Recommended protocols based on a survey of current practice in genotoxicity testing laboratories: II. Mutation in Chinese hamster ovary, V79 Chinese hamster lung and L5178Y mouse lymphoma cells.

255

Mutatton Research, 246 (1991) 255-284 © 1991 Elsewer Science Pubhshers B V (Biomedical Division) 0027-5107/91/$03 50 ADONIS 002751079100053L

MUT 02955

Recommended protocols based on a survey of current practice in genotoxicity testing laboratories: II. Mutation in Chinese hamster ovary, V79 Chinese hamster lung and L5178Y mouse lymphoma cells E.R. N e s t m a n n

a,

R.L. Brillinger a, J.P.W. G i l m a n and S.H.H. Swierenga d

b,

C.J. R u d d c

a CanTox lnc, 627 Lyons Lane, Sutte 200, Oakvtlle, Ont. L6J 5Z7 (Canada), b Bwmedwal Scwnces, Unwerstty of Guelph, Guelph, Ont NI G 2 WI (Canada), c SRI Internattonal, 333 Ravenswood A venue, Menlo Park, CA 94025 (U S A ) and a Bureau of Drug Research, Health Protectwn Branch, Health and Welfare Canada, Tunney's Pasture, Ottawa. Ont KIA OL2 (Canada)

(Recewed 5 February 1990) (Revision recetved 26 April 1990) (Accepted 15 May 1990)

Keywords Recommended protocol, Current usage, Point mutation. CHO, V79, L5178Y mouse lymphoma

Summary Laboratory protocols and gmdehnes have been developed for the performance of point mutation assays using Chinese hamster ovary (CHO) cells, V79 cells, and L5178Y mouse lymphoma cells. Since only minor differences in the treatment of CHO and V79 cells exast, these two assays could be combined in one procedural guideline. A second protocol was developed for the mouse lymphoma assay in order to incorporate concerns and methods specific to that cell type and genetic locus. The protocols were based primarily on current laboratory practices as determined by responses to a detailed questionnaire completed by North-American and European governmental, university and contract laboratories involved with in vitro mutation testing. This report identifies those modifications to previously described methodologies which are being used on a regular basis, provides recommendations, and also serves to clarify confusing or inconsistent practices.

The three mutation assays described utilize three different mammalian cell lines and involve various differences in protocol, but operate under the same basic principles. In each, treatment with a test chemical is carried out to induce a forward mutation at a specific locus. The cultures then are incubated to allow phenotypic expression of any

Correspondence Dr. S H H. Sw~erenga, Bureau of Drug Research, Health ProtecUon Branch, Health and Welfare Canada, Tunney's Pasture, Ottawa, Ont K1A 0L2 (Canada)

reduced mutants, and finally, the cells are allowed to form macroscopic cultures in the presence of an agent that allows for only the growth of the mutant cells. The three cell types discussed here are Chinese hamster ovary (CHO), Chinese hamster lung (V79) and mouse lymphoma L5178Y. The L5178Y mouse lymphoma clone used in the mutation assay is the TK-/--3.7.2C strain which is heterozygous at the thymidine klnase (TK) locus. The T K enzyme is responsible for incorporating exogenous thymldlne, via a salvage pathway, into the cell in the form of thymidine

256

monophosphate (Cole and Arlett, 1984). Analogues of thymidine, such as trIfluorothymldlne (TFT), can also be phosphorylated by TK, which leads to cell toxicity Forward mutation at this locus results in a loss of TK activity and subsequent resistance to TFT, which is used as the selective agent to kill wild-type cells (Clive et al., 1987). TK -~- mutants are not killed by T F T and are able to survive due to the ability to synthesize purInes de novo. Both CHO (Chinese hamster ovary) and V79 (Chinese hamster lung) cells are used to monitor mutation events at the X-linked hypoxanthme guanine phosphoribosyl transferase ( H G P R T ) locus. The H G P R T enzyme normally converts the purlnes hypoxanthme and guanine Into nucleotides via a purlne nucleotide salvage pathway, which is used in preference to the de novo purine biosynthetic pathway. Purlne analogues, such as 6-thioguanine (TG), are also substrates for the H G P R T enzyme and are converted to toxic rlbophosphorylated derivatives (Li et al., 1987). H G P R T mutants of C H O and V79 cells do not have a functional purlne salvage pathway and rely on de novo purine biosynthesis for surv,val Thus, H G P R T - mutants are able to survive and develop Into mutant colonies in concentrations of purine analogues that kill wild-type cells. The H G P R T gene is located on the X-chromosome of both humans and rodents (Bradley et al., 1987), which may be advantageous for interspecies extrapolation As well, a human disease, the L e s c h - N y h a n syndrome, IS known to arise from the H G P R T - genotype (Hsle et al., 1981). In order to discover what protocols are being used in research and testing laboratories and to deterrmne the modifications being made to published mammalian cell mutation procedures, 77 responses to a detailed questionnaire were obtained from laboratories in Canada, the United States and Europe A list of the participants in this effort is presented in Appendix A. The frequency of usage of the technique varied widely: some labs reported conducting mutation assays five times per year or less, while others, as many as 300 times in a year. These responses were incorporated Into the recommended protocol in two ways: (i) the most common answer (on a percentage basis) to each question was used as the

recommended procedural step, and (n) alternative responses were often included in the Comments column of the protocol or in the discussion As often as possible, each step in the protocol was representative of a separate question on the questionnaire Published reference sources frequently cited by the respondents were also consulted, and the resultlng protocol is a combination of the ideas presented in several publications, modified by the practical expertise of respondents to the questionnalre If a clear majority was apparent in the responses to the questionnaire, this was the procedural step Included in this protocol When answers vaned widely, the literature was consulted. For steps not addressed in the questionnaire, the most commonly cited references were consulted The comments section contains less popular techmque~ and hints for improving data quahty. Unless cited in the text of the protocol, the procedure and accompanying comments are derived from responses to the questionnaires a n d / o r from the references most commonly cited by the respondents, as listed at the end of this section. More detailed information pertaining to the origin(s) of specific recommendations and an example of the complete point mutation questionnaire can be obtained from S. Swlerenga. There are two distinct protocols presented m the following section, one describing the steps Involved for a mutation assay in either C H O or V79 cells, and the other representing the procedure for mouse lymphoma L5178Y cells. Since C H O and V79 are derived from the same animals species and are usually selected for mutation at the same locus (HGPRT), the recommended protocols are very similar, and are combined and discussed separately from the L5178Y assay In areas where differences exist, these differences are described m the procedure or in the accompanying comments. There are examples available in the published hterature of similar guidelines for the use of CHO, V79 and L5178Y cells m mutation assays For both C H O and L5178Y, guidelines have been recommended by a Subcommittee of the American Society for Testing of Materials (ASTM) m collaboration with the Enwronmental Mutagen Society. These guidelines were based on a con-

257 sensus of the Subcommittee members in order to aid in the performance, understanding and analysis of the CHO (Li et al., 1987) and T K (Clive et al., 1987) assays. For the V79 line, a review and analysis of the literature pertaining to the V79 assay is described m a report to the U.S. EPA Gene-Tox Program prepared by a Work Group within the Program (Bradley et al., 1981). The important distinction between these publications, as well as the others that were consulted, and the

one presented herein is that this report is based on current usage and method modification capture. As such, it provides valuable information pertaining to the methods and modifications of preference in laboratories utilizing the protocols on a regular bas~s, at the time of the survey. Furthermore, it is more detailed than a guideline and can be used as a step-by-step protocol at the laboratory bench if desired.

Procedure for C H O / V 7 9 mutation assay

Comments

1. O. Cell maintenance and quahty control 1.1. Assay cell type. Two cell lines derived from the Chinese hamster can be used for detection of mutation events at the H G P R T locus, namely CHO and V79 cells. The Chinese hamster ovary (CHO) subclone CHO-K1-BH 4 is the most widely used CHO strain, and can be obtained from A.W. Hsie at Oak Ridge National Laboratory. V79 cells, which can be obtained from E.H.Y. Chu at the University of Michigan or from ATCC, are derived from the lung of male Chinese hamsters.

1.1. The CHO-K1-BH 4 subclone has the same characteristics as the parent CHO line (well characterized, readily synchronizable, stable, easily identified karyotype) as well as having a low rate of spontaneous mutation at the H G P R T locus. Other CHO subclones are available for detection of mutation at other loci. Some examples include CHO-1B-2 (APRT locus) and CHO-AT3-2 (TK and A P R T loci) (Carver et al., 1980), and AA-8 (APRT locus) (Thompson et al., 1983). The CHO wild-type line enables the use of several genetic markers, including resistance to thioguamne, ouabain, emetine, methylglyoxal bisguanylhydrazone and 5,6-dichlororibofuranosyl benzimidazole (Gupta and Singh, 1982).

Desirable characteristics of the V79 cell line include a rapid growth rate, high cloning efficiency, stable karyotype, and reproducible charactenstics following cryopreservatlon. Other loci on the V79 cell have been used for detection of mutations, most notably that for ouabain resistance (Langenbach et al., 1978).

1.2. Cell mamtenance. Cell cultures are normally maintained in a 75-cm2 flask. Subculturlng should be done every 2 - 3 days for CHO cells and every 3 - 7 days for V79 cells to prevent cultures from becoming confluent. The conditions for subculturing should be constant with a pH of 7.2-7.4, a CO 2 level of 5% and relative humidity of 95100%.

1.2. Checks for viability can accompany the subculture procedure (see Section 6.5). There is no strict hmit on the number of subculture passages per culture.

258 Cells can be frozen and then stored in a hquld nitrogen freezer ( - 1 9 6 ° C ) in ahquots of at least 106 cells/ml of complete medium with 10% DMSO. To use, aliquots are thawed rapidly in a 37°C waterbath, then diluted as necessary in medium. Subculturlng should take place approximately 24 h after thawing.

1 3. Cell hne quahty control To ensure maintenance of cell properties, it is recommended that determination of colony forming efficiency, spontaneous mutant frequency and karyotype analysis be conducted for each stock culture. Historical results should be relied upon for comparison for these parameters. As gmdehnes, colony forming efficiency should range from 50 to 100%, spontaneous mutant frequency at the H G P R T locus from 0 to 2 0 × 1 0 6 mutants for C H O c e l l a n d f r o m 0 to 100 × 10 6 for V79 cells, and the karyotype should show 22_+ 1 chromosomes. Checks for mycoplasma should be done approximately every 3 months. The response to positive control chemical(s) should be monitored as well by inclusion m each assay (see Section 4.1) Stocks should be rejected if responses for any of these parameters vary significantly compared to historical values and the results cannot be attributed to other experimental variables.

1 3 Growth curve records are maintained to provide an historical basis for comparison of these parameters. Other parameters that can be monitored include saturation density, reclonlng, and verification of genetic markers. Genetic markers that can be verified include T G and 8-azaguanxne resistance, H A T G sensitivity, specific activity of lmmunopreclpitated H G P R T (should be _< 5-10% of the wild-type value), and the Incorporation of radioactive hypoxanthine (should be _< 5-10% of the wild-type value)

1.4 Cleansing of cultures The spontaneous mutant frequency increases with subculturmg, and to prevent high backgrounds, cleansing of the cultures should be done as required. This is done by culturing cells in H A T G medium (hypoxanthtne, aminopterm, thyrmdme and glycine), either on a regular basis or just prior to preparing cells for treatment.

