ELSEVIER Mutation Research 312 (1994) 235-239

Environmental Mutagenesis

Mammalian cell gene mutation assays working group report

C.S. Aaron a,. (Chair), G. Bolcsfoldi b, H.-R. Glatt c, M. Moore 0, y . Nishi e, L. Stankowski f, J. Theiss g (Rapporteur) , E. Thompson h (Rapporteur)

a The Upjohn Company, Investigative Toxicology (7228-300-5), Kalamazoo, MI 49001, USA b AB Astra, Safety Assessment, S-151 85, S6dertiilje, Sweden

c University ofMainz, Mainz, Germany d USEPA (MD-68A), Research Triangle Park, NC 27711, USA

e Life Sciences Research Laboratory, Japan Tobacco Inc., Yokohama, Kanagawa 227, Japan f Pharmakon Research International, P.O. Box 609, Wacerly, PA 18471, USA

g Parke-Davis Pharmaceutical Research, 2800 Plymouth Road, Ann Arbor, MI 48105, USA h The Procter and Gamble Company, Miami Valley Laboratory, P.O. Box 398707, Cincinnati, OH 45239-8707, USA

(Received 28 January 1994; accepted 3 February 1994)

Abstract

As part of the International Workshop on Standardization of Genotoxicity Test Procedures, in Melbourne, 27-28 February 1993, various international guidelines were examined with respect to protocol issues in the area of mammalian cell gene mutation assays. The working group on mammalian cell gene mutation assays discussed a wide range of protocol issues related to study design; in most cases the recommendations are reasonably consistent with existing guidelines. Agreement was reached on several issues as follows. The upper limit of concentration for testing non-toxic substances should be 10 mM or 5 mg/ml , whichever is lower. For testing toxic substances the criteria of an acceptable upper limit of concentration should yield 10-20% survival. Any of several established mammalian cell mutation assays (L5178Y TK +/-, C H O / H P R T , AS52/XPRT, V79 /HPRT) can be used to evaluate mutagenesis in mammalian cells; the ouabain (Na/K-ATPase) system is not an acceptable mutation assay for routine evaluation of mutagenesis in mammalian cells. Ability to recover small colonies must be convincingly demonstrated when using the L5178Y TK +/- mouse lymphoma assay. In the mouse lymphoma assay (L5178Y TK÷/- ) , colonies in positive controls and at least two (if available) representative positive doses of the test compound should be sized if a positive response is seen; in the event of a negative response due to the test compound, colony sizing of the positive control is necessary to validate the conduct of the assay. Testing both in the presence and absence of $9 metabolic activation is necessary. It was not possible to come to a firm conclusion about the length of treatment. There was a general agreement that extended treatment times (> 2 cell cycles) often bear more disadvantages than advantages and should only be used with adequate justification. It is not necessary to repeat clear positive or clear negative tests

* Corresponding author. This article has been reviewed by the U.S.E.P.A. and approved for publication. Approval does not signify the contents necessarily reflect the views or policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

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236 C.S. Aaron et al. / Mutation Research 312 (1994) 235-239

when the assay has been adequately performed; this recommendation differs significantly from the UK guidelines. If treatment groups are not replicated, the numbers of doses tested should be increased; this recommendation differs significantly from the UK guidelines. Each laboratory should establish a historical database for the performance of a given assay in that laboratory.

Key words: Mammalian cell gene mutation assay; L5178Y TK÷/-; Standardisation

I. Introduction

Mammalian cell gene mutation assays have been used for many years and the diversity of the available systems attests to the varied methods found to grow mammalian cells and detect muta- tions (Cole et al., 1990; Li et al., 1991). As part of the International Workshop on Standardization of Genotoxicity Test Procedures, in Melbourne, 27-28 February 1993, various international guide- lines (Kirkland, 1994a) were examined with re- spect to protocol issues in the area of mammalian cell gene mutation assays. These assays generally detect cells which are able to survive in the pres- ence of a selective agent only when a newly arising mutation occurs. The assays share certain characteristics, such as the need to allow time for expression of mutations after treatment, but dif- fer significantly with respect to the cell types used, growth conditions of the cells and charac- teristics of the mutant colonies. Nevertheless, the working group felt that mutations can be de- tected reliably with several of these assays and that mammalian cell mutation assays offer advan- tages over other in vitro assays in some respects. However, the context for these assays, i.e., when they are to be used, was beyond the scope of the deliberations of the working group and therefore this aspect was not further discussed in Mel- bourne.

