ASTM E1262-88(2003)
(Guide)Standard Guide for Performance of the Chinese Hamster Ovary Cell/Hypoxanthine Guanine Phosphoribosyl Transferase Gene Mutation Assay
Standard Guide for Performance of the Chinese Hamster Ovary Cell/Hypoxanthine Guanine Phosphoribosyl Transferase Gene Mutation Assay
SIGNIFICANCE AND USE
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.
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 () 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 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 2003)
Standard Guide for
Performance of Chinese Hamster Ovary Cell/Hypoxanthine
Guanine Phosphoribosyl Transferase Gene Mutation Assay
This standard is issued under the fixed designation E 1262; 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 the original treated population are monitored for the loss of
functionalHGPRT,presumablyduetomutations.Resistanceto
1.1 This guide highlights some of the more relevant bio-
a purine analogue, 6-thioguanine (6TG) (or less desirably,
logical concepts as they are currently understood, and summa-
8-azaguanine (8AG)), is employed as the genetic marker.
rizes the critical technical aspects for acceptable bioassay
HGPRT catalyzes the conversion of the nontoxic 6TG to its
performances as they currently are perceived and practiced.
toxic ribophosphorylated derivative. Loss of the enzyme or its
The Chinese hamster ovary cell/hypoxanthine guanine phos-
activity therefore leads to cells resistant to 6TG.
phoribosyl transferase (CHO/HGPRT) assay (1) has been
2.2 Because HGPRT is an enzyme of the purine nucleotide
widely applied to the toxicological evaluation of industrial and
salvage pathway, loss of the enzyme is not a lethal event.
environmental chemicals.
Different types of mutational events (base substitutions, frame-
1.2 This guide concentrates on the practical aspects of cell
shifts, deletions, some chromosomal type lesions, and so forth)
culture, mutagenesis procedures, data analysis, quality control,
should theoretically be detectable at the hgprt locus. The
and testing strategy. The suggested approach represents a
CHO/HGPRT assay has been used to study a wide range of
consensus of the panel members for the performance of the
mutagens, including radiations (2-4), and a wide variety of
assay. It is to be understood, however, that these are merely
chemicals (1), and complex chemical mixtures (5).
general guidelines and are not to be followed without the use
of sound scientific judgement. Users of the assay should
3. Characteristics of CHO Cells
evaluate their approach based on the properties of the sub-
3.1 Different CHO cell lines/subclones are appropriate for
stances to be tested and the questions to be answered.
theCHO/HGPRTassay.TheCHO-K1-BH4celllinedeveloped
1.3 Deviation from the guidelines based on sound scientific
and extensively characterized by (6) is probably the most
judgement should by no means invalidate the results obtained.
widely employed. The CHO(WT) cell line and its derivative,
1.4 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-
the cell lines employed, a number of general characteristics are
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
3.1.2 Cultures in logarithmic phase of growth should have a
the X-linked hypoxanthine-guanine phosphoribosyl transferase
population doubling time of 12 to 16 h.
(hgprt) locus (coding for the enzyme, HGPRT) in Chinese
3.1.3 The modal chromosome number should be 20 or 21,
hamster ovary (CHO) cells. Cells originally derived from
as is characteristic of the particular cell line/subclone used.
Chinese hamster ovary tissue are exposed to a test article and,
3.1.4 Cultures should be free from microbial and myco-
following an appropriate cell culture regimen, descendants of
plasma contamination.
3.2 The cell properties that are critical for the assay should
This guide is under the jurisdiction of ASTM Committee F04 on Medical and
be routinely monitored as part of the quality control regimen.
Surgical Materials and Devices and is the direct responsibility of Subcommittee
Routine quality control procedures should include testing of
F04.16 on Biocompatibility Test Methods.
serum and media for each new purchase, as well as myco-
Current edition approved Sept. 10, 2003. Published September 2003. Originally
approved in 1988. Last previous edition approved in 1996 as E 1262 – 88 (1996).
plasma and karyotype checks at least once yearly, preferably
The boldface numbers in parentheses refer to the list of references at the end of
once every three months.
this guide.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
E1262–88 (2003)
4. Mutagenesis Procedures ment. Cytotoxicity is usually expressed as relative CE which is
the ratio of the CE of the treated cells to that of the solvent
4.1 The mutagenesis protocol can be divided into three
control. Viability determination should take into account any
phases: mutagen treatment, expression, and selection.