1 4 Ahquots of cleansed cultures can be frozen and stored for future use if desired. A m m o p t e r m inhibits the de novo synthesis of nucleotides, therefore cells depend on the 3 exogenous purlnes in the m e d m m for incorporation via the salvage pathway Spontaneously arising H G P R T - mutants cannot use these substrates and therefore do not survive. A disadvantage of repeated treatment, however, is the possibility of selection of H G P R T cells more resistant to the amlnopterm treatment Cleansing, with H A T G , y~elds an increased fraction of H A T G and G T G resistant cells over time. As a result reclomng is required.

1.5. Medium and reagents. H a m ' s F12 medium is most commonly used for C H O cell cultures, and can be obtained from Glbco or K C. Biolog~cals For V79 cells, minimal essential medium (WII-

1 5 Funglzone can also be added to both H a m ' s F12 and MEM. Antibiotics such as gentamycln, kanamycin and amphotericin can be used in conjunction with or instead of p e n / s t r e p .

259 liams, Eagle, Dulbecco or Alpha MEM), available from Gibco, is recommended. When stored at 4°C, Ham's F12 has a shelf life of approximately 6 months to 1 year, while MEM should only be used for 8-12 weeks. Either gentamycin (25-50 /~g/ml) or p e n / s t r e p (100 units penicilhn/ml and 100 /zg streptomycin/ml) are routinely added to Ham's F12 medmm. To MEM, supplements include pen/strep (100 units penicillin/ml and 100 #g streptomycin/ml) and L-glutamlne (2.0 mM). Fetal bovine serum (FBS), obtained from Gibco, is added at concentrauons of upto 10%, to culture cells. FBS should be stored at - 2 0 ° C and can be kept for 6 months to 1 year. It is advisable to conduct tests for growth-promoting activity of each new lot of serum, usually by determination of colony-forming efficiency a n d / o r growth rate, and to ensure that it has no adverse effects on the assay.

1.6. Test chemtcal solubzhty. Testing for the limit of solubility should be conducted prior to beginning the assay in case the concentration range tested is affected. Common organic solvents include DMSO, acetone and ethanol. Solvents for water-soluble test chemicals include Fischers or RMPI 1640 medium, water, saline and Hank's balanced salt solution. In addition to solublhty determination, addition of test chermcal and solvent to the treatment medium should be carried out to observe changes in pH, precipitate formation, osmolarity and reaction with the vessel. Changes in pH, measured using a pH meter or colour changes, should be noted. If necessary, 1.e., if the pH is either very acidic or very basic, titration with NaOH or HC1 can be used to neutralize. The following steps should be carried out before commencing a mutation assay: (1) Selection of concentration range (Section 3.0). (ii) Solubility of test chemical, pH changes (Section 1.6). (iii) Preparatton of media: Culture medium (Section 2.1), Treatment medium, with and without $9 (Section 5.3), Selection medium (Section 8.2).

The addition of antibiotics to cell cultures can cover up mycoplasma infections and should be used with caution. If desired, pre-tests for growth promoting ability (e.g., colony forming efficiency, growth rate, cell density) and for adverse effects on the assay can be conducted for each new batch of medium.

Although FBS is the most common, virtually any sera can be used for this protocol.

26O

2.0 Preparation of cultures 2 1. Cells are seeded m 5 - 1 0 ml of culture m e d t u m with 10% fetal b o v i n e serum (FBS). Either flasks or & s h e s can be used for culturing

21

CHO culture medmm 450 ml Ham's F12 50 ml FBS (10%)

V79 culture medmm 450 ml Dulbecco's MEM 50 ml FBS (10%)

T h e culture m e d i u m can be stored at 8°C for 7 - 1 0 d a y s without loss of activity. A n y type of r m m m a l m e d i u m (e.g.. H a m ' s F12, Eagle's, A l p h a or D u l b e c c o ' s M E M ) can be used for these cells, t h o u g h the ones specified a b o v e are the m o s t p o p u l a r . Serum c o n c e n t r a u o n can range from 5 to 10%. T h e size of culture vessels also ranges, usually either 100-mm dishes or 25-cm 2 flasks for C H O , a n d 100-mm dishes or 75-cm 2 flasks for V79. H y p o x a n t t u n e - f r e e culture m e d i u m , with 5% FBS, can be p r e p a r e d a n d used for selection m e d m m after a d d i n g only 6 - t h l o g u a n m e (see Section 8.2). Initial seeding d e n s i t y d e p e n d s on the d u r a t i o n of i n c u b a t i o n , which can range f r o m 4 to 24 h for C H O cells a n d 18 to 48 h for V79. T h e following table c o n t a i n s the i n f o r m a t i o n necessary to deterrmne initial seeding density:

Incubation period (h) Doubhng time (h) Treatment concentration (cells/vessel) Volume of suspension (ml) lmtlal seeding density (cells/vessel)

CHO

V79

24 12-16 1 X 10 6 a 5 5 × 105 ~

24 12-16 1 × 10 6 10 2 5 × 105

Higher seeding densltxes can be used (106-2×106 cells) yielding more cells for treatment

2.2. Cells are i n c u b a t e d for 24 h, u p o n which time there should b e a p p r o x i m a t e l y 1 x 1 0 6 cells p e r culture vessel. Cells should b e m e x p o n e n t i a l g r o w t h p h a s e u p o n the l m t i a t l o n of treatment. 2.3. Number of culture vessels requtred. Cultures are r e q m r e d for b o t h the m u t a t i o n assay a n d the p r e l i m i n a r y c y t o t o x i c l t y assay, which is perf o r m e d a n d c o m p l e t e d b e f o r e the m u t a t i o n assay can be started (refer to Section 3.0). F o r the c y t o t o x i c i t y assay, two culture vessels are r e q m r e d for each c h e m i c a l c o n c e n t r a t i o n (one each for presence a n d a b s e n c e of $9) over the test range, a n d two are r e q u i r e d for the solvent c o n t r o l ( + / -

2.3. The n u m b e r of cultures required for a typical e x p e r i m e n t , with 10 doses tested m the p r e h m l n a r y c y t o t o x i c i t y assay a n d 4 c o n c e n t r a tions used m the m u t a t i o n assay, w o u l d be. Cytotoxaclty test = 2 ~ × (10 chemical levels + 1 negative control ) = 22 cultures

261 $9). For the mutation assay, a total of 4 vessels should be used for each treatment level and for each positive and negative control tested, two each in the presence and absence of metabolic actwation.

Mutation assay = 2 a × (4 chenucal levels+ 1 negatwe control + 1 positive control) × 2 rephcates = 24 cultures a

+/-$9

If preliminary cytotoxicity tests (Section 3.0) indicate that the test chemical significantly reduces the survival of culture cells it becomes necessary to increase the number of cultures to ensure that an adequate number of colomes are formed to assess mutagenioty. [For example if survival is 100% and background mutant frequency is 2 × 10 -6, then 4 cultures, seeded at 1 × 106 cells per vessel, have 106 × 4 × 2 × 10 -6 = 8 colonies. If treatment doubles the mutant frequency but yields a survival of 50% then 8 colonies are still observed.] 3.0. Selectton of treatment levels A minimum of 4 treatment levels should be selected, usually on the basis of preliminary cytotoxicity tests that determine colony-forming efficiency (CFE). Cytotoxicity tests must be carried out over a range of test compound concentrations prior to beginning the experiment.

3.0. For V79 cells, a decrease in cell counts, either alone or in conjunction with C F E determination, is sometimes used to determine cytotoxicity.

3.1. Prehmmary cytotoxtclty test 3.1.1. Cultures, prepared as described in Section 2.0, are treated with a range of chemical concentrations (refer to Sections 5.1-6.3).

3.1.1. A possible range of concentrations could start at a high dose of 3-10 m M and decrease m half-log amounts (i.e., 3, 1, 0.3, etc.). Untreated solvent controls are essential for the calculation of survival of the treated cultures relative to controls. Solvent controls are also recommended for inclusion to observe possible related toxicity and their effects on background levels.

3.1.2. Aliquots of treated cultures, approximately 100 V79 or 200-5000 C H O cells are inoculated in 4 - 5 ml of minimal medium (F12 or D - M E M ) in 60-mm dishes. Triplicate plates are prepared for each concentration.

3.1.2. The number of cells inoculated depends partly on expected survival.

3.1.3. After 37°C incubation for 7 days, the developed colonies are rinsed with sahne, fixed, stained and counted using a calibrated electronic counter or hand counter (refer to Section 8.1).

3.1.3. For C H O cells, fixation ~s usually done with 3.7% formalin, and crystal violet solution (0.1%) is used to stain. For V79 cells, methanol is commonly the fixation agent and Giemsa stain is recommended.

262

3 2 Determmatton of concentratton range Survival relative to untreated controls (1.e, the relative CFE) is determined for each dose level tested (refer to Section 7.3). Recommended treatment concentrations should include at least 4 doses that gwe cell survivals of ranging between 100 and 10% survival relative to control cell survival.

3.2. Survival at a mlmmum of 4 doses should be found at the end of the experiment, and the use of more than 4 is recommended to take into account possible losses due to cytotoxlcity, contamlnatlon, etc. Survival curves, plotting the log of the relative CFE (refer to Section 7.2) against a linear dose scale, can be used to determine the concentration range. Cultures that have less than 10% of the cells surviving are not usually useful for evaluation of mutant frequencies because substantial variability in apparent spontaneous mutant frequencies can occur. In order to avoid toxic effects on the cultures, organic solvent concentrations should not exceed 1% (v/v) Non-toxic, highly soluble chemicals should be tested at a maximum concentration of 0.1-10 mg/ml.

4.0 Controls 4.1. Posttwe controls If possible, positive controls should include known carclnogens/mutagens which are structurally sxmilar to the test compound.

4.1 Commonly used positive controls for both C H O and V79 include: Direct acting (no acuvauon needed) Ethyl methanesulfonate (EMS) [200-500/~g/ml] N-methyl-N'-nltro-N-nltrosoguamdme(MNNG) Promutagens (requmng actwauon) Dimethylmtrosamme (DMN) 7,12-Dlmethylbenzanthracene (7,12-DMBA) 2-Acetylammofluorene(2-AFF) Benzo[a]pyrene 3-Methylcholanthrene (3-MCA) [5 p.g/ml] It may be advantageous to include two positive controls, one with an anticipated strong response (e.g., EMS) and one with a moderate response (e.g., 2-AAF), in order to provide a basis for comparison. An attempt to use both promutagens and direct-acting mutagens should be made.

4.2. Negatwe controls. Solvent controls should always be included in the assay, and the use of untreated controls with no solvent is also recommended. Negative controls both with and without $9 metabolic activation should be run.

