ASTM E1262-24 中国仓鼠卵巢细胞次黄嘌呤鸟嘌呤磷酸核糖转移酶基因突变检测的性能
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This international standard was developed in accordance with nternationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards,Guides and Recommendaions issued by the World Trade Organization Technical Barriers to Trade(TBT)Committee.
Designation:E1262-24
Standard Guide for
Performance of Chinese Hamster Ovary Cell/Hypoxanthine
Guanine Phosphoribosyl Transferase Gene Mutation Assay¹
This standard is issued under the fixed designation E1262;the number immediately following the designation indicates the year of
original adoption or,in the case of revision,the year of last revision.A number in parentheses indicates the year of last reapproval.A
superscript epsilon(e)indicates an editorial change since the last revision or reapproval.
1.Scope
1.1 This guide highlights some of the more relevant bio-
logical concepts as they are currently understood,and summa-
rizes the critical technical aspects for acceptable bioassay
performances as they currently are perceived and practiced.
The Chinese hamster ovary cell/hypoxanthine guanine phos-
phoribosyl transferase(CHO/HGPRT)assay (1)²has been
widely applied to the toxicological evaluation of industrial and
environmental chemicals.The method is limited to detection of
small-scale genetic interactions.Therefore,when this method
is used for genotoxicity assessment it is recommended that an
in vitro clastogenicity assay(for example,chromosomal
aberration,micronucleus)is considered to detect large-scale
(for example,chromosomal)DNA damage.
1.2 This guide concentrates on the practical aspects of cell
culture,mutagenesis procedures,data analysis,quality control,
and testing strategy.The suggested approach represents a
consensus of the panel members for the performance of the
assay.It is to be understood,however,that these are merely
general guidelines and are not to be followed without the use
of sound scientific judgement.Users of the assay should
evaluate their approach based on the properties of the sub-
stances to be tested and the questions to be answered.
1.3 Deviation from the guidelines based on sound scientific
judgement should by no means invalidate the results obtained.
1.4 The values stated in SI units are to be regarded as
standard.No other units of measurement are included in this
standard.
1.5 This standard does not purport to address all of the
safety concerns,if any,associated with its use.It is the
responsibility of the user of this standard to establish appro-
priate safety,health,and environmental practices and deter-
mine the applicability of regulatory limitations prior to use.
!This guide is under the jurisdiction of ASTM Committee F04 on Medical and
Surgical Materials and Devices and is the direct responsibility of Subcommittee
F04.16 on Biocompatibility Test Methods.
Current edition approved Dec.1,2024.Published December 2024.Originally
approved in 1988.Last previous edition approved in 2018 as E1262-88(2018).
DOI:10.1520/E1262-24.
2 The boldface numbers in parentheses refer to the list of references at the end of
this guide
1.6 This international standard was developed in accor-
dance with internationally recognized principles on standard-
ization established in the Decision on Principles for the
Development of International Standards,Guides and Recom-
mendations issued by the World Trade Organization Technical
Barriers to Trade(TBT)Committee.
2.Significance and Use
2.1 The CHO/HGPRT assay detects forward mutations of
the X-linked hypoxanthine-guanine phosphoribosyl transferase
(hgprt)locus(coding for the enzyme,HGPRT)in Chinese
hamster ovary(CHO)cells.Cells originally derived from
Chinese hamster ovary tissue are exposed to a test article and,
following an appropriate cell culture regimen,descendants of
the original treated population are monitored for the loss of
functional HGPRT,presumably due to mutations.Resistance to
a purine analogue,6-thioguanine(6TG)(or less desirably,
8-azaguanine(8AG)),is employed as the genetic marker.
HGPRT catalyzes the conversion of the nontoxic 6TG to its
toxic ribophosphorylated derivative.Loss of the enzyme or its
activity therefore leads to cells resistant to 6TG.
2.2 Because HGPRT is an enzyme of the purine nucleotide
salvage pathway,loss of the enzyme is not a lethal event.
