ASTM E1280-97(2003)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
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Designation:E1280–97 (Reapproved 2003)
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 (e) 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 2.1.1 clastogen—any agent that is capable of inducing
chromosome breaks.
1.1 The purpose and scope of this guide is to present
2.1.2 gene mutation—any heritable change whose physical
background material and to establish criteria by which proto-
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extent is restricted to the limits of a single gene.
cols and procedures for conducting the L5178Y/TK -3.7.2C
2.1.3 mutagen—any physical or chemical agent capable of
mouselymphomamutagenicityassay(commonlyreferredtoas
inducing a mutation.
the mouse lymphoma assay, (MLA)) can be properly under-
2.1.4 mutation—any heritable change in the genetic mate-
stood and evaluated. This guide is also intended to aid
rial, not caused by genetic segregation or genetic recombina-
researchers and others to gain a better understanding of the
tion, and that is transmitted to daughter cells.
critical elements involved with mammalian cell mutagenicity
2.2 Definitions of Terms Specific to This Standard:
testing. More specifically, this guide is intended to provide for
2.2.1 chromosome mutation—a mutation resulting from a
researchers the accomplishment of the following goals:
structural change to a chromosome involving the gain, loss, or
1.1.1 Provide an understanding of the critical procedures
relocation of chromosome segments. Chromosome mutations
(steps)intheperformanceofthismammaliancellmutagenicity
can be either intrachromosomal or interchromosomal.
test.
2.2.2 relative suspension growth (RSG)—used to measure
1.1.2 Provide generalized criteria by which researchers can
the cytotoxicity of a given treatment based on the growth of
evaluate if they are properly performing, utilizing, and inter-
cells in suspension culture relative to the untreated or solvent
preting this assay.
control(s). RSG is calculated according to the method of Clive
1.1.3 Provide criteria by which individuals responsible for
and Spector (1).
evaluating MLA data can determine if the experiments have
2.2.3 relative total growth (RTG)—used as a means to
been properly performed and interpreted.
measure the relative toxicity to cells (survival) following
1.1.4 Provide a basis from which new procedures and
treatment in the mouse lymphoma assay. RTG is calculated
developments in testing procedures can be evaluated.
according to the method of Clive and Spector (1) and includes
1.1.5 Provide an understanding of the types of genetic
RSG as well as the ability to form colonies in the clonal phase
damage (that is, gene and chromosome mutation) that may be
of the assay.
detected in this mammalian cell mutagenicity test.
2.3 Symbols:
2. Terminology
2.3.1 BrUdR—5-bromo-28-deoxyuridine.
2.3.2 BUdR—bromouracil deoxyriboside.
2.1 Definitions:
2.3.3 CAS—chemical abstract service.
2.3.4 DMSO—dimethylsulfoxide.
2.3.5 MLA—mouse lymphoma assay.
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 Sept. 10, 2003. Published September 2003. Originally Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
approved in 1989. Last previous edition approved in 1997 as E1280–97. this guide.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
E1280–97 (2003)
2.3.6 NADP—nicotinamide-adenine dinucleotide phos- Baltimore Biological Laboratories (BBL); see Ref. (8)) and
phate. often with additional serum. Each investigator should deter-
2.3.7 TFT—trifluorothymidine. mine serum and agar concentrations that yield the best cloning
2.3.8 THMG—thymidine + hypoxanthine + methotrexate + conditionsintheirlaboratory.Seereferencesin4.1foragarand
glycine. serumconcentrationsastheyvarybetweenlaboratories.Serum
2.3.9 VC—viable count(s). concentration is often adjusted to 20% in the cloning medium
since this concentration has been reported to provide the
3. Significance and Use
highest cloning efficiency for L5178Y cells (9); however, this
3.1 This guide is limited to procedures used solely for the optimum may vary among lots of horse serum and among
testing of substances to determine their mutagenicity and does
laboratories.
not apply to other methods and uses such as exploring
4.1.4 Selective Media—There are two types of selective
mechanisms of mutation.
media routinely used in the MLA: cloning medium supple-
3.2 Recentevidencesuggeststhatthisassaymeasuresadual
mentedwithTFTtopermitquantitationandcharacterizationof
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genetic end point; therefore, some discussion of the relation-
TK mutants;andTHMGcleansingmediumwhichkeepsthe
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shipsbetweenmammaliancellmutagenicitytestingresultsand
spontaneous TK mutant frequency at a minimum, thereby
theresultsobservedbothinpuregenemutationalassaysandin
optimizing the assay sensitivity.