4.2 Noncarcmogenlc analogues of the test compound should be used as negative controls if avadable, though thas is not commonly done. The highest concentration of the solvent used in the test chemical preparations should be used in the negative control treatments, and the same volume should be used.

5.0 Treatment of cultures The remainder of the assay should be carried out for each concentration of test chermcal, and for all positive and negative controls. Each treatment should be conducted in duplicate

5.0. The number of rephcate cultures may be increased if variation in background mutant frequencies is high or if the compound is hkely to be a weak mutagen.

263 5.1. Actwatton. Cultures should be treated with and without Aroclor 1254-induced rat-liver homogenate S9-mix. Negative controls should also revolve + / $9 treatments. Normally, S9-mix is added to the treatment medium in a ratm of 1 part $9 mix to 4 parts medium.

S9-mix consists of rat-liver $9, induced by Aroclor 1254, which has been buffered and supplemented with the essential co-factors N A D P and glucose 6-phosphate. $9 can be stored for at least 1 year at temperatures below - 8 0 ° C without loss of activity. Once it has thawed, $9 should never be re-frozen for subsequent use (Venitt and Parry, 1984). 5.2. Addttton of treatment medtum. For each culture prepared in Section 2.0, replace culture medium with 4 - 5 ml of treatment medium containmg $9 metabolic activation as required.

5.1. C H O cells lack the ability to metabolize m a n y chemicals to their mutagenic form, in a manner sinular to m a m m a h a n cells (Venitt and Parry, 1984). A 1 : 4 ratio of $9 rmx to treatment medium is usually tolerated, however, if excessive toxicity is noted during the preliminary cytotoxicity tests, the amount may vary. Alternatives to $9 activation for C H O cells is the use of a host-mediated system (O'Neill et al., 1977b; Hsie et al., 1978), or a cell-mediated system with rat hepatocytes (Langenbach et al., 1978; Jones and Huberman, 1980). The latter type of activation may represent more closely metabolic processes in intact cells (Jones and Huberman, 1980).

5.2. Treatment medtum. 10-60 #1 test chenucal at the appropriate concentration, dissolved m solvent (refer to Section 3 1) 1 ml $9 rmx, ,f reqmred Serum-free rmmmal medmm (F12 or D-MEM), to a final volume of 5 ml

The addition of 5-10% FBS to the treatment medium is common, however, the presence of serum in the treatment medium can reduce the mutagemc activity of chemicals that have an affinity for serum proteins, and its use during the treatment phase of the experiment should be avoided. Serum is used to maintain normal cell growth and division, thus, m cases when the treatment duration exceeds 5 h, the inclusion of serum (5-10% a) may be necessary. 5.3. Treatment duratmn. Cultures are incubated m the presence of the test chemical for a period of 1-5 h. The duration with and without $9 activation is usually the same.

5.3. Decomposition of the test compound can occur and may limit the duration of treatment. The incubation length should be adjusted accordlng to the half-hfe of the compound, if known. Incubation duration in the presence of $9 should not generally exceed 5 h since $9 is often toxic to cells. If possible, an effort should be made to use the same treatment duration as in other in vitro mammalian cell assays so that a basis of comparison is maintained.

6.0. Post-treatment 6.1. Following the treatment period, cultures are washed once with a calcium/magnesium-free saline solution containing disodium E D T A (0.02%).

6.1. The presence of E D T A (or EGTA) in the sahne wash, and the absence of calcium and magnesium, facilitate the action of trypsin and, thus, the detachment procedure. Saline G and Dulbec-

264

co's phosphate-buffered sahne (PBS) are other alternauve washes. More than one wash may be required to remove the chemical. Vacuum aspiration can be used to effioently remove the medium before rinsing.

6.2. Trypsmtzanon. Cells are detached from the culture vessel by a 3 - 4 min incubation at 37°C w~th 1 ml of a trypsin (0.05% porcine pancreatlc trypsin) and E D T A solution (0.02%).

6.2 Alternatively, 1 - 2 ml of trypsin (0 05% in s a l i n e / E D T A rinse solution) is added to the vessel for 1 nun and removed. Cells are incubated for 1 - 3 min at 37°C in order to allow cells to detach. Trypsin cleaves the proteins responsible for the attachment of cells to the culture vessel. The role of E D T A / E G T A ~s to chelate calcmm, magnesmm and serum proteins which may inhibit the activity of trypsin. E D T A / E G T A can be omitted, but longer incubations, higher trypsin concentrations, a n d / o r thorough washes prior to treatment may be necessary to achieve good cell recovery.

6.3. The cell density is deterlmned using a hemocytometer or electronic counter. 6.4. One half of the culture (approximately cells) is replated in either 10 ml of m e d m m (F12, 5-10% FBS) in 100-mm dishes or in 5 ml of m e d m m (F12, 5-10% FBS) per 60-mm dish, to be used in the expression phase of the experiment (Section 7.0). An aliquot is removed from the remaining culture preparation in order to determine the cell density and initial survival rate. 10 6

6.4. The determination of the initial cell survival is most accurate and reproducible if done ~mmediately following treatment, and tins is the recommended procedure. However, trypslmzat~on ~mmedlately following treatment sometimes increases cytotoxxcity, and a 24 or 48 h recovery period is sometimes used. In tins case, following rinsing, 5 ml of fresh minimal medium should be added and cells should be incubated for the additional 24 or 48 h until trypslnizatlon. In this case, the relative numbers of cells m the dishes will be a better indicator of survival than will be the cloning efficiency. Another possibility is to treat the cells in suspension, thereby evading the problem of trypsinlzat~on post-treatment.

6.5. Determtnatlon of mtttal survtval 6.5.1. The cell density of the aliquot is determined. 6.5.2. Ahquots contaImng 200 cells are added to 5 ml of culture medium in 60-mm dishes, in tnphcate for each treatment.

6.5.2 Plating density can be increased if low survival is expected.

265

6.5.3. Following a 7-day incubation period, colonies are fixed, stained, and counted (refer to Section 9.1). Colony-forming efficiency (CFE) is calculated, as described in Section 7.3. 7.0. Expresston 7.1. The culture prepared in Section 6.4, containmg approximately 1 × 10 6 cells, is incubated at 37°C for the expression period of 7 - 9 days for CHO cells and 6 - 7 days for V79 cells, during which time 2-3 subcultures are made.

7.1. During the 7-day expression period, preexisting enzyme activity is lost and the mutant phenotype becomes fully expressed. There are normally 10-14 generations during this time, with a population doubling time of 12-16 h for CHO cells and 12-16 h for V79 cells.

7. 2. Preparanon of subcultures 7.2.1. Cultures are trypsinlzed and aliquots removed for cell density determination. 7.2.2. Cultures are plated in 10 ml of culture medium in either 100-mm dishes or 75-cm2 flasks at a seeding density of 1 × 106 cells/vessel.

7.2.2. 2 )< 106 cells can also be subcultured in 75- or 150-cm2 flasks.

7.2.3. Subcultures are prepared every 2-3 days of the expression period, for example on Days 1, 3 and 5.

7.2.3. Frequency of subcultunng can vary depending on cell density. One subculturing should occur after every 2-3 doubling times (i.e., when cell density equals approximately 4 )< 1 0 6 cells/vessel).

7.3. Determmatton of colony-formmg effwtency The colony-forming efficiency (CFE) is normally determined at the last subculture. This is done using the same method as the survival test (refer to Section 6.5). The relatwe CFE for each treatment concentration is calculated as a percentage of the untreated solvent control CFE. 7.4. On Day 7 of the expression period, the cells are washed with a calcium/magnesium free saline solution containing disodium EDTA (0.02%) and treated with a t r y p s i n / E D T A solution to detach them from the vessel in preparation for plating for selection.

8.0. Selection 8.1. Cell density, is determined using an aliquot of the final subculture preparation.

7.3. Calculatton of CFE Absolute CFE =

number of coloniesformed number of cellsplated × 100%

Relatwe CFE = absolute CFE (treatment) × 100% absolute CFE (control)

266

8 2 Approximately 2 × 105 cells are 100-ram dishes containing 10 ml of medium. At least 5 dishes are prepared ment culture (total of 10 dishes per level).

seeded m selectmn per treattreatment

8.2. Selectton medtum

(5% FBS)

470 ml hypoxanthlne-free minimal m e d m m (F12 or D-MEM) 25 ml dialyzed FBS 5 ml of a 1 m M 6-thloguanlne standard stock solution

The standard 1 mM stock solution of 6-thloguanine Is prepared by addition of 167.2 /zg 6thloguamne per 1 ml of ddute NaOH for C H O cultures or N a : C O 3 for V79 cultures. Dialyzed serum is used to ehrrunate competenon between TG and the purme bases m the serum. Hypoxanthlne is eliminated for the same reason. 10 rephcate plates are commonly prepared for each treatment level m the V79 assay Cells can be selected in a sermsohd agar medmm (106 cells/30 ml agar/100 mm plate) (LI and Shlmlzu, 1983). One advantage of this method ~s the reduction of metabohc competmon between wild type and mutant cells and a resulting htgher mutant frequency.

8.3

Selectmn plates are incubated for 7 days.

84. After incubation, stained and counted.

colonies

are

fixed,

Note. It ~s recommended that each experiment be repeated once, and possibly again ff eqmvocal results are obtained. The conditions should be duplicated as precisely as possible, with slight modifications to the dose range as applicable.

8 3 Incubation can exceed 7 days if colony formation appears to be slow At least 14 days are required for colony formation of C H O cells m semisolid medium because many more cells are needed to make detectable colonies than for attached cultures.

8.4. After Incubation, colonies are rinsed m water or saline, fixed, stained and counted. After fixing and staining, plates can be stored for counting if desired.

9.0. Data collectton and calculations 9.1. Counting colomes. Colony counts are usually done by eye, although electronic counters are sometimes used. An acceptable criterion for minimum colony stze can be those with a diameter of 0.5 mm or greater or those with a mimmum of 50 cells per colony.

9.1 A description of the mimmal acceptable colony size should be included in the methodology.

9.2 Calculatton of mutant frequency. The mutant frequency (MF) is estimated for each

9.2

267 treatment by the following calculation: number of mutant colomes MF= (total cells seeded on all rephcate plates) × absolute CFE The C F E value used is calculated from data obtained at the end of the expression period (refer to Section 7.2 above). M F is usually expressed as the frequency per 106 viable, or clonable, cells.

The use of these units are deceptive unless 10 6 cells or more are present m each culture during the entire experiment. If 90% of the cells are killed by the treatment, leaving 105 cells at the beginning of the expression period, the significance of the data is limited to 105 cells even if they grow back up to 10 6 cells. approximately

10.0. Data analysts 10.1. Spontaneous mutant frequency. The normal acceptable range of N F values for untreated or solvent controls is 0 - 2 0 mutant colonies per 106 clonable cells for C H O cultures and 0-100 mutant colonies per 10 6 clonable cells for V79 cultures.