Different types of mutational events(base substitutions,frame
shifts,deletions,and so forth)can be detectable at the hgprt
locus.The assay does not appear to detect large-scale chromo-
somal damage.The CHO/HGPRT assay has been used to study
a wide range of mutagens,including radiations(2-4),and a
wide variety of chemicals(1)and complex chemical mixtures
(5).
3.Characteristics of CHO Cells
3.1 Different CHO cell lines/subclones are appropriate for
the CHO/HGPRT assay.The CHO-K1-BH4 cell line developed
and extensively characterized by Ref (6) is probably the most
widely employed.The CHO(WT)cell line and its derivative,
CHO-AT3-2,are used to monitor mutations at other gene loci
in addition to hgprt (7,8 ).While there are differences among
the cell lines employed,a number of general characteristics are
critical for the performance of the assay:
3.1.1 The cloning efficiency(CE)of the stock cultures
should not be less than 70%.The CE of untreated or solvent
control experimental cultures should not be less than 50%.
Copyright OASTM Intermational,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959.United States
2
E1262-24
3.1.2 Cultures in logarithmic phase of growth should have a
population doubling time of 12 to 16h.
3.1.3 The modal chromosome number should be 20 or 21,
as is characteristic of the particular cell line/subclone used.
3.1.4 Cultures should be free from microbial and myco-
plasma contamination.
3.2 The cell properties that are critical for the assay should
be routinely monitored as part of the quality control regimen.
Routine quality control procedures should include testing of
serum and media for each new purchase,as well as myco-
plasma and karyotype checks at least once yearly,preferably
once every three months.
4.Mutagenesis Procedures
4.1 The mutagenesis protocol can be divided into three
phases:mutagen treatment,expression,and selection.
4.2 Mutagen Treatment:
4.2.1 Cell Plating—Cells should be in exponential phase
when plated for treatment.Several media(for example,Ham's
F12,alpha-MEM)that are known to be optimal for cell growth
can be used.Cells should be seeded at an appropriate cell
density to allow exponential growth as well as quantitation of
induced responses.A common practice is to plate 0.5×10⁶
cells in a 25cm²flask,or 1.5×10⁶cells in a 75 cm²flask,on
the day before treatment.
4.2.2 Chemical Handling—The solubility of the test article
in an appropriate medium should be determined before treat-
ment.Commonly used solvents are,in the order of preference,
medium,water,dimethylsulfoxide,ethanol,and acetone.
Generally,the nonaqueous solvent concentration should not
exceed 1%and should be constant for all samples.As part of
the solubility test,an aliquot of the test chemical should be
added to the treatment medium to note any pH changes,the
presence of any chemical precipitation,and any apparent
reaction of the chemical or solvent with the culture vessel.The
solvent of choice should not have any undesirable reactions
with the test article,culture vessel,or cells.
4.2.3 Addition of Test Article to Cells—Test samples are
prepared and added to each flask.Dilutions of the test article
should be such that the concentration of solvent(when a neat
test article extract is not used)remains constant for all samples.
Cells are generally treated with the test article for at least 3 h.
For treatment times of 3 to 5h,serum-free medium can be
used.As serum is required to maintain cell division,medium
containing serum should be used for a prolonged treatment
period(for example,16h or longer).Serum requirement for
treatment periods between 5 and 16h should be determined on
a case-by-case basis.
4.2.4 Exogenous Activation Systems—Aroclor 1254-
induced rat liver homogenate(S9)is the most commonly used
exogenous metabolic activating system for the assay.As the
availability of Aroclor 1254 has greatly diminished,other
standard inducers of rat liver S9(for example,β-naptho-
flavone and phenobarbital combination)are commonly used.
When S9 is used,cofactors for the mixed function monooxy-
genases should be present.Calcium chloride(CaCl₂),which
enhances the mutagenicity of nitrosamines and polycyclic
hydrocarbons( 9,10 ),appears to be another useful addition.
