ASTM E1262-88(2013)
(Guide)Standard Guide for Performance of Chinese Hamster Ovary Cell/Hypoxanthine Guanine Phosphoribosyl Transferase Gene Mutation Assay
Standard Guide for Performance of Chinese Hamster Ovary Cell/Hypoxanthine Guanine Phosphoribosyl Transferase Gene Mutation Assay
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, frameshifts, deletions, some chromosomal type lesions, and so forth) should theoretically be detectable at the hgprt locus. 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).
SCOPE
1.1 This guide highlights some of the more relevant biological concepts as they are currently understood, and summarizes the critical technical aspects for acceptable bioassay performances as they currently are perceived and practiced. The Chinese hamster ovary cell/hypoxanthine guanine phosphoribosyl transferase (CHO/HGPRT) assay (1) 2 has been widely applied to the toxicological evaluation of industrial and environmental chemicals.
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 substances 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 appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
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Designation: E1262 − 88 (Reapproved 2013)
Standard Guide for
Performance of Chinese Hamster Ovary Cell/Hypoxanthine
Guanine Phosphoribosyl Transferase Gene Mutation Assay
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.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope hamster ovary (CHO) cells. Cells originally derived from
Chinese hamster ovary tissue are exposed to a test article and,
1.1 This guide highlights some of the more relevant bio-
following an appropriate cell culture regimen, descendants of
logical concepts as they are currently understood, and summa-
the original treated population are monitored for the loss of
rizes the critical technical aspects for acceptable bioassay
functionalHGPRT,presumablyduetomutations.Resistanceto
performances as they currently are perceived and practiced.
a purine analogue, 6-thioguanine (6TG) (or less desirably,
The Chinese hamster ovary cell/hypoxanthine guanine phos-
8-azaguanine (8AG)), is employed as the genetic marker.
phoribosyl transferase (CHO/HGPRT) assay (1) has been
HGPRT catalyzes the conversion of the nontoxic 6TG to its
widely applied to the toxicological evaluation of industrial and
toxic ribophosphorylated derivative. Loss of the enzyme or its
environmental chemicals.
activity therefore leads to cells resistant to 6TG.
1.2 This guide concentrates on the practical aspects of cell
culture, mutagenesis procedures, data analysis, quality control, 2.2 Because HGPRT is an enzyme of the purine nucleotide
and testing strategy. The suggested approach represents a salvage pathway, loss of the enzyme is not a lethal event.
consensus of the panel members for the performance of the Different types of mutational events (base substitutions,
assay. It is to be understood, however, that these are merely
frameshifts, deletions, some chromosomal type lesions, and so
general guidelines and are not to be followed without the use forth)shouldtheoreticallybedetectableatthehgprtlocus.The
of sound scientific judgement. Users of the assay should
CHO/HGPRT assay has been used to study a wide range of
evaluate their approach based on the properties of the sub-
mutagens, including radiations (2-4), and a wide variety of
stances to be tested and the questions to be answered.
chemicals (1), and complex chemical mixtures (5).
1.3 Deviation from the guidelines based on sound scientific
3. Characteristics of CHO Cells
judgement should by no means invalidate the results obtained.
1.4 The values stated in SI units are to be regarded as
3.1 Different CHO cell lines/subclones are appropriate for
standard. No other units of measurement are included in this theCHO/HGPRTassay.TheCHO-K1-BH4celllinedeveloped
standard.
and extensively characterized by (6) is probably the most
widely employed. The CHO(WT) cell line and its derivative,
1.5 This standard does not purport to address all of the
CHO-AT3-2, are used to monitor mutations at other gene loci
safety concerns, if any, associated with its use. It is the
in addition to hgprt (7, 8). While there are differences among
responsibility of the user of this standard to establish appro-
thecelllinesemployed,anumberofgeneralcharacteristicsare
priate safety and health practices and determine the applica-
critical for the performance of the assay:
bility of regulatory limitations prior to use.
3.1.1 The cloning efficiency (CE) of the stock cultures
2. Significance and Use
should not be less than 70%. The CE of untreated or solvent
control experimental cultures should not be less than 50%.
2.1 The CHO/HGPRT assay detects forward mutations of
theX-linkedhypoxanthine-guaninephosphoribosyltransferase
3.1.2 Culturesinlogarithmicphaseofgrowthshouldhavea
(hgprt) locus (coding for the enzyme, HGPRT) in Chinese
population doubling time of 12 to 16 h.
3.1.3 The modal chromosome number should be 20 or 21,
as is characteristic of the particular cell line/subclone used.
This guide is under the jurisdiction ofASTM Committee F04 on Medical and
Surgical Materials and Devicesand is the direct responsibility of Subcommittee
3.1.4 Cultures should be free from microbial and myco-
F04.16 on Biocompatibility Test Methods.
plasma contamination.