10.1. A spontaneous M F higher than 20 for C H O or 100 for V79 may increase the chance of obtaining false negative results for weak mutagens, whose response may be masked by the high background. This can be prevented by frequent cleansing of cultures (refer to Cell Maintenance and Quality Control).

10.2. Crtterta for a poslttoe response The factors considered in the assessment of the mutagenlc activity of a test compound differ slightly for the C H O assay and the V79 assay. For C H O results, the following factors are considered by respondents to be indicatwe of a positive response: (1) dose-related increase in the M F (refer to Section 10.2.1); (li) statistical significance (refer to Section 10.2.2); (iii) reproducibility of results; (iv) results outside acceptable range of historical negative controls; a n d / o r (v) increase in absolute number of mutant colonies with respect to controls.

10.2 For CHO, the factors most commonly considered in combination are the presence of a dose response and statistically significant increases in MF. Consideration is sometimes given to increases (statistically significant or 2- or 3-fold increases) in absolute numbers of mutant colonies as compared to negative controls.

For V79 results, the following characteristics are considered: (1) dose-related increase m the M F (refer to Section 10.2.1); (li) reproduciblhty; (iii) 3-fold increase m M F over controls; a n d / o r (iv) increase in the absolute number of mutant colonies with respect to controls.

For V79, either the existence of a dose response or a m i m m u m 3-fold increase over background is evidence of a mutagemc action. The effects must be shown to be reproducible. As for C H O tests, some consideration should also be given to the effect of treatment on absolute mutant counts.

10.2 1. Dose responses. Dose responses can be assessed using statistical a n d / o r graphical methods. Linear and non-hnear dose responses can be observed by plotting the induced number of mutations as a function of the integrated dose over time. 10.2.2. Stattsttcal analysts. One of the most commonly used statistical tests for the C H O assay is a transformation test by Snee and Irr (1981). A

10.2.2. Other possible statistical tests include linear regression and the chl-square test. It should be made clear that the significance of the data is

268

Student's t-test can be used to interpret the results of the transformation (Snee and Irr, 1981). Other types of stat~shcal analyses include the analysis of variance (ANOVA), and the Student's t-test. For the V79 assay, the most common statistical test ~s the Student's t-test.

related to the m i m m u m numbers of mutant cells (expressed or unexpressed) in each culture This is a function of the total number of cells surviving treatment and the potency of the mutagen

10.3 Criteria for rejectton. An experiment should be rejected if any of the following condinons occur: 0) spontaneous mutant frequency outside the acceptable range (see Section 10.1); (1i) failure of either positive or negative controls: (in) absolute C F E of the negative (solvent) controls below an acceptable standard; (iv) lack of reproducibihty, (v) the presence of extensive contammatlon.

10.3. (1) The acceptable range for spontaneous mutant frequency should be a standard set by each laboratory based on experience and should fall m the range of 0 - 2 0 for C H O and 0-100 for V79. (n) Deviation from hlstoncal values exceedmg 20% should be questioned. (iu) The rmnlmum acceptable C F E for solvent controls should be a standard set by each laboratory based on experience. Until such a standard is estabhshed, the minimum 50% level should be used. (iv) Rephcate tests show conflicting results. Other reasons for rejection include excess tox~clty, a dose range that does not reach 80-90% toxicity, and poor cell growth or wabihty, and too few replicates or dose levels remaining at the end of the study.

11.0 Report preparation The report should include the following mformarion, in tabular, graphical or discussion format. (1) Concentrations of test chemical, positive controis and solvents. 01) Details of the methodology (e.g., seeding densities, treatment times, frequency of subculturmg, incubation durations, number of treated cells etc.). (li0 Absolute and relative C F E for each treatment condmon m the p r e h m m a r y cytotoXlclty assay. (iv) Absolute C F E for each treatment condition at selection. (v) Absolute numbers of mutant colonies. (v0 M F per 10 6 clonable cells. (vii) Results of statistical analyses.

Procedure for mouse iymphoma L5178Y mutation assay

Comments

10. Cell maintenance and quahty control 1.1 Assay cell type. A strata of L5178Y mouse l y m p h o m a cells, heterozygous at the thymldine kmase (TK) locus are used m this assay to detect

1.1 Some valuable characteristics of the mouse lymphoma T K + / cell line include the ability to grow m suspension, relatively short gen-

269 mutation events at the T K locus. Cells can be obtained from D. Clive (Wellcome Research hmit the Laboratories, 3030 Cornwallis Road, Research Triangle Park, NC).

eration time, stable karyotype and a good cloning efficiency. Other loci that can be used in this strain are H G P R T and ouabain resistance (Cole and Arlett, 1984).

1.2. Cell mamtenance. Cultures are usually grown in suspension in either 25- or 75-cmz plastic tissue culture flasks or 250-500 ml erlenmeyer flasks. Checks for viability should be conducted on cultures periodically.

1.2. Cultures can be centrifuged after thawing at 200-300 × g for 10 min. Cells in erlenmeyer flasks should be kept on a rotory shaker. Cell density should not exceed 2 × 10 6 cells/ml and is best kept to less than 1.2 x 10 6 cells/ml.

Freezing of cells using liquid nitrogen and storage at - 1 9 6 ° C can be done m aliquots of 1-2 × 10 6 cells/ml of complete medium with 10% DMSO. To use, aliquots should be thawed quickly in a 37°C waterbath and diluted as necessary. The first subculture should be done approximately 24 h following thawing. 1.3. Cell hne quality control. The spontaneous mutant frequency and the colony-forming efficiency should be verified in conjuction with each assay conducted. The routine maintenance of growth curve records is recommended and can be used as a basis for comparison in stock tests. Acceptable limits should be based on historical experience pertaining to these parameters, and generally fall in the range of 70-100% for colony forming effioency and 2-10 × 10 -5 for spontaneous mutant frequency. Mycoplasma checks should be conducted every 3 months for each cryopreserved stock culture. The response to positive control(s) should be observed by using accompanying controls for each assay (see Section 4.1) Stocks should be rejected if the values obtained for any of these determinations is outside of the historically acceptable ranges.

1 3. Other culture characteristics that can be monitored periodically are saturation density, karyotype and growth rate. Mutant colonies can be checked for retention of trifluorothymidine (TFT) resistance by picking several colonies, growing in unselective medium and then rechallenging with T F T (Bradley et al., 1987).

1 4. Cleansing of cultures. To prevent high backgrounds arising from spontaneous mutations, cells lacking T K can be killed by culturing cells medium with added T H M G (methotrexate, hypoxanthine, thymidine and glycine), either on a regular basis or just prior to preparing cells for treatment. T H M G is normally present in the culture medmm for 24 h, then cells are resuspended in medium without methotrexate but with thymidme, hypozanttun and glycine (THG) for 1-3 days.

1.4. Aliquots of cleansed cultures can be frozen and stored for future use if desired. Methotrexate forces the cells to be dependent on the T K salvage pathway of thymidine monophosphate (TMP) synthesis. Spontaneously arising T K - / - mutants cannot use the exogenous purlne substrates and therefore do not survive.

270

1.5 Medium and reagents The most common m e d m m used to culture L5178Y cells is Fischers, and R P M I 1640 is also widely used, both of winch can be purchased from Gibco. At a temperature of 4°C, the recommended maximum storage duratton for these media is 26 weeks. Pre-tests conducted on each new batch are recommended m order to verify the growth-promoting capabilities of the medium, usually by determmatton of the growth rate a n d / o r colony forming efficiency. Supplements to the medium usually include sodium pyruvate (220 ktg/ml), L-glutamlne (2.0 mM) and p e n / s t r e p (100 units penaclllln/ml, 0.050-0.1% Pluronlc F68 and 100 t~g streptomycm/ml).

1 5 Medium pre-tests for adverse effects on the assay may also be conducted for new lots.

Heat-inactivated horse serum at a concentration of 10% is used to culture L5178Y cells. Serum should be stored at - 2 0 ° C and can be used for up to one year after receipt. The growth-promoting abilities of the serum should be verified once for each batch, usually by assessing colony forming efficiency a n d / o r growth rate. Pre-tests should also include observations of adverse effects on the assay.

1 6 Test chemical solubtho' C H O / V 7 9 Mutauon Assay

See Section 1.6,

The following steps should be carried out before commencing mutation assay: (0 Selection of concentration range (Section 3.0); (i0 Solubility of test chemical, p H changes (section 1.6), (ifi) Preparation of media, F)oP medium (Section 2 1), Cloning medium (Section 8.2).

2.0 Preparation of cultures 2.1 Cells are stored and maintained m Fl0 P medium containing 10% horse serum Seeding cells in preparation for treatment involves a 50% dilution of the stock culture with Flscher's m e d m m with no horse serum (Fop), thus reducing the serum concentration to 5%. Flasks (200 ml) or polypropylene tubes (50 ml) can be used for seeding cells.

2 1. Flop medium 450 ml F~scher's medmm+ supplement 50 ml horse serum (10%) An alternative to 1=1op medium is R P M I 1640. R P M I medium has an approximate 6-fold higher concentration of phosphate than Fischer's medium does. Tins can cause problems during the selection phase since phosphate can speed up the mact~vation of T F T by serum, however, the addition of extra T F T a n d / o r thorough reactivation of the serum (56°C for 30 min) can overcome these

271 problems. The higher nutrient composition in R P M I enables the use of a lower concentration of serum (1.e., 5%). If cells are mcubated for 24 h before treating, the seeding density should be reduced to approximately 0.3 × 106 cells/ml. Mmrotiter plates are sometimes used to seed cells for treatment, as described by Cole and Arlett (1984).

2.2. Seeding density should be 6 × 105 cells/ml in 10 ml medium if cells are prepared immediately prior to treatment. A 30-min incubation is recommended to allow cells to re-establish growth (Cole and Arlett, 1984). 2.3. Number of culture vessels requtred Section 2.3, C H O / V 7 9 Mutation Assay.

2.2. If cells are to be incubated prior to treatment, consideration must be given to the generatlon time and the final cell density required per culture. Refer to Section 7.2 for a description of cell density determination.

See

3.0. Preparatton of test chemical 3.1. Selectton of treatment groups. A minimum of 4 treatment levels should be selected, usually on the basis of a prehminary cytotoxicity test to determine cell counts. Cytotoxicity tests must be carried out over a range of test compound concentratlons prior to beginning the experiment. The cell cycle delay must also be monitored during the cytotoxic~ty tests since delays to the cell cycle, caused by the test chemical, can result in false negative conclusions regarding the data. 3.1 1.

3.1. Determination of the colony-forming efficiency or observation of morphology, alone or in conjunction with cell counts, can also be used to determine the effect of treatment on cytotoxacity. False-negative conclusions can result when a cell cycle delay is mistaken for a negative response.

Prehmmary cytotoxwtty test

3.1.1.1 Cultures, prepared as described in Section 2.0, are treated with a range of chemical concentrations (refer to Sections 5.0-5.4).

