and validating the
inactivation more rapidly in RPMI-based cloning medium if
L5178Y mouse lymphoma assay, Clive and associates (1)
the serum is improperly heat inactivated, thereby critically
routinely used Fischer’s medium; however, other laboratories
decreasing TFT-selection stringency in the mutant selection
have recently validated the assay with RPMI 1640 medium
plates. This can be overridden by a combination of increased
(3-5). Either medium can be used; however, it is important to
TFT concentration, extra attention to the proper heat inactiva-
note several differences between them. The most important of
tion of the horse serum (that is, ensure that the serum reaches
theseisthelargedifferenceinphosphateconcentration,afactor
56°C prior to initiating the 30 min incubation; Mayo, unpub-
which can affect the stringency of trifluorothymidine (TFT)
lished data) (2, 11), and stringent screening of serum lots prior
selection in RPMI medium (6) if proper precautions concern-
to routine use in the assay.
ing heat inactivation and quality of horse serum are not taken
NOTE 1—Historically, 5-bromo-2`-deoxyuridine (BUdR; BrUdR) has
(7); (see 4.1.4.1). Secondly, the effective concentrations of
−/−
beenutilizedwiththisassaytoselectforTK cells.TFThasbeenshown
cleansingmediumcomponentsisdependentonthetypeofbase
tobeamoreeffectiveselectiveagent,andtheuseofBUdRisdiscouraged
medium used (see 4.1.4.2). It is recommended that critical
(10).
components (for example, horse serum) be heat-inactivated
either separately or after combination. Fischer’s medium is
photosensitive in liquid formulations!
The sole source of supply of the apparatus known to the committee at this time
is BASF Wyandotte Corp., Wyandotte, MI 48192. If you are aware of alternative
4.1.1 Base Medium—A base medium is generally prepared
suppliers, please provide this information to ASTM International Headquarters.
from powdered formulation or is purchased as a 10× or 1×
Your comments will receive careful consideration at a meeting of the responsible
liquid. Some laboratories prepare 2× medium which can be technical committee, which you may attend.
E1280 − 97 (2008)
4.1.4.2 THMG Cleansing—Cleansing medium (growth me- (forexample,Aroclor-1254–inducedratliverS9 (14);Aroclor-
diumsupplementedwithTHMG)isonemethodusedtoridthe 1254–induced hamster or mouse S9 (15)).
−/−
stock culture of spontaneously accumulated TK mutants. It 4.2.1 Sources—Preparations designed to provide metabolic
is composed of: methotrexate (M), to block folate-dependent activation may be prepared from a variety of sources depend-
thymidylate synthase production of thymidine monophosphate ing on the needs of a particular assay. Factors which may vary
(TMP), thus forcing the cells into dependency on the TK include, for example, species, sex, tissue, age, method of
salvage pathway of TMP synthesis; thymidine (T) and hypox- induction, and method of preparation.
anthine (H), to bypass the folate block in TK-competent cells; 4.2.2 Cofactor Mixes for Enzyme Preparations—should be
−/−
and glycine (G) as a methyl group source. In TK mutant shown to support enzyme activity, as measured either directly
cells, the exogenous thymidine cannot be phoshorylated, and or by a biological effect. Commonly used cofactors include
thesecellsdiefromTMPdeficiency.Following24-hgrowthin NADPin conjunction with either sodium isocitrate or glucose-
the cleansing medium, the stock culture is centrifuged and the 6-phosphate (3, 5, 14-16).
cells are washed free of unbound methotrexate and resus- 4.2.3 MetabolicActivities—Themetabolicactivationsystem
pended in growth medium supplemented with THG (that is, to be used should be capable of converting appropriate known
THMG without methotrexate) for 1 to 3 days.This permits the promutagens to mutagens while causing little or no toxicity or
cells to fully recover from the remaining bound methotrexate
mutagenicity to the mouse lymphoma cells in the solvent
and resume synthesis of TMP and purines by the folate- control culture(s).
dependentpathways.Cellsshouldbeallowedtototallyrecover
from the metabolic stress of the cleansing procedure (about 2
5. Test Method
to 3 days) before being used in a test.
5.1 Test Principle—The mouse lymphoma assay utilizes a
+/−
4.1.4.3 While it has been suggested that the cleansing
strain (TK -3.7.2C clonal line) of L5178Ymouse lymphoma
procedure be performed on a weekly basis, some laboratories
cells that has been made heterozygous at the TK locus (17).
may find a less frequent cleansing schedule acceptable, pro-
These cells contain the TK enzyme and are sensitive to the
viding a low background mutation frequency is maintained.
cytostaticandcytotoxiceffectsofappropriateconcentrationsof
Other alternatives include: freezing populations of freshly
TFT (10). Forward mutations to the single functional TK gene
cleansed cells and thawing them a few days prior to use; using
can result in the loss ofTK activity and thus the acquisition of
cultures grown for a very low inoculum (ca. 600 cells/culture;
TFT-resistance. These mutant cells can be quantitated after an
however, this method suffers from potential genetic drift
appropriateexpressionperiodbycloninginasoftagarmedium
problemswhichcouldalterthiswell-characterizedcellline);or
supplementedwiththeselectiveagent,TFT (10, 18).Anumber
maintaining an uncleansed population of cells and cleansing a
of protocols have been described (1, 14-19). The assay has
portion of these cells prior to use. In these cases the exposure
been adapted to detect a wide variety of mutagens including
+/−
of TK cells to methotrexate, which, in the absence of THG
those requiring exogenous metabolic activation.
is known to induce mutations, can be reduced to a minimum.
5.2 Description of Test System:
For specific concentrations of the ingredients and cell popula-
+/−
5.2.1 Cell Line—The MLAuses theTK -3.7.2C heterozy-
tions used in the cleansing step, refer to references in 4.1.Itis
gote of L5178Ymouse lymphoma cells (17).This cell line has
important to note for those laboratories utilizing RPMI 1640
been cytogenetically characterized by banded karyotype at the
medium, that slightly higher concentrations of THMG and
230 to 300-band level of resolution (20 and 21). The chromo-
THG are required, as noted in the literature.
some 11 homologs, the known location of the TK gene in the
4.1.5 Quality Control of Media—The quality of culture
mouse (22), have been shown to possess a centromeric
media is a common cause of problems with the MLA. A
heteromorphism that distinguishes the chromosomes 11a and
number of factors are known to contribute to variations in
11b (small and large centromeres, respectively) (23). Through
medium quality, the principal ones being water quality and
−/−
bandedkaryotypeanalysisofalargenumberofTK mutants,
exposure of liquid Fischer’s medium to excessive light. An-
thispropertyhasallowedtheprovisionalmappingofthesingle
other identified source of assay problems is the lot and source
functional TK gene to
...
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