cytogeneticassaysisnecessary.However,itisnottheintentof
4.1.4.1 TFT Selection—Cloning medium supplemented
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this guide to discuss other relationships between this mamma-
with TFT is used to arrest growth of TK cells and to allow
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liancellmutagenicitytestingresultsandtheresultsobservedin
clonalgrowthofTK cells.TheoptimalconcentrationofTFT
other tests for mutagenicity and carcinogenicity.
may vary among laboratories, but is usually in the range of 1
to 5 µg/ml. Those laboratories utilizing RPMI 1640 medium
4. Test Materials
may find it necessary to use a TFT concentration at the higher
4.1 Media—Fischer—(2) successfully adapted L5178Y
end of this range. Each laboratory should establish the efficacy
mouse leukemic cells to growth in suspension culture using
oftheirTFTselectionbyappropriatemeans.Differentiallotsof
F10 (Gibco H-11) medium. In developing and validating the
horse serum vary in their ability to inactivate TFT, possibly
L5178Y mouse lymphoma assay, Clive and associates (1)
resulting from varying amounts of the enzyme thymidine
routinely used Fischer’s medium; however, other laboratories
phosphorylase. This enzyme, in the presence of inorganic
have recently validated the assay with RPMI 1640 medium
phosphate, converts TFT to an inactive form. The approxi-
(3-5). Either medium can be used; however, it is important to
mately sixfold higher level of inorganic phosphate present in
note several differences between them. The most important of
RPMI 1640 medium (relative to Fischer’s medium) drives this
theseisthelargedifferenceinphosphateconcentration,afactor
inactivation more rapidly in RPMI-based cloning medium if
which can affect the stringency of trifluorothymidine (TFT)
the serum is improperly heat inactivated, thereby critically
selection in RPMI medium (6) if proper precautions concern-
decreasing TFT-selection stringency in the mutant selection
ing heat inactivation and quality of horse serum are not taken
plates. This can be overridden by a combination of increased
(7); (see 4.1.4.1). Secondly, the effective concentrations of
TFT concentration, extra attention to the proper heat inactiva-
cleansingmediumcomponentsisdependentonthetypeofbase
tion of the horse serum (that is, ensure that the serum reaches
medium used (see 4.1.4.2). It is recommended that critical
56°C prior to initiating the 30 min incubation; Mayo, unpub-
components (for example, horse serum) be heat-inactivated
lished data) (2, 11), and stringent screening of serum lots prior
either separately or after combination. Fischer’s medium is
to routine use in the assay.
photosensitive in liquid formulations!
NOTE 1—Historically, 5-bromo-28-deoxyuridine (BUdR; BrUdR) has
4.1.1 Base Medium—A base medium is generally prepared
−/−
beenutilizedwiththisassaytoselectforTK cells.TFThasbeenshown
from powdered formulation or is purchased as a 103 or 13
tobeamoreeffectiveselectiveagent,andtheuseofBUdRisdiscouraged
liquid. Some laboratories prepare 23 medium which can be
(10).
usedforavarietyofmediapreparations.PluronicF68 mustbe
4.1.4.2 THMG Cleansing—Cleansing medium (growth me-
added to the base medium to facilitate growth in suspension
diumsupplementedwithTHMG)isonemethodusedtoridthe
culture. Other supplements usually include antibiotics, sodium
−/−
stock culture of spontaneously accumulated TK mutants. It
pyruvate, and occasionally, glutamine. Refer to references in
is composed of: methotrexate (M), to block folate-dependent
4.1 for suggested concentrations.
thymidylate synthase production of thymidine monophosphate
4.1.2 Growth Medium—Growth medium is prepared by
(TMP), thus forcing the cells into dependency on the TK
supplementing the base medium with horse serum, usually
salvage pathway of TMP synthesis; thymidine (T) and hypox-
10% by volume.
anthine (H), to bypass the folate block in TK-competent cells;
4.1.3 Cloning Medium—Cloning medium is growth me-
−/−
and glycine (G) as a methyl group source. In TK mutant
dium further supplemented with agar (Noble, purified, or
cells, the exogenous thymidine cannot be phoshorylated, and
thesecellsdiefromTMPdeficiency.Following24-hgrowthin
The sole source of supply of the apparatus known to the committee at this time
the cleansing medium, the stock culture is centrifuged and the
is BASF Wyandotte Corp., Wyandotte, MI 48192. If you are aware of alternative
cells are washed free of unbound methotrexate and resus-
suppliers, please provide this information to ASTM International Headquarters.