3.1.1.1. A possible range of concentrations could involve doses that are one half-log apart, e.g., 10000, 3000, 1000, 300, 100, down to 0.01

~g/mL 3.1.1.2. After the 4-h treatment phase, cells are resuspended m 10 ml of F10P and incubated at 37°C, with shaking for an additional 24-48 h. 3 1.1.3. Cell counts (refer to Section 7.2) are determined and compared to solvent controls. 3.1.1.4 follows:

The relative growth is calculated as

suspension growth of treated culture Relative growth suspenstongrowth of control culture × 100

272

3 1 2. Determmatton of concentratton range A minimum of 4 doses should be selected for testing, ranging from about 10 to 90% relative growth, as compared to controls

3.12. Non-toxic soluble test chemicals are normally tested using a concentration range up to 10.0 m g / m l , sometimes beyond the limit of solubility. In the eventuality of preopltate formation, attempts may be made to dissolve the precipitate, using dispersion methods or an alternate solvent, or the preparation may be discarded. In order to avoid cell toxicity caused by the solvent, a maximum bolvent concentration of 1% ~s recommended. Conditions of physiological stress can cause the apparent reduction of mutants in cultures of mammahan cells (Brusick, 1986) High doses of a compound that cause a reduction of pH (below 6.0) or increase the osmolarity (above 400 m O s m / k g water) of the culture medmm can lead to artifactual increases m mutation, in chromosomal events and in cell transformation (Bruslck, 1986: Clfone et al., 1985; Galloway et al., 1987). These parameters should be measured as part of the dose selectmn process and also at the end of the test for each dose as part of quality control. This effort will reduce the number of spurious reports of positive result due to artifacts

4,0. Controls See Section 4.0, C H O / V 7 9 Mutation Assay.

5 0 Treatment of cultures The remainder of the assay should be carried out for each concentration of test chemical, and for all positive and negative controls. Each treatment should be conducted in duplicate.

5.1 Number of cells. Each treatment vessel should contain 10 ml of the prepared cell suspension at a cell density of 6 X 105 cells/ml, for a total of 6 × 1 0 6 cells/vessel. 5 2. Activation. Cultures should be treated with and without Aroclor 1254-induced rat-hver homogenate $9 mix. Negative controls should also involve + / $9 treatments Normally, 4 ml of $9 max are added to 6 ml of the cell suspension. If $9 is not added to the treatment, 4 ml of Fop should be added instead.

5.2 The concentration of horse serum has been diluted to 3% Serum concentration should be reduced during treatment to minimize interaction with the test chemical or metabohtes.

273 S9-mix consists of rat-liver $9, induced by Aroclor 1254, which has been buffered and supplemented with the essential co-factors N A D P and glucose 6-phosphate. 5.3. Addttion of treatment medium. To the 10 ml of cell suspension plus $9 or serum-free medium, a maximum of 100/~1 of the test chemical should be added, dissolved at the appropriate concentrations in the chosen solvent. For solvent controls, the same volume of solvent only should be added. 5.4 Treatment duratton. Cultures are incubated in either a roller drum or shaker at 37°C in the presence of the test chemical for a period of 1-5 h. The duration with and without $9 is usually the same. 6.0. Post-treatment 6.1. Following the treatment period, cultures are centrifuged at 200 × g for 10 min and resuspended in 10 ml of F10P. This is repeated a total of 3 times, with 20 ml of F10P used m the final resuspension, for a final cell concentration of 0.3 )< 106 cells/ml. 6.2.

Determmatton of mitlal survwal ( CFE )

6.2.1. A 1-ml aliquot is removed from the resuspended culture (Cole and Arlett, 1984), and the cell density of the aliquot is deterrmned. 6.2.3. One 300-ml flask per treatment level, labelled accordingly, is sterilized and 100 ml of cloning medium (refer to Section 8.2) is added to each flask and stored in a 37°C water bath. Cells are inoculated into the appropriate flask to produce a final density of 6 cells/ml, and flasks are incubated with shaking for 15 mln. 6.2.4. One third of the culture preparation is poured into each of three 100-mm plates using a sterilized 50-ml graduated cylinder. 6.2.5. Plates are left to sohdlfy, then placed m a 37°C incubator (5% CO 2, 95% humidity) for 10-12 days to allow for colony formation.

6.2. An alternative to the initial survival deternunation described here is the measurement of total growth. The number of divisions that occur between the initiation of and recovery from the treatment phase is determined by measuring the cell density during the expression period (Mitchell et al., 1988). This may be a more accurate indication of cytotoxac effects since some ceils survwe more readdy under crowded tissue-culture conditions than they do in CFE-type conditions. Doserelated induction of cell cycle delay must be considered if this method is used.

274

6.2 6. Colonies are c o u n t e d as d e s c r i b e d m Sect×on 9 1. T h e C F E is c a l c u l a t e d as d e s c r i b e d m Section 9.3. 70. Expression 7 1. The culture p r e p a r e d in Section 6.1, conraining a p p r o x a m a t e l y 6 × 10 6 cells, is i n c u b a t e d at 37°C for the e x p r e s s i o n p e r i o d of 2 days, d u r ing which time subcultures are m a d e on b o t h days.

71. S u b c u l t u r l n g is d o n e to m a i n t a i n exp o n e n t i a l g r o w t h a n d allow for m a x i m u m expression of the m u t a n t p h e n o t y p e .

7.2 Determmatton of cell counts. Cell c o u n t s are d e t e r m i n e d i m m e d i a t e l y following t r e a t m e n t a n d at each subculture. 72 1. Single cell s u s p e n s i o n s are p r e p a r e d either b y v o r t e x i n g at low speed or b y vigorous plpettmg.

7.2.1 T h e cells often grow m small c l u m p s a n d m u s t be d i s s o c i a t e d into single cells to e n a b l e a c c u r a t e d e t e r m i n a t i o n of cell numbers. I n c u b a tion at 37°C for 10 mln in 0.1% trypsin is a n o t h e r m e t h o d of p r o d u c i n g single cell suspensions

7 2.2. Cell c o u n t s can be d e t e r m i n e d using a h e m o c y t o m e t e r or electronic counter.

7.2.2 T h e use of a h e m o c y t o m e t e r enables a check on the p h y s i c a l c o n d i t i o n of the cells as well as a c o u n t (Cole a n d Arlett, 1984).

7 3. Preparation of subcultures. 24 a n d 48 h following t r e a t m e n t , cultures are d i l u t e d in F10e m e d m m to a d e n s i t y of 3 × 105 c e l l s / m l , m a i n taining a final v o l u m e of 1 0 - 2 0 ml. Cultures are i n c u b a t e d at 37°C in a shaker b a t h or roller d r u m b e t w e e n subcultures.

73 If cell d e n s i t y is less than 3 × 105 c e l l / m l , no s u b c u l t u r e is necessary.

7.4 Cell survwal deterrnmatton. C F E is detern'nned at the last s u b c u l t u r e as well as after t r e a t m e n t . This is d o n e using the m e t h o d des c r i b e d b e l o w in Section 8.1 to 8.11.

7.4. C F E can be d e t e r m i n e d at each subculture as well as i m m e d i a t e l y following t r e a t m e n t .

80. Selectton / cloning 8.1 F o r each culture, two 300-ml flasks a n d one 125-ml flask are needed. Also, one 6000-ml e r l e n m e y e r flask, a n d one 500, 100 a n d 50 ml g r a d u a t e d c y h n d e r m u s t be sterilized. All glassware should b e sterilized a n d s t o r e d at 37°C until used. 82 C l o n i n g m e d i u m ( C M ) is p r e p a r e d b y first p r e w a r m m g F l s c h e r ' s m e d i u m with 20% horse s e r u m (F20 P) a n d p o u r i n g it into the large

8.2 C l o m n g m e d i u m ( C M ) - 5 0 0 0 ml 3538 ml Flscher's medmm 1000 ml horse serum (20%), heat-reactivated (56°C, 30 ram) 462 ml 4% Noble agar. melted at 95°C

275 erlenmeyer flask. Noble agar is added with mixing to the F20P. CM can be stored at 37°C until needed.

An alternative is the use of R M P I 1640 medium, with 10-20% heat inactivated horse serum and 0.24% purified agar. Each culture requires 250 ml of CM, and an excess amount should be prepared.

8.3. Following the 48-h subculture, the cell density of the cultures should be 3 x 105 cells/ml. Cultures are incubated in a shaker or roller drum for 30 min prior to cloning to allow them to adapt to the fresh m e d m m added during the final subculture. 8.4 While the cells are incubating, 100-ml volumes of CM are poured into each of the 300-ml flasks, and 50 ml into the 125-ml flasks. These flasks are returned to the 37°C water bath. One of each of the larger flasks is also labelled ' T F T ' (trifluorothymidine) and will contain the selection culture. The other 100-ml flask is labelled ' V C ' (viable cells) and will be used to determine the final CFE.

8.4. The CM should not be distributed to all flasks at once since clumping of the agar is likely.

8.5. Cells are seeded at a concentration of 3 x 104 cells/ml. This is achieved by centrifuging 10 ml (3 x 10 6 cells) of each culture at 200 x g for 10 min, decanting all supernatant except 1 - 2 ml. The cells are then transferred to one of the 100-ml T F T flasks containing CM, with swirling.

8. 5. The centrifuge tube should be rinsed using the c e l l / C M mixture, and the rinsate returned to the flask.

8.6. Cultures are placed m a shaker incubator at 37°C for 30 rain to mix cells in the medium.

8. 6. The concentration of cells in the selection flasks is 3 x 10 4 cells/ml.

8. 7. Another dilution is prepared by transfern n g 1 ml of the mixed T F T culture to the corresponding 50-ml flask containing CM. The flasks are mixed for 15 min. Serial dilutions can also be done in tubes containing 9 and 4 ml of medmm. 8.8. During incubation of the smaller flasks, 1.0 ml of T F T stock solution is added to each of the 100-ml T F T flasks, and these are placed m the shaker incubator.

8.8. T F T stock solution (1 # g / m l ) 50 #g tnfluorothyrmdme (TFT) Add sahne, to a final volume of 50 ml

Other agents used to dissolve T F T include water and serum-free medium. T F T can be frozen and stored for up to 3 months. Stock solutions should be protected from exposure to light and unused portions should not be re-frozen.

276

8.9. A further dilution of the TFT-free mixture is prepared by transferring 1.0 ml from each of the 50-ml flasks to the corresponding 100-ml VC flask. These mixtures are incubated for 15 mm with agitation.

8.9. Cell density of the VC cultures is 6 cells/ml.

8.10 A total of six 100-mm plates per treatment level are labelled with the culture number, and 3 of these were also labelled ' T F T ' and the other 3 were labelled ' V C ' The VC plates are poured first using a graduated cylinder to measure 33 ml into each of the plates from the VC flask. The same graduated cylinder can be used for the T F T plates, adding 33 ml of the TFT culture to each. 8.11, Plates are left to sohdlfy, then are stored in a 37°C incubator (5% CO 2, 95% hunudity) for 10-12 days to allow the development of colonies.

Note. It is recommended that each experiment be repeated once, and possibly more ff equivocal results are obtained. The conditions should be duplicated as precisely as possible, with slight modifications to the dose range as applicable. 90. Data collection and calculations 9 1. Countmg colomes. Colonies are usually counted using an electromc counter (e.g., Artek Model 880 R) although counts can be done manually. The minimum colony size counted is 0.3 mm m diameter with the Artek 880.