In preparing for the workshop in Melbourne, available regulatory guidelines (Kirkland, 1994a) were reviewed in an effort to evaluate protocol issues and identify areas of agreement and dis- agreement among them. The degree of detail present in the regulatory guidelines concerning gene mutation assays in mammalian cells was usually limited. However, five topic areas (dose setting, test systems of choice, study design, repe- tition of tests and criteria of final results) were

selected for discussion by the Working Group on Mammalian Cell Gene Mutation Assays. In gen- eral, the group felt that the protocols for conduct of specific gene mutation assays in mammalian cells have been worked out independently and that many aspects of protocol design are not highly contested. Consensus (as noted) was reached in many areas with respect to elements of protocol design for assays for mutation induc- tion using mammalian cells in culture. A number of issues of a generic nature, common to all in vitro assays was discussed in the section on in vitro issues in this series (Kirkland, 1994b).

2. Dose setting

The upper limit of concentration for testing non-toxic substances should be 10 mM or 5 mg/ml , whichever is lower (consensus). For test- ing toxic substances the criteria of an acceptable upper limit of concentration should yield 10-20% survival (consensus). Each assay uses somewhat different methods for estimating viability, how- ever, the choice of upper dose limit was not felt to be dependent on the method used for deter- mining the viability. For many concentra- t ion/toxicity curves the steepness of the effect, i.e. the relative increase in toxicity with increasing concentration of compound, is such that an arbi- trary level of toxicity may be difficult to predict with absolute assurance prior to the experiment and thus the suggested 10-20% level of survival is understood to be a guideline. Furthermore, cases may arise, particularly in those systems with high relative increases in mutant frequencies, for which excessive toxicity occurs above doses giving 20% survival. In such cases, it may be acceptable to choose a high dose which yields more than 20% survival. The acceptable metric for establish-

C.S. Aaron et aL / Mutation Research 312 (1994) 235-239 237

ing the upper concentration in the case of toxic agents should be chosen with regard to the method used for the assay but will normally be either relative total growth or colony forming efficiency determined immediately after treat- ment. Other methods such as LDH release or various staining methods were not felt to be ade- quately characterized to substitute in determining dose selection. In the case of compounds produc- ing steep dose/toxicity relationships, the interval between doses may be reduced. The number of concentrations to be tested was felt to be highly dependent on the number of replicates per con- centration and the number of viable cells (follow- ing treatment) per replicate. Finally, the doses used in the experiment should be chosen so as to span a wide concentration range from toxic to non-toxic while emphasizing the higher toxicity levels.

3. Test system

Mammalian cell mutation assays based on commonly used rodent cell lines were discussed in depth and a consensus was reached that any of several established mammalian cell mutation as- says (L5178Y TK +/-, C H O / H P R T , AS52 / XPRT, V 7 9 / H P R T ) assays can be used to evalu- ate mutagenesis in mammalian cells. Each of the assays clearly has advantages and disadvantages and no particular assay was judged superior for testing purposes (consensus). However, several assays ( 'ouabain' locus and those using primary cells) were either judged to be too insensitive or incompletely validated for routine use (con- sensus). While not specifically addressed in the charge to the working group, the location of the H P R T gene on the X-chromosome was noted to result in insensitivity to compounds which pro- duce their effect through a clastogenic mecha- nism and this factor will influence the role of the V 7 9 / H P R T and C H O / H P R T assays in any overall testing strategy devised using them; this point was not further pursued by the working group.