pended in growth medium supplemented with THG (that is,
Your comments will receive careful consideration at a meeting of the responsible
technical committee, which you may attend. THMG without methotrexate) for 1 to 3 days.This permits the
E1280–97 (2003)
cells to fully recover from the remaining bound methotrexate toxicity or mutagenicity to the mouse lymphoma cells in the
and resume synthesis of TMP and purines by the folate- solvent control culture(s).
dependentpathways.Cellsshouldbeallowedtototallyrecover
5. Test Method
from the metabolic stress of the cleansing procedure (about 2
5.1 Test Principle—The mouse lymphoma assay utilizes a
to 3 days) before being used in a test.
+/−
strain (TK -3.7.2C clonal line) of L5178Ymouse lymphoma
4.1.4.3 While it has been suggested that the cleansing
cells that has been made heterozygous at the TK locus (17).
procedure be performed on a weekly basis, some laboratories
These cells contain the TK enzyme and are sensitive to the
may find a less frequent cleansing schedule acceptable, pro-
cytostaticandcytotoxiceffectsofappropriateconcentrationsof
viding a low background mutation frequency is maintained.
TFT (10). Forward mutations to the single functional TK gene
Other alternatives include: freezing populations of freshly
can result in the loss of TK activity and thus the acquisition of
cleansed cells and thawing them a few days prior to use; using
TFT-resistance. These mutant cells can be quantitated after an
cultures grown for a very low inoculum (ca. 600 cells/culture;
appropriateexpressionperiodbycloninginasoftagarmedium
however, this method suffers from potential genetic drift
supplementedwiththeselectiveagent,TFT(10, 18).Anumber
problemswhichcouldalterthiswell-characterizedcellline);or
of protocols have been described (1, 14-19). The assay has
maintaining an uncleansed population of cells and cleansing a
been adapted to detect a wide variety of mutagens including
portion of these cells prior to use. In these cases the exposure
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those requiring exogenous metabolic activation.
of TK cells to methotrexate, which, in the absence of THG
5.2 Description of Test System:
is known to induce mutations, can be reduced to a minimum.
+/−
5.2.1 Cell Line—TheMLAusestheTK -3.7.2Cheterozy-
For specific concentrations of the ingredients and cell popula-
gote of L5178Ymouse lymphoma cells (17).This cell line has
tions used in the cleansing step, refer to references in 4.1. It is
been cytogenetically characterized by banded karyotype at the
important to note for those laboratories utilizing RPMI 1640
230 to 300-band level of resolution (20 and 21). The chromo-
medium, that slightly higher concentrations of THMG and
some 11 homologs, the known location of the TK gene in the
THG are required, as noted in the literature.
mouse (22), have been shown to possess a centromeric
4.1.5 Quality Control of Media—The quality of culture
heteromorphism that distinguishes the chromosomes 11a and
media is a common cause of problems with the MLA. A
11b (small and large centromeres, respectively) (23). Through
number of factors are known to contribute to variations in
−/−
bandedkaryotypeanalysisofalargenumberofTK mutants,
medium quality, the principal ones being water quality and
thispropertyhasallowedtheprovisionalmappingofthesingle
exposure of liquid Fischer’s medium to excessive light. An-
functional TK gene to the terminal two-band region of chro-
other identified source of assay problems is the lot and source
mosome11binthiscellline (23).Itisrecommendedthatthese
of agar (8) and the problem of the use of a dirty autoclave to
cellsbeobtainedfromD.Cliveinordertominimizeinterlabo-
sterilize the agar. Serum requires particular precautions with
ratory variability.
RPMI medium (7, 11); see 4.1.4.1. For these reasons, rigorous
5.2.2 Mutational End points—This cell line forms two
methods for media quality control should be established for
classes of TK-deficient (TFT-resistant) mutants based on the
eachlaboratorytoaddresstheabilitytosupport:(1)suspension
6 criterion of colony size in soft agar cloning medium supple-
growth of both low (#1000 cells/mL) and high (>1 310
mented with a selective concentration of TFT. Both large and
cells/mL) cell inocula, (2) high cloning efficiencies under
small TFT-resistant colonies are totally and heritably TK-
nonselective conditions, (3) adequate recoveries of small and
deficient by direct enzyme assay
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