9.1 There 1s some evidence that the formation of small versus large colonies may be indicative of chromosome breakage versus point mutation events, respectively (Doerr et al., 1989). With the use of an Artek model 880 automatic colony counter, modified with a 10-turn potentlometer, colony size distributions can be recorded (Doerr et al., 1989).

9.2 Calculation of mutant frequency The mutant frequency (MF) is estimated for each treatment by the following calculation:

9.2. The dilution factor for the methodology described here is 2 × 10 4 for the selection cultures (1/50 × 1/100 = 2 × 1 0 - 4 ) .

MF =

m e a n n u m b e r of colonies on T F T p l a t e s m e a n n u m b e r of c o l o m e s on V C p l a t e s x ddut~on factor

The MF is usually expressed as the frequency per 106 survivors.

9 3. Calculation of CFE. The colony-forrmng efficiency (CFE) is calculated as follows (L1 et al.,

9.3. The CFE for solvent controls should ideally be a minimum of 75%. Low CFE values may

277

1987) for both initial and final survival: Absolute CFE =

Relative CFE =

number of colomes formed × 100% number of cells plated absolute CFE (treatment) × 100% absolute CFE (control)

be due to the temperature, viscosity or pH of the cloning medium, lack of an adequate single cell suspension prior to addition of CM, insufficient incubation duratton during the selection phase, ~mproper pH or temperature dunng incubation, low quality serum and agar, or overgrowth of the culture prior to cloning.

10.0. Data analysts 10.1 Spontaneous mutant frequency. The recommended acceptable range of spontaneous mutant frequency is 20-100 per 10 6 clonable cells.

101. The spontaneous MF range can vary shghtly between laboratories. Turner et al. (1984) report an historical range of 25-115 mutant colonies per 10 6 cells. The acceptable range should be determined based on laboratory experience, and should be used as a guideline for rejection of an experiment once established (see Section 10.4).

10.2. Criteria for a posttwe response. The following factors are considered by respondents to be indicative of a positive response: (i) dose-related increase in MF; (ii) a minimum 2-fold increase in M F over solvent controls; (hi) statistical slgmficance (refer to Section 10.3); (iv) an absolute increase in numbers of mutant colonies for at least one dose (Cole and Arlett, 1984); a n d / o r

10.2. The 2 factors most often reqmred to be able to conclude that a positive response occurred are a minimum 2-fold increase in MF and a dose response.

10.3. Stattstwal analysis. The most commonly used statistical analyses are the analysis of variance (ANOVA) or linear regression. 10.4. Crtterta for rejection. An experiment should be rejected if any of the following conditions occur: (i) failure of positive or negative controls; (ii) spontaneous mutation frequency outside acceptable range; (iii) poor growth or viability (low CFE).

10.4. (ii) the spontaneous MF should be within a range of 20-100 mutants/106 cells; (iii) A CFE below 50% ts usually reason for rejection of the experiment. Other reasons for rejection include excess toxicity or contarmnation of cultures.

11. O. Report preparatton The report should include the following information, in tabular, graphical or discussion format: (i) Concentrations of test chemical, positive controls and solvents. (ii) Details of the methodology (e.g., seeding densities, incubation durations, media and solvents used, etc.). (iii) Values of absolute cell counts in both cytotoxicity and mutagenicity assays. (iv) Cell growth and CFE. (v)

278

M F per 1 0 6 clonable cells (vl) Results of statistical analyses.

Discussion The 3 cell hnes discussed in this report, namely CHO, V79 and L5178Y, in order of frequency of use, are the most commonly employed cell types for use m mutation assays according to the 77 respondents surveyed. The two recommended standardized protocols described above, one for C H O and V79 combined and one for L5178Y, represent asslrmlatlons of the procedural steps most commonly used by respondents to the questionnaire and of the procedures described m a number of primary references. This discussion mchides more detaded characteristics of the assays as well as the rationale for some of the recommendations in the protocols. The 3 cell types are discussed individually, and some comparison also is revolved. Further information, including complete data sets of responses to the questlonna~re, can be obtained upon request.

(I) C H O / H G P R T assay The most frequently cited references for the C H O / H G P R T assay were several from O'Neill and Hsie (24/33 respondents, or 73%). The majorlty of users (25/33, or 76%) stated that minor modifications were made to their chosen protocol, while only 6 / 3 3 (18%) conducted the test exactly as described m their primary reference and only 2 / 3 3 (6%) included major modifications. In a study of 16 animal carcinogens and 2 noncarcmogens, the C H O / H G P R T assay detected 16 mutagens and 2 nonmutagens for a 100% correlation (Hsie et al., 1981). Among respondents, 8 / 1 3 (62%) cited a 100% correlation of pos~twe mutagenicity with known carcinogens, though most of these have tested only a small number of compounds The laboratory that tested the largest number of known carcinogens tested reported an approximate 90% correlation. (II) V79 / HGPR T assay The most popular reference source for users of the V79 assay was Bradley et al. (1981), cited by 9/21 (43%) of the respondents. Each of these

respondents stated that minor modfftcauons were made to this pubhshed protocol. The only other reference c~ted by more than one respondent was Jenssen (1984), used by 2/21 (10%). When responses for all of the references cited were combined, 17/21 (81%) reported using minor modifications to the published protocol and the remainder conducted the test exactly as c~ted. In addition to studying the H G P R T locus, V79 cells can be used to detect mutation at the N a + / K + ATPase locus by using the selective agent ouabaln (Bradley et al., 1981). The advantages assocmted with this locus are its relatwe stabdity and the fact that it ~s well-characterized However, there are also some disadvantages, including a lower induced mutant frequency (MF), the existence of some cells for which N a + / K + ATPase actwity is required for survwal and its ability to detect only single-base changes (Bradley et al., 1981). Only 15% of respondents (3/20) using V79 cells reported m o m t o n n g for mutation at this locus, usually in conjunction with H G P R T Only 6 respondents to the questionnaire prowded information regarding the sensltwity of the V 7 9 / H G P R T assay. Of these, the responses ranged from 83 to 100% correlation for positive m u t a g e m c observations induced by known carcinogens. The two laboratories with the highest number of test results for carcinogens (19 and 20 tests) reported correlations of 100% and 95%, respectively.

(Ili) Mouse lymphoma L1578Y assay The most commonly cited author for the L5178Y assay was D. Chve, primary or contributing author for 20/26 (77%) of the references used by respondents. Upon combining the 26 references cited by respondents, 2 0 / 2 6 (77%) were used with minor modifications, 2 / 2 6 (8%) with major modifications, and the remainder (15%) were conducted as described in their primary reference. In addition to the T K locus, mouse lymphoma cells can be used to examine mutation at the H G P R T locus which requires a longer period of time for expression of the mutant phenotype (7-9

279

days rather than 2 days). Use of the H G P R T locus m L5178Y cells is not very common - only 2 / 2 6 respondents (8%) stated that they select for H G P R T - mutants. The mouse lymphoma assay has a umque characterist~c associated with it that is not shared by either the CHO or V79 assay. It has been observed that there are often two size groupings of mutant colonies for many mutagenic compounds. Using a 10-turn potentiometer attached to an Artek model 880 colony counter, several investigators have observed a biomodal curve upon plotting MF versus size setting (Clive et al., 1979; Turner et al., 1984; Doerr et al., 1989), corresponding to so-called small and large colomes. Small colonies have liquid and soft-agar growth impairment, and also decrease in frequency with increasing time between treatment and cloning, perhaps due to a dilution factor caused by their slow growth relative to large colonies (Clive et al., 1979). The characteristics of the small and large colonies have been shown to be heritable as demonstrated by picking colonies and measuring growth rate, colony forming efficiency and colony size (Clive et al., 1979). An hypothesis has been suggested for the existence of small and large colonies. The possibility of random occurrence has been ruled out since the frequency varies widely. In unselected medium, cultures used to assess the viable count have a very low percentage of small colonies. In contrast, a relationship between the presence of both large and small colonies following treatment with known carcinogens has been demonstrated. Using a potentlometer attached to an electronic colony counter, colony size distribution can be graphed, aod peaks representative of both small and large colonies are often observed with compounds active in the mouse lymphoma assay. It has been suggested that small colonies may arise from the reduction of chromosome damage and that large ones arise from mutations (Clive et al., 1979; Turner et al., 1984; Doerr et al., 1989). The observation that the majority of small-colony cells have chromosome abnormalities (unspecified) on the T K chromosome, number l l b (Clive et al., 1987), supports this hypothesis. Doerr et al. (1989) have recently demonstrated a rolationship between small-colony mutants and the existence of chromosome damage. For 11 of

12 compounds tested using the mouse lymphoma assay, clear dose-responses were observed for small-colony MF, chromosome aberrations (i,e., breaks and rearrangements) and micronucleus formation. The authors concluded that determining the size distribution and small colony M F can give an indication of the extent of clastogenic~ty of a test compound in addition to its mutagenlcity as measured by the large-colony frequency. The numbers of compounds used in this invesUgation was low (only 12), and further information is reqmred before definitive recommendaUons can be made in this area. However, investigators with the capability to collect information on size distributions of mutant colonies are encouraged to do so in order to add to the existing database. Users of the mouse lymphoma assay were asked to c~te the percent correlation for positive mutagemcity with known carcinogens. The responses ranged from 75 to 100%, with 6 / 1 3 respondents (46%) observing 100% correlation. Two of these respondents, having tested 24 and 25 known carcinogens, cited 80% and 100% correlation, respectively. Another laboratory reported testing 100 compounds and finding that 80-90% of the carcinogens and 50% of the noncarcinogens were positive mutagens.