Current edition approved Oct. 1, 2013. Published October 2013. Originally
approved in 1988. Last previous edition approved in 2008 as E1262–88 (2008).
3.2 The cell properties that are critical for the assay should
DOI: 10.1520/E1262-88R13.
be routinely monitored as part of the quality control regimen.
Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
this guide. Routine quality control procedures should include testing of
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1262 − 88 (2013)
serum and media for each new purchase, as well as myco- 4.2.5 Estimation of Cytotoxicity—Plating CHO cells imme-
plasma and karyotype checks at least once yearly, preferably diately after treatment for cytotoxicity determination is gener-
once every three months. 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-
4. Mutagenesis Procedures
ment.CytotoxicityisusuallyexpressedasrelativeCEwhichis
4.1 The mutagenesis protocol can be divided into three
the ratio of the CE of the treated cells to that of the solvent
phases: mutagen treatment, expression, and selection.
control. Viability determination should take into account any
loss of cells during the treatment period, cell trypsinization
4.2 Mutagen Treatment:
procedures, and the overnight incubation period.
4.2.1 Cell Plating—Cells should be in exponential phase
4.2.6 Positive and Solvent Controls—An appropriate nega-
when plated for treatment. Several media (for example, Ham’s
tive control is treatment of cells with the solvent used for the
F12,alpha-MEM)thatareknowntobeoptimalforcellgrowth
test article. Positive controls, both direct-acting and indirect-
can be used. Cells should be seeded at an appropriate cell
acting, should also be included to demonstrate the adequacy of
density to allow exponential growth as well as quantitation of
the experimental conditions to detect known mutagens. An
induced responses. A common practice is to plate 0.5×10
2 6 2
untreated control may also be included to evaluate the effects
cells in a 25-cm flask, or 1.5×10 cells in a 75-cm flask, on
of the solvent on mutagenicity. Commonly used positive
the day before treatment.
controls are ethyl methane sulfonate (EMS) and N-methyl-N'-
4.2.2 Chemical Handling—The solubility of the test article
nitro-N-nitrosoguanidine (MNNG) as direct-acting mutagens,
in an appropriate medium should be determined before treat-
and benzo(a)pyrene (BaP) and dimethylnitrosamine (DMN) as
ment. Commonly used solvents are, in the order of preference,
promutagens that require metabolic activation.
medium, water, dimethylsulfoxide, ethanol, and acetone.
4.3 Expression of Induced Mutations:
Generally, the nonaqueous solvent concentration should not
4.3.1 After mutation at the hgprt locus, the mutant pheno-
exceed 1% and should be constant for all samples.As part of
typerequiresaperiodoftimebeforeitiscompletelyexpressed
the solubility test, an aliquot of the test chemical should be
(expression requires the loss of pre-existing enzyme activity).
added to the treatment medium to note any pH changes, the
Phenotypic expression is presumably achieved by dilution of
presence of any chemical precipitation, and any apparent
the pre-existing HGPRT enzyme and mRNA through cell
reaction of the chemical or solvent with the culture vessel.The
division and macromolecular turnover. At the normal popula-
solvent of choice should not have any undesirable reactions
tion doubling times of 12 to 16 h for CHO cells, an expression
with the test article, culture vessel, or cells.
period of 7 to 9 days is generally adequate (11, 12).
4.2.3 Addition of Test Article to Cells—Stock solutions of
4.3.2 The most widely employed method for phenotypic
the test samples are prepared and aliquots are added to each
expression allows exponential growth of the cells for a defined
flask. Dilutions of the test article should be such that the
time period after mutagen treatment. CHO cells can be
concentrationofsolventremainsconstantforallsamples.Cells
subcultured with 0.05% trypsin with or without EDTA.
are generally treated with the test article for at least 3 h. For
Aliquotsof1×10 cellsaresubculturedat2or3dayintervals
treatmenttimesof3to5h,serum-freemediumcanbeused.As
in 100-mm diameter tissue culture dishes or 75 cm t-flasks.
serum is required to maintain cell division, medium containing
Either complete medium or hypoxanthine-free medium can be
serum should be used for a prolonged treatment period (for
employed, with either dialyzed or nondialyzed serum. It is
example, 16 h or longer). Serum requirement for treatment
important to ensure that the medium employed will allow a
periods between 5 and 16 h should be determined on a
population doubling time of 12 to 16 h.
case-by-case basis.