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) that are known to be optimal for cell growth
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
6 the experimental conditions to detect known mutagens. An
induced responses. A common practice is to plate 0.5 3 10
untreated control may also be included to evaluate the effects
2 6 2
cellsina25–cm flask,or1.5 3 10 cellsina75–cm flask,on
of the solvent on mutagenicity. Commonly used positive
the day before treatment.
controls are ethyl methane sulfonate (EMS) and N-methyl-N8-
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. Gen-
4.3 Expression of Induced Mutations:
erally, the nonaqueous solvent concentration should not exceed
4.3.1 After mutation at the hgprt locus, the mutant pheno-
1 % and should be constant for all samples. As part of the
type requires a period of time before it is completely expressed
solubility test, an aliquot of the test chemical should be added
(expression requires the loss of pre-existing enzyme activity).
to the treatment medium to note any pH changes, the presence
Phenotypic expression is presumably achieved by dilution of
of any chemical precipitation, and any apparent reaction of the
the pre-existing HGPRT enzyme and mRNA through cell
chemical or solvent with the culture vessel. The solvent of
division and macromolecular turnover. At the normal popula-
choice should not have any undesirable reactions with the test
tion doubling times of 12 to 16 h for CHO cells, an expression
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 3 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, alternative methods of subculturing involving suspen-
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
directly to the cofactor mixture. One volume of the S9/cofactor
for suspended colony growth (16).An advantage of the former
mixture is added to 4 volumes of the treatment medium. Other
is that after the colonies are fixed and stained, the plates can be
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
4.2.5 Estimation of Cytotoxicity—Plating CHO cells imme- period of 6 to 8 days and the number of colonies counted after
diately after treatment for cytotoxicity determination is gener- fixing (for example, with 10 % formalin or 70 % methanol),
ally expected to yield the most accurate results. Otherwise, and staining (for example, with 10 % Giemsa or crystal violet).
cytotoxicity can be estimated on the day after treatment. Soft agar colonies are usually counted in situ after a culturing
Aliquots of the cells are plated to allow for colony develop- period of 10 to 14 days.
E1262–88 (2003)
4.4.2 Reliable selection has been established in 7. Data Analysis
hypoxanthine-free medium containing dialyzed serum and 10
7.1 Due to the possibility of stochastic fluctuation, only
µM 6TG. Fetal bovine serum, newborn bovine serum, or calf
samples with no fewer than 100 000 viable cells after treat-
serum can be used, providing that the serum has been ad-
ment should be used for data analysis. Judgement on mutage-
equately tested and shown to support the desirable character-
nicity should be made based on the following information:
istics of CHO cells as described here. Dialyzed serum is
7.1.1 Dose response relationship.
usually necessary to eliminate the competition between 6TG
7.1.2 Significance of response (in comparison to the nega-
andpurinebasesintheserum.Ithasbeenfoundthataselection
tive control).
cell density of 2 3 10 or fewer cells per 100 mm dish for
7.1.3 Reproducibility of the results.
attached colony growth (14, 15) and 10 or fewer cells per 100
7.2 Exact statistical analysis is difficult because the distri-
mm dish (in 30 mLof agar) for agar colony growth (16) allows
bution of the number of mutant colonies depends on the
essentially 100 % recovery of mutant cells.
complex processes of cell growth and death after mutagen
treatment. While other appropriate methods can be used, the
5. Data Presentation
following two approximate methods are used commonly:
5.1 Results from the assay should include the following
7.2.1 Weighted Regression Analysis—A weighted regres-
experimental data:
sion analysis where the weights are proportional to the ob-
5.1.1 Concentrations and solvents used for the test article
served number of mutant colonies divided by the square of the
and positive controls. observed mutant frequency (17). This weighting scheme was
5.1.2 Absolute and relative cloning efficiencies (CE) in the derived by assuming that the variance of the observed mutant
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.
mation procedure with which the observed mutant frequency is
5.1.4 Absolute CE at selection for each treatment condition.
transformed using the following equation:
5.1.5 Mutant frequency (MF) values, expressed as mutants
b
per 10 cells.
Y 5 ~X1a! (1)
5.1.5.1 Mutant Frequency (MF) Values—MF values equal
the number of mutant colonies divided by the number of
where:
clonable cells.
Y = transformed mutant freq
...
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