ASTM E1262-88(1996)

NOTICE: This standard has either been superceded and replaced by a new version or discontinued.
Contact ASTM International (www.astm.org) for the latest information.
Designation: E 1262 – 88 (Reapproved 1996)
Standard Guide for
Performance of the 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
functional HGPRT, presumably due to mutations. Resistance to
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-
2 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, etc.) should
culture, mutagenesis procedures, data analysis, quality control,
theoretically be detectable at the hgprt locus. The CHO/
and testing strategy. The suggested approach represents a
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.
the CHO/HGPRT assay. The CHO-K1-BH4 cell line developed
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.
Current edition approved July 29, 1988. Published September 1988.
The boldface numbers in parentheses refer to the list of references at the end of
this guide.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
NOTICE: This standard has either been superceded and replaced by a new version or discontinued.
Contact ASTM International (www.astm.org) for the latest information.
E 1262 – 88 (1996)
serum and media for each new purchase, as well as myco- ally expected to yield the most accurate results. Otherwise,
plasma and karyotype checks at least once yearly, preferably cytotoxicity can be estimated on the day after treatment.
once every three months. Aliquots of the cells are plated to allow for colony develop-
ment. Cytotoxicity is usually expressed as relative CE which is
4. Mutagenesis Procedures
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
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
cells in a 25–cm flask, or 1.5 3 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-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
concentration of solvent remains constant for all samples. Cells
subcultured with 0.05 % trypsin with or without EDTA.
are generally treated with the test article for at least 3 h. For
Aliquots of 1 3 10 cells are subcultured at 2 or 3 day intervals
treatment times of 3 to 5 h, serum–free medium can be used. 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
4.4 Mutant Selection:
(9, 10), appears to be another useful addition. However, the
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
of mutant cells to form colonies. In general, cells are plated in
glucose-6-phosphate (5 mM), potassium chloride (30 mM),
magnesium chloride (10 mM), and CaCl (10 mM). S9 is added tissue culture dishes for attached colony growth (11), or in agar
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),
NOTICE: This standard has either been superceded and replaced by a new version or discontinued.
Contact ASTM International (www.astm.org) for the latest information.
E 1262 – 88 (1996)
and staining (for example, with 10 % Giemsa or crystal violet). totoxic test articles, the highest concentration has generally
Soft agar colonies are usually counted in situ after a culturing been 1 to 10 mg/mL, or to the limit of solubility.
period of 10 to 14 days.
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-
and purine bases in the serum. It has been found that a selection
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 mL of 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
concurrent cytotoxicity assay.
frequency is a constant multiple of that which would occur if
5.1.2.1 Absolute CE—Absolute CE equals the number of
the number of mutant colonies on each selection plate per
colonies formed divided by the number of cells plated.
treatment conforms to a Poisson distribution. A test compound
5.1.2.2 Relative CE—Relative CE equals CE (treatment)
is considered to exhibit a mutagenic response if the slope of the
divided by CE (solvent control).
mutant induction as a function of test concentrations is greater
5.1.3 Actual number of mutant colonies observed for each
than zero at the 0.01 level according to the t-test (18).
treatment condit
...