4.3.3 Besides the normal growth of cells as monolayer
4.2.4 Exogenous Activation Systems—Aroclor 1254-
cultures,alternativemethodsofsubculturinginvolvingsuspen-
induced rat liver homogenate (S9) is the most commonly used
sion (8), unattached (13), and division arrested (14) cultures
exogenous metabolic activating system for the assay.When S9
have also been successful. The use of a particular subculture
is used, cofactors for the mixed function monooxygenases
regimen in the expression period should be substantiated by
should be present. Calcium chloride (CaCl ), which enhances
data demonstrating the achievement of optimal expression.
the mutagenicity of nitrosamines and polycyclic hydrocarbons
(9, 10), appears to be another useful addition. However, the 4.4 Mutant Selection:
need for CaCl has yet to be documented for a wide variety of 4.4.1 Conditions for the selection of mutants must be
chemicals. A commonly used cofactor mixture consists of defined to ensure that only mutant cells are able to form
sodium phosphate (50 mM, pH 7.0 to 8.0), NADP (4 mM), colonies and that there is no significant reduction in the ability
glucose-6-phosphate (5 mM), potassium chloride (30 mM), of mutant cells to form colonies. In general, cells are plated in
magnesiumchloride(10mM),andCaCl (10mM).S9isadded tissueculturedishesforattachedcolonygrowth (11),orinagar
directlytothecofactormixture.OnevolumeoftheS9/cofactor for suspended colony growth (15).An advantage of the former
mixture is added to 4 volumes of the treatment medium. Other isthatafterthecoloniesarefixedandstained,theplatescanbe
exogenous systems (for example, hepatocytes, S9 from other counted at a later date. An advantage of the latter is that
animal species or produced using different enzyme induction metabolic cooperation between wild type and mutant cells is
conditions, and other cofactor mixtures) can also be used reduced, allowing selection of a higher cell number per plate.
depending on the intent of the experiment. For attached colonies, the cells are in general cultured for a
E1262 − 88 (2013)
period of 6 to 8 days and the number of colonies counted after particularly important if the response is negative. For noncy-
fixing (for example, with 10% formalin or 70% methanol), totoxic test articles, the highest concentration has generally
andstaining(forexample,with10%Giemsaorcrystalviolet). been 1 to 10 mg/mL, or to the limit of solubility.
Soft agar colonies are usually counted in situ after a culturing
7. Data Analysis
period of 10 to 14 days.
4.4.2 Reliable selection has been established in
7.1 Due to the possibility of stochastic fluctuation, only
hypoxanthine-free medium containing dialyzed serum and 10
samples with no fewer than 100 000 viable cells after treat-
µM 6TG. Fetal bovine serum, newborn bovine serum, or calf
ment should be used for data analysis. Judgement on mutagen-
serum can be used, providing that the serum has been ad-
icity should be made based on the following information:
equately tested and shown to support the desirable character-
7.1.1 Dose response relationship.
istics of CHO cells as described here. Dialyzed serum is
7.1.2 Significance of response (in comparison to the nega-
usually necessary to eliminate the competition between 6TG
tive control).
andpurinebasesintheserum.Ithasbeenfoundthataselection
7.1.3 Reproducibility of the results.
cell density of 2×10 or fewer cells per 100 mm dish for
7.2 Exact statistical analysis is difficult because the distri-
attached colony growth (14, 16) and 10 or fewer cells per 100
bution of the number of mutant colonies depends on the
mmdish(in30mLofagar)foragarcolonygrowth (15)allows
complex processes of cell growth and death after mutagen
essentially 100% recovery of mutant cells.
treatment. While other appropriate methods can be used, the
following two approximate methods are used commonly:
5. Data Presentation
7.2.1 WeightedRegressionAnalysis—Aweightedregression
5.1 Results from the assay should include the following
analysis where the weights are proportional to the observed
experimental data:
number of mutant colonies divided by the square of the
5.1.1 Concentrations and solvents used for the test article
observed mutant frequency (17). This weighting scheme was
and positive controls.
derived by assuming that the variance of the observed mutant
5.1.2 Absolute and relative cloning efficiencies (CE) in the
frequency is a constant multiple of that which would occur if
concurrent cytotoxicity assay.
the number of mutant colonies on each selection plate per
5.1.2.1 Absolute CE—Absolute CE equals the number of
treatment conforms to a Poisson distribution.Atest compound
colonies formed divided by the number of cells plated.
isconsideredtoexhibitamutagenicresponseiftheslopeofthe
5.1.2.2 Relative CE—Relative CE equals CE (treatment)
mutant induction as a function of test concentrations is greater
divided by CE (solvent control).
than zero at the 0.01 level according to the t-test (18).
5.1.3 Actual number of mutant colonies observed for each
7.2.2 Power Transformation Procedure—A power transfor-
treatment condition.
mationprocedurewithwhichtheobservedmutantfrequencyis
5.1.4 AbsoluteCEatselectionforeachtreatmentcondition.
transformed using the following equation:
5.1.5 Mutant frequency (MF) values, expressed as mutants
6 β
Y 5 ~X1α! (1)
per
...
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