(IV) Compartson of assays The two protocols described in this report, one conducted with either CHO or V79 cells and one with mouse lymphoma L5178Y cells, have in common 7 primary steps. These are the preparation of cells for treatment (Section 1), preliminary cytotoxicity test (Section 2.2), treatment (Section 4), determination of initial CFE (Section 5), expression (Section 6), determination of final CFE (Section 6.3), and selection (Section 7). The types of data collected are also s~milar and include calculation of the MF, CFE values, reporting of the absolute mutant colony numbers, assessment of a dose-response, and sometimes a statistical analysis. However, the methods for these procedures vary in several key areas, including primary mutation locus, ~nitial seeding density and expression time. There are some parameters m these assays which are flexible and can be varied shghtly if required. However, with the accumulation of experience, an

280 effort should be made to establish a workable protocol with httle variation, Varlatton m culture conditions can arise unexpectedly, and the less uncertainty associated with the protocol itself, the better the chances are of improving the reproduclbIhty and reliability of the results. The maintenance of uniform growth rates, CFEs and low spontaneous MFs is important to allow for quality control and confidence in experimental results (Bradley et al., 1981). Seeding densities at any stage (e.g., pre-treatment, expression, selection) can be increased or decreased if survival is higher or lower than expected. However, in C H O cells an mhibttion in the growth of mutant colomes has been observed to occur above the selection seeding density of 3 × 105 wild-type cells/plate (O'Neill et al., 1977a). The seeding densities used should be reported. The optimal expression time should be established for each laboratory. During the expression period, the damage induced in mutant cells must be fixed in the D N A of these cells, and the levels of the wild-type enzyme (i.e., H G P R T or TK) and the R N A coding for the enzyme, must decrease to a neghglble level to allow for maxtmum expression of the mutant phenotype (Bradley et al., 1981). Testing well-characterized positive carcmogens/ mutagens, with known optimal expression time and MF, ts recommended to establish the duration of expression time required. The necessary use of exogenous metabolic acttvation can lead to problems with varymg responses since batches of $9 do not always have identical characteristics. New lots of $9 should always be screened using compounds with known mutagenic activities and also using solvent controls It is also good practice to mclude positive promutagens in each experiment requiring $9 so that the mutagenlc potency of the test compound can be determined relative to the standard mutagen (Bradley et al., 1981). Another source of variation is the amount of serum used for culture, treatment and selection medta. As for $9, new lots of serum should be screened prior to use since characteristics such as effecttveness and competittveness with the treatment or selection agents can lead to experimental variation. New lots of medium should also be

screened for their abihty to support culture growth at historically acceptable levels, to enable high CFE, and to produce healthy cultures possessing normal rates of spontaneous forward and reverse mutations (Chve et al., 1987). A method of c o m p a n n g mutagemc potenctes of various compounds was suggested by Clive et al. (1979) for mouse lymphoma data, but could be used for C H O and V79 results as well. The mutagenlc potency can be calculated by takmg into account the MF, treatment duration and compound concentration: Mutagemc potency MF( x 10 6 surv,vors)Xtreatment duratxon (h) treatment concentration ( btg/ml ) During these assays, two mechantsms of mutation can occur, t.e., forward and reverse mutation. Forward mutation results in loss of the wild-type phenotype and is the endpolnt that is detected. Reverse mutation can occur spontaneously in mutant cells and the resulting phenotype ts that of the original wild-type. The necessary change required to produce a forward mutation at either the H G P R T or T K locus is not specific (Hsie et al., 1981). Theoretically, each of the forward mutation assays ~s able to detect a vartety of mutation events including base substitutions, framesinfts, deletions and some chromosomal lesions (Bradley et al., 1981; Lt et al., 1987), winch makes this one of the strengths of this type of assay since a range of potential mutagens with a variety of effects can be detected. In contrast, spontaneous reversion from H G P R T - to H G P R T + (for CHO and V79) or from TK to TK + (for L5178Y) involves a very specific type of mutation, and these events occur with a low frequency, approximately 10 -s to 10 v revertants/CHO cell (O'Neill et al., 1977a: Hsle et al., 1981), < 2 × 10 7 revertants/V79 cell (Hodgklss et al., 1980), and 1 2 × 10 v revertants/ L5178Y cell (Chve et al., 1979). A distraction should be drawn between the capabilities of the H G P R T and TK loci to permit recovery of chromosomal mutations. Both locl are equally capable of permitting the recovery of gene (pomt) mutations involving base substitutions, framesinfts, deletions and rearrangments within the gene. However, chromosomal (multiple gene) mutations or

281 multilocus deletions are better recovered at the T K locus rather than at the H G P R T locus (DeMarinl et al., 1989; Moore et al., 1987). One area that deserves comment pertains to the recommendation in the protocols for both assays concerning the importance of recording, reporting and assessing the effect of the test compound on the absolute number of mutant colonies, and not only the MF. Very few of the published protocols cited or the respondents to the questionnaire discussed this aspect. Since the M F represents the number of mutant colonies per number of surviving cells, an increase in MF can arise from an increase in the number of mutants (with constant survival level), a decrease in survival (with constant mutant level), or an increase in the number of mutants with a s~multaneous loss of survival. If survival decreases, which usually happens as specified in the protocols, false positive conclusions can be made. This is commonly known as the 'distilled water effect', in which the calculation of mutant frequencies per surviving cells can produce an apparent positive response for an agent that causes cell death but not mutation (Green and Muriel, 1976). It is important that the actual mutant counts be reported or can be calculated from the data reported in order to allow for a complete assessment of the results. The advantages associated with each of the three cell types CHO, V79 and L5178Y as reported by the respondents to the questionnaire vary shghtly. For the CHO assay, the most frequently cited strengths included reproducibility (48%), ease (30%), sensitivity (22%), specificity (22%), and the ability to use the same cell type to test for effects on other endpoints such as chromosome aberrations and sister-chromatid exchange (17%). Users of the V79 assay cited the following advantages: ease (50%); high survival (28%); extensive vahdation (28%); and a relatively short t~me required to complete the assay (22%). Finally, for the L5178Y system, common responses regarding the strengths of the assay included high sensitivity (45%), reproducibility (41%), short assay time (32%), easier handling of suspension cultures (27%), simplified protocol (27%) and the fact that several types of mutations can be detected (23%). Respondents were asked to list the shortcom-

ings of the three respective tests. As for the advantages, the frequently cited answers varied somewhat, and even sometimes contradicted commonly listed advantages. For CHO, the most common disadvantages were the large cost and time associated with the assay (33%), the absence of intrinsic metabolism (21%), low sensitivity to weakly positive mutagens (13%), and the fact that highly cytotoxic agents are difficult to assess (13%). Similar problems are reportedly associated with the use of V79 cultures: the costly and t~me-consuming procedure (61%), the requirement for exogenous metabolic activation (56%), the variability of results (28%), and low sensitivity (17%). The most commonly reported weakness for the mouse lymphoma assay was the prevalence of false-positives (39%), followed by the difficulty of assessing very cytotoxic agents (11%), the frequent observation of weak-positwe responses (11%), the absence of a single, well-defined optimal $9 concentration (11%), and the complications associated with the existence of small and large colonies, for example possible increases in the number of false-positives observed (11%). On exanunation of the advantages and disadvantages associated with the mutation assay, as reported by the respondents, a number of contradictory results were evident. Certain of these apparent contradictions may be explained by the ability of a given laboratory to cope with large volumes of mutation testing and the concurrent experience associated with frequent testing. Other differences may arise from variation in the types of compounds tested and unique problems, and solutions, that may have arisen as a result. Based on the the number of laboratories using the three assays discussed here, it appears that the CHO and mouse lymphoma assays are the most popular. The number of laboratories using either C H O or V79 cells (i.e., momtoring for mutation at the H G P R T locus) exceeds the number using L5178Y cells (56% versus 28%). However, the total number of mouse lymphoma assays conducted yearly exceeds the number conducted with either CHO or V79. According to respondents, a total of approxamately 1140 mouse lymphoma tests are conducted per year (56%) compared to 520 (25%) with CHO and 380 (19%) with V79. The protocols for CHO and V79 are very slmi-

282

lar and could be combined in this report. The abdity to use CHO cells to carry out chromosome aberrat,on and Slster-chromatld exchange assays ts advantageous m terms of comparison of results. One slgmficant disadvantage of the L5178Y assay is the frequent occurrence of false-positives which reduces its cred~bdtty as a screening test. However, the potential use of L5178Y cells to examine the frequency of both mutations and clastogenic~ty. by using large and small colony mutant frequenctes, may prove to be a valuable tool. References Amacher, D E , S Palllet and V A Ray (1979) Point mutattons at the thyrnldme kmase locus m L5178Y mouse lymphoma cells, I Apphcat~on to geneUc toxicological testing, Mutation Res, 64, 391-406 Bradley, M . O , B, Bhuyan, M C Francis, R Langenbach, A Peterson and E. H u b e r m a n (1981) Mutagenes,s by chemical agents in V79 Ctunese hamster cells A rev,ew and analysts of the hterature A report of the Gene-Tox Program, Mutation Res, 87, 81-142 Brus~ck, D (1986) Genotoxlc effects m cultured m a m m a h a n cells produced by low pH treatment cond,t,ons and mcreased ion concentration, Environ M u t a g e n , 8, 879-886. Carver, J H , G M. Adair and D L Wandres (1980) Mutagemc,ty testing m m a m m a h a n cells, II Vahdat,on of mult,ple drug-resistance markers hawng practical apphcation for screening potentml mutagens, Mutation R e s , 72, 207 230 Cffone, M . A , B Myhr, A Elche and G Bolcsfoldl (1987) Effect of p H shifts on the mutant frequency at the thymldine kanase locus in mouse l y m p h o m a L5178Y T K +/ cells, Mutation R e s , 189, 39-46 Clive, D , and J F,S Spector (1975) Laboratory procedure for assessing speclflC locus mutations at the T K locus m cultured L5178Y mouse lymphoma cells, Mutation R e s , 31, 17-29. Chve, D , K.O. Johnson, J F S Spector, A.G Batson and M M M Brown (1979) Vahdatlon and characterization of the L 5 1 7 8 Y / T K +/ mouse lymphoma mutagen assay system, Mutation Res., 59, 61-108. Chve, D., W Caspary, P E. Karby, R Krehl, M Moore, J Mayo and T J . Oberly (1987) Guide for performing the mouse l y m p h o m a assay for m a m m a h a n cell mutagemc~ty, Mutatton R e s , 189, 143-156 Cole, J., and C F. Arlett (1984) The detect,on of gene mutations m cultured m a m m a h a n cells, m S Vemtt and J M. Parry (Eds), Mutagemcity Testing A Practical Approach, IRL Press, Washington, pp. 233-273 D e M a n m , D.M., H E Brockman, F J de Serres, N H Evans, L F Stankowskl Jr. and A W Hsle (1989) Speofic-locus mutations mduced m eukaryotes (espectally m a m m a h a n cells) by radiation and chenucals a perspective, M u t a u o n R e s , 220, 11-29 Doerr. C L , K Harnngton-Brock and M M Moore (1989)