However,the need for CaCl₂has yet to be documented for a
wide variety of chemicals.A commonly used cofactor mixture
consists of sodium phosphate (50 mM,pH 7.0 to 8.0),NADP
(4 mM),glucose-6-phosphate (5 mM),potassium chloride(30
mM),magnesium chloride(10 mM),and CaCl₂(10 mM).S9 is
added directly to the cofactor mixture.One volume of the
S9/cofactor mixture is added to four volumes of the treatment
medium.Other exogenous systems(for example,hepatocytes,
S9 from other animal species or produced using different
enzyme induction conditions,and other cofactor mixtures)can
also be used depending on the intent of the experiment.
4.2.5 Estimation of Cytotoxicity—Plating CHO cells imme-
diately after treatment for cytotoxicity determination is gener-
ally expected to yield the most accurate results.Otherwise,
cytotoxicity can be estimated on the day after treatment.
Aliquots of the cells are plated to allow for colony develop-
ment.Cytotoxicity is usually expressed as relative CE which is
the ratio of the CE of the treated cells to that of the solvent
control.Viability determination should take into account any
loss of cells during the treatment period,cell trypsinization
procedures,and the overnight incubation period.
4.2.6 Positive and Solvent Controls—An appropriate nega-
tive control is treatment of cells with the solvent used for the
test article.Positive controls,both direct-acting and indirect-
acting,should also be included to demonstrate the adequacy of
the experimental conditions to detect known mutagens.An
untreated control may also be included to evaluate the effects
of the solvent on mutagenicity.Commonly used positive
controls are ethyl methane sulfonate (EMS)and N-methyl-N-
nitro-N-nitrosoguanidine(MNNG)as direct-acting mutagens,
and benzo(a)pyrene (BaP)and dimethylnitrosamine(DMN)as
promutagens that require metabolic activation.
4.3 Expression of Induced Mutations:
4.3.1 After mutation at the hgprt locus,the mutant pheno-
type requires a period of time before it is completely expressed
(expression requires the loss of pre-existing enzyme activity).
Phenotypic expression is presumably achieved by dilution of
the pre-existing HGPRT enzyme and mRNA through cell
division and macromolecular turnover.At the normal popula-
tion doubling times of 12 to 16h for CHO cells,an expression
period of 7 to 9 days is generally adequate(11,12 ).
4.3.2 The most widely employed method for phenotypic
expression allows exponential growth of the cells for a defined
time period after mutagen treatment.CHO cells can be
subcultured with 0.05%trypsin with or without EDTA.
Aliquots of 1×10⁶cells are subcultured at 2 or 3 day intervals
in 100 mm diameter tissue culture dishes or 75 cm²t-flasks.
Either complete medium or hypoxanthine-free medium can be
employed,with either dialyzed or nondialyzed serum.It is
important to ensure that the medium employed will allow a
population doubling time of 12 to 16h.
4.3.3 Besides the normal growth of cells as monolayer
cultures,alternative methods of subculturing involving suspen-
sion( 8),unattached (13),and division arrested (14)cultures
have also been successful.The use of a particular subculture
regimen in the expression period should be substantiated by
data demonstrating the achievement of optimal expression.
4.4 Mutant Selection:
E1262-24
3
4.4.1 Conditions for the selection of mutants must be
defined to ensure that only mutant cells are able to form
colonies and that there is no significant reduction in the ability
of mutant cells to form colonies.In general,cells are plated in
tissue culture dishes for attached colony growth(11),or in agar
for suspended colony growth (15 ).An advantage of the former
is that after the colonies are fixed and stained,the plates can be
counted at a later date.An advantage of the latter is that
metabolic cooperation between wild type and mutant cells is
reduced,allowing selection of a higher cell number per plate.
For attached colonies,the cells are in general cultured for a
period of 6 to 8 days and the number of colonies counted after
fixing (for example,with 10%formalin or 70%methanol),
and staining(for example,with 10%Giemsa or crystal violet).
Soft agar colonies are usually counted in situ after a culturing
period of 10 to 14 days.
4.4.2 Reliable selection has been established in
hypoxanthine-free medium containing dialyzed serum and 10
μM 6TG.Fetal bovine serum,newborn bovine serum,or calf
serum can be used,providing that the serum has been ad-
equately tested and shown to support the desirable character-
istics of CHO cells as described here.Dialyzed serum is
usually necessary to eliminate the competition between 6TG
and purine bases in the serum.It has been found that a selection
cell density of 2×10⁵or fewer cells per 100 mm dish for
attached colony growth (14,16 ) and 10⁶or fewer cells per 100
mm dish(in 30 mL of agar)for agar colony growth(15 )allows
essentially 100%recovery of mutant cells.