Mtcronucleus, chromosome aberratton, and small-colony T K mutant analysts to quant~tate chromosomal damage m L5178Y mouse lymphoma cells, Mutation Res.. 222, 191 203 Galloway, S M , D A Deasy, C.L Bean, A.R. Kraynak, M J Armstrong and M O Bradley (1987) Effects of high osmotic strength on chromosome aberration, stster-chromatld exchanges and D N A strand breaks, and the relation to toxicity, Mutation R e s . 189. 15-25 Green, M H L , and W J Munel (1976) Mutagen testmg using T R P + reversion in Eschertchta cob, Mutation R e s , 38. 3--32 Gupta, R S, and B Smgh (1982) Mutagemc responses of five independent genetic loct m C H O cells to a variety of mutagens, Development and charactensttcs of a mutagen screenmg >ystem based on selection for multiple drug resistant markers, Mutation R e s , 94, 449-466 HodgkJss, R J , J Brennand and M. Fox (1980) Carcinogenesis, 1, 175 187, cited m Bradley et al. (1981) Hsle, A , R Machanoff, D B Couch and J M, Holland (1979) Mutagemclty of dimethylmtrosamme and ethyl methanesulfonate as determined by the host-mediated C H O / H G P R T assay, Mutation R e s , 51, 77-84 Hste, A W , D A Casctano, D B Couch, D F Krahn, J P O'Neill and B.L Whltfteld (1981) The use of Chmese hamster ovary cells to quantify specific locus mutation and to determine mutagemc,ty of chemicals, A report of the Gene-Tox Program, Mutation Res, 86, 193-214 Jones, C A , and E. H u b e r m a n (1980) A sensitive hepatocytemedmted assay for the metabohsm of mtrosanunes to mutagens for m a m m a h a n cells, Cancer Res, 40, 406-411 Langenbach, R , H J Freed and E H u b e r m a n (1978) Liver cell-medmted mutagenes,s of mammalian cells by hver carcinogens, Proc Natl Acad Scl (U S A ), 75, 2864-2867 Ll, A . P , and R W Shlmzu (1983) A modif, ed agar assay for the quant~tatton of mutat,on at the hypoxanthme guanine phosphonbosyl transferase gene locus m Chmese hamster ovary cells, Mutation R e s , 111,365-370, ctted m LJ et al (1987) LI, A P , J H Carver, W N Choy, A W Hsie, R S Gupta, K S Loveday, J P O'Nedl, J C Riddle, L.F. Stankowskl and L L Yang (1987) A guide for the performance of the Chinese hamster ovary c e l l / h y p o x a n t h m e - g u a n m e phosphonbosyl transferase gene mutat,on assay, Mutation R e s , 189, 135 141 Moore, M M , D H . Brock, D M D e m a n m and C L Doerr (1987) Dffferentml recovery of reduced mutants at the TK and H G P R T Ioc, m m a m m a h a n cells, m Moore, M M , D M D e M a n m , F J de Serres and K R Tindall (Eds), M a m m a h a n Cell Mutagenesls, Banbury Report 28, Cold Springs Harbor Laboratory, pp 93-108 O'Nedl, J P , and A.W Hsle (1979) Phenotyplc expression t,me of mutagen-mduced 6-thloguamne reststance m Chinese hamster ovary cells ( C H O / H G P R T system), Mutat,on R e s . 59, 109-118 O'Nedl, J P , P A Bnmer, R Machanoff, G P Hlrsch and A W Hste (1977a) A quantitative assay of mutat,on mducation at the hypoxanthme-guanme phosphonbosyl transferase locus m Chinese hamster ovary cells ( C H O / H G P R T

283 system)- Development and defimUon of the system, Mutatmn Res, 45, 91-101 O'Neill, J.P., D B. Couch, R Machanoff, J,R. San Sebastian, P A. Bnmer and A.W Hsw (1977b) A quanUtatlve assay of mutatmn induction at the hypoxantlune-guamne phosphorlbosyl transferase locus tn Clunese hamster ovary cells (CHO/HGPRT system): Uuhzatton with a variety of mutagemc agents, Mutauon Res., 45, 103-109 Snee, R.D, and J D. Irr (1981) Design of a statistical method for the analysts of mutagenests at the hypoxanthlne-guanine phosphonbosyl transferase locus of cultured Chinese hamster ovary cells, Mutation Res., 85, 77-93

Thompson, L H., A.V Carrano, E Salazar, J.S Felton and F T. Hatch (1983) Comparatwe genotoxac effects of the cooked-food-related mutagens Trp-P-2 and IQ in bacterm and cultured mammahan cells, Mutation Res, 117, 243257. Turner, N T., A G Batson and D Chve (1984) Procedures for the L5178Y/TK+/--TK - / - Mouse Lymphoma Cell Mutagemclty Assay, m B.J Kflbey, M Legator. W Nichols and C Ramel (Eds.), Handbook of Mutagemoty Test Procedures, 2nd edn., Elsevter, Amsterdam, pp. 239-268

APPENDIX A LISTING OF RESPONDENTS TO QUESTIONNAIRE Name of participant

Representing

Region

Aeschbacher, H -U Anderson, D Back, Andrea Balwlerz, P. Basler, A Berry, D. Bradford, J C. Bradley, E. Bradley, M O Bridges. B.A. Bruslck, D. Bryant, D F Chang, C.C Clive, Donald Covadonga, Caballo Cross, M F. Du Frain, R J Ehas, Z. Fenwtck, R G. Flowers, L Gamott, M , and Soelter, S. Ghckman, B W Henderson, L Hitchins, Vtctona Huang, Shlu Jenssen, Dag Jones, Carol A Ketels, K Kirby, P E Lakhamlsky, T Lt, A P Llu, Vivtan Lee Madle, S Majeska, Janess B Marshall, M Marzm, P. Matheson, Dale, W Meltz, M.L. Mtller, M.W

Nestec Ltd., Dept. de Recherche, Lab Biologique BIBRA Mlcrobtol Assoc C.D Searle and Co Bundesgesundhettsamt USDA Western Regional Center Sterhng Winthrop Research Inst Inst du Cancer de Montreal Merck Sharp and Dohme Research Labs MRC Cell Mutation Umt, University of Sussex Hazelton Laboratories Inc Dept of Btochermstry, McMaster University Mtctugan State Umvers~ty Burroughs Wellcome Co Centro Naoonal de Ahmentacton y Nutnoon Central Toracology Laboratory Genetic Toxicology, Alhed Corp INRS Dept of Blochenustry Monsanto Co EHL

Switzerland UK Bethesda, MD Skokte, IL FRG Berkeley, CA Rensselaer, NY Montreal, Que. West Point, PA U.K Kensmgton, MD Harmlton, Ont. East Lansing, M1 Research Triangle Park, NC Madrid, Spain Macclesfield, Ches Morrtstown, NJ France Halifax, NS St. Louts, MO

C D Searle and Co Dept of Biology, York Umverslty Huntington Research Centre Center for Devices and Radaologlcal Health EHRT Unw of Stockholm, Wallenberg Laboratory Argonne Nattonal Lab ITT Research Institute SITEK Research Labs Laboratotre MTC Monsanto Co. EHL Mutagenesls Section, Health Protection Branch Federal Health Office Stauffer Chemical Co. Southwest Foundation for Biomedical Research InstltUt Pasteur de Lille Stauffer Chemical Co Umv. of Texas Health Sciences Centre Umverstty of Rochester

Skokle, IL Toronto, Ont UK. Rockvdle, MD Research Triangle Park, NC Stockholm, Sweden Argonne, IL Chicago, IL Rockvllle, MD Brussels St Lores, MO Ottawa, Ont. Berhn Frarmngton, CT San Antomo, TX Lille Frammgton, CT San Antomo, TX Rochester, NY

284 A P P E N D I X A (continued) N a m e of participant

Representing

Region

Mitchell, A D Moore, M Nalsm~th, R W Nestec, D Oldham, J. Oleson, F B (Jr) Olive, Peggy O'Donnovan, Mr O'Neill, J P Pezzuto, John Phdhps, B J, Probst, G S Rogers, C Romagna, F Rudd, Colette San, R , Rosin, M and Stlch, H Santa-Maria, A n a Sarnf, Awm Schremer, C A Shma, A K Slamenova, D Slesmskl, R S Thompson, Larry Tong, C Traul, K A Tu, Alice S Waldren, D Charles Zehkoff, Judith

Genesys Research Inc Genetic Toxicology Division, U S EPA Pharmakon Research lnternauonal Inc Nestec Ltd McNeil Pharmaceuticals Bristol Myers BC Cancer Research Centre The Boots C o m p a n y PLC Um'~ of Vermont PCRPS College of Pharmacy, U m v of llhnols BIBRH Lilly Research Labs Foods Directorate, Health and Welfare Canada Prechmcal Toxicology SRI International

Mountain View, CA Research Triangle Park, NC Waverley, PA Switzerland Spring House, PA Syracuse, NY Vancouver, BC Thurgarto, Nottingham Burlington, VT Chicago, IL UK Greenfield, IN Ottawa, Ont Switzerland Menlo Park, CA

BC Cancer Research Centre Centro Naclonal de Ahmentacton y Nutrlclon E 1 duPont de Nemours and Co Mobil Off Corp Lake Jackson Research Center, Dow Chemical C o m p a n y Slovak Acad Sciences, Cancer Research Inst Bushy Run Research Center, Union Carbide Corp Lawrence Llvermore National Lab [No laboratory given] Toxicology Laboratory Exxon Corporation Arthur D Little lnc U m v of Colorado N Y U Medical Center, A J Lanza Labs

Vancouver, BC Madrid. Spain Newark, DE Princeton, NJ Freeport, TX Czechoslovakia Export, PA Llvermore. CA Fort Lee, NJ East Millstone, NJ Cambridge MA Denver, CO Tuxedo, NY

Total of 77 responses, of which 6 submitted 2 questionnaires for &fferent cell types, and 2 who preferred to remain anonymous.

Recommended protocols based on a survey of current practice in genotoxicity testing laboratories, IV. Chromosome aberration and sister-chromatid exchange in Chinese hamster ovary, V79 Chinese hamster lung and human lymphocyte cultures.

Recommended protocols based on a survey of current practice in genotoxicity testing laboratories: study evaluation.

Recommended protocols based on a survey of current practice in genotoxicity testing laboratories: study design.

Genotoxicity of 2,4,6-trichlorophenol in V79 Chinese hamster cells.

Genotoxicity of 1-nitronaphthalene in Chinese hamster V79 cells.

c 3T3 mouse fibroblast and Syrian hamster embryo cell cultures.

Mouse Fyn induces pseudopodium formation in Chinese hamster ovary cells.

Dolichol metabolism in Chinese hamster ovary cells.

Effect of tumor promoting agents on mutation frequencies in cultured V79 Chinese hamster cells.

Recommended protocols based on a survey of current practice in genotoxicity testing laboratories: I. Unscheduled DNA synthesis assay in rat hepatocyte cultures.

Induction of mutation in V79 Chinese hamster cells by tetrachlorohydroquinone, a metabolite of pentachlorophenol.

Pesticide induced ouabain resistant mutants in Chinese hamster V79 cells.

Characterization of a mutator gene in Chinese hamster ovary cells.

Fibronectin receptors are functional on mitotic Chinese hamster ovary cells.

Characterization of GLUT3 protein expressed in Chinese hamster ovary cells.

Regulation of polyamine transport in Chinese hamster ovary cells.

Action of cyclic nucleotide analogues in Chinese hamster ovary cells.

Mutation induction in Chinese hamster lung V79 cells by five alk-2-enals produced by lipid peroxidation.

Legionella pneumophila inhibits protein synthesis in Chinese hamster ovary cells.

Gene targeting in Chinese hamster ovary cells is conservative.

Transfer RNA isoacceptors in cultured Chinese hamster ovary cells.

Impact of graphene oxide on viability of Chinese hamster ovary and mouse hepatoma MH-22A cells.

Genotoxicity of 1-methylpyrene and 1-hydroxymethylpyrene in Chinese hamster V79-derived cells expressing both human CYP2E1 and SULT1A1.

High level expression of wild type and variant mouse glucocorticoid receptors in Chinese hamster ovary cells.