5.Data Presentation
5.1 Results from the assay should include the following
experimental data:
6.1.2 The mean mutant frequency of the solvent controls in
each experiment should fall within the range from 0 to 20
mutants per 10⁶clonable cells.A higher mutant frequency may
preclude detection of weak mutagens.Under such conditions,
data acceptability should be evaluated on a case-by-case basis.
6.1.3 The positive control must induce a statistically signifi-
cant response at a magnitude appropriate for the mutagen under
the chosen experimental conditions.
6.1.4 The highest test article concentration should,if
possible,result in a significant cytotoxic response(for
example,10%to 30%survival,where survival is the percent
of the treated population that is viable after treatment).This is
particularly important if the response is negative.For non-
cytotoxic test articles,the highest concentration has generally
been 1 to 10 mg/mL,or to the limit of solubility.
7.Data Analysis
7.1 Due to the possibility of stochastic fluctuation,only
samples with no fewer than 100000 viable cells after treatment
should be used for data analysis.Judgement on mutagenicity
should be made based on the following information:
7.1.1 Dose-response relationship.
7.1.2 Significance of response (in comparison to the nega-
tive control).
7.1.3 Reproducibility of the results.
7.2 Exact statistical analysis is difficult because the distri-
bution of the number of mutant colonies depends on the
complex processes of cell growth and death after mutagen
treatment.While other appropriate methods can be used,the
following two approximate methods are used commonly:
7.2.1 Weighted Regression Analysis—A weighted regression
5.1.1 Concentrations and solvents used for the test article analysis where the weights are proportional to the observed
and positive controls number of mutant colonies divided by the square of the
5.1.2 Absolute and relative cloning efficiencies(CE)in the observed mutant frequency(17).This weighting scheme was
concurrent cytotoxicity assay. derived by assuming that the variance of the observed mutant
5.1.2.1 Absolute CE—Absolute CE equals the number of frequency is a constant multiple of that which would occur if
colonies formed divided by the number of cells plated. the number of mutant colonies on each selection plate per
5.1.2.2 Relative CE—Relative CE equals CE(treatment) treatment conforms to a Poisson distribution.A test compound
divided by CE(solvent control). is considered to exhibit a mutagenic response if the slope of the
5.1.3 Actual number of mutant colonies observed for each mutant induction as a function of test concentrations is greater
treatment condition. than zero at the 0.01 level according to the t-test(18).
5.1.4 Absolute CE at selection for each treatment condition. 7.2.2 Power Transformation Procedure—A power transfor-
5.1.5 Mutant frequency(MF)values,expressed as mutants mation procedure with which the observed mutant frequency is
per 10⁶cells. transformed using the following equation:
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5.1.5.2 Number of Clonable Cells—The number of clonable Y =transformed mutant frequency,
cells equals the cells plated multiplied by the absolute CE at X =observed mutant frequency,and
selection. a,β=constants.
7.2.2.1 Data transformed by this method appears to satisfy
6.Criteria for Data Acceptability the assumptions of homogeneous variance and normal distri-
6.1 Generally,for the data of a given assay to be acceptable, bution( 18).Comparison to negative control values and dose-
the following criteria should be met: response relationships are examined with Student's t-test and
6.1.1 The absolute CE of the negative controls should not be an analysis of variance(ANOVA)using the transformed
less than 50%.Absolute CE values lower than 50%would values.Computations can be done with computer programs
indicate suboptimal culturing conditions for the cells. readily available.
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1ThisinternationalstandardwasdevelopedinaccordancewithnternationallyrecognizedprinciplesonstandardizationestablishedintheDecisiononPrinciplesfortheDevelopmentofInternationalStandards,GuidesandRecommendaionsissuedbytheWorldTradeOrganizationTechnicalBarrierstoTrade(TBT)Committee.Designation:E1262-24St...
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