The established mammalian cell gene muta- tion assays substantially differ in properties which

are critical for an appropriate test design. For example, in systems with low background mutant frequencies and high relative increases in mutant frequencies in cells treated with reference muta- gens (such as the V79 and C H O / H P R T assays) the number of cells at risk is a critical factor. In systems with high background mutant frequencies and lower relative increases in mutant frequen- cies in treated cells (such as the L5178Y/TK system), optimal exposure concentrations are es- pecially important .

Ability to recover small colonies must be con- vincingly demonstrated when using the L5178Y TK +/- mouse lymphoma assay (consensus). Colonies in positive control and two (if available) positive doses of the test compound should be sized if a positive response is seen (consensus); colony sizing of test compound is not necessary in the case of experiments with compounds giving negative results. The positive control substance should be one capable of producing both small and large colonies in the LS178Y TK +/- mouse lymphoma assay. The properties of the experi- mental compound under test should be consid- ered carefully if a base analogue or a compound related to the selective agent is tested in any of the mammalian cell gene mutation assays includ- ing the mouse lymphoma assay. Thus, perfor- mance of the selection sys tem/agent must be confirmed when testing chemicals structurally re- lated to the selective agent.

4. Study design

Testing both in the presence and absence of $9 metabolic activation is necessary (consensus). The use of $9 metabolic activation is routinely used in testing for genotoxic activity and is de- scribed in several of the reports from this work- shop as well as in the literature, however, it is worth emphasizing that the choice of metabolic activation may take into account the structure of the test material (if known) as well as any avail- able information on the metabolism of the com- pound. Some differences in the method of prepa- ration and use of $9 metabolic activation exist between the various assays, therefore, it will be

238 C.S. Aaron et al. / Mutation Research 312 (1994) 235-239

necessary to document the characteristics of the preparation and use of the specific $9 metabolic activation system used in an experimental system.

The choice of metabolic activation conditions should be justified; usually an induced rat liver $9 system in the range of 2 -10% in the final mixture is used although other concentrations can be used with adequate justification. Treatment time in the presence of $9 and in the absence of $9 is nor- mally different due to the toxicity of $9. Further- more, duration of treatment is dependent on test system. Thus, it was not possible to come to a firm guideline about the length of treatment, although the dose range-finding data used to set upper limit doses should be based on a similar treatment regimen to that used in the experi- ment. Extended treatment times (> 2 cell cycles) often bear more disadvantages than advantages and should be justified on a case by case basis; many members of the panel felt that extended treatment times were generally inappropriate in mammalian cell gene mutation assays.

5. Replication of tests

With regard to the replication of assays, it is not necessary to repeat clearly positive or clearly negative tests when the assay has been ade- quately performed (consensus); repeating clearly positive or negative experiments was judged to be optional. In the experience of the group, few cases of discrepant results, i.e., cases in which a replicate assay gave fundamentally different re- sults, were cited, therefore the need for repeating these assays was not supported. Adequate perfor- mance of the assays implies that sufficient doses, i.e. three or more, and sufficiently rigorous doses, i.e. a high dose giving between 10 and 20% sur- vival, were used. Furthermore, it is not required to test replicate cultures provided sufficient vi- able cells are passed and plated at the high dose to ensure that negative cultures in the high dose selection plates are not due to toxicity. The work- ing group agreed to the following suggested guid- ance for several of the most widely used systems in terms of the numbers of viable cells to be used for mutation estimation (V79 and C H O / H P R T :

10 6 cells per treatment group; L5178Y TK+/- : 3 x 105 cells per culture; AS52/XPRT: 2 x 105). There was general agreement that this number of viable cells must have been present at the point in the assay corresponding to the minimum cell mass. The group emphasized the importance of the choice of dose interval (previously men- tioned) as being crucial for reaching a firm con- clusion, particularly when a steep dose/toxicity function is observed. The working group reached consensus that if treatment groups are not repli- cated, the numbers of doses tested should be increased.

6. Criteria for evaluation of final results

Each laboratory should establish a historical database for the performance of a given assay in that laboratory (consensus). An acceptable exper- iment will have control data consistent with the historical data base for that laboratory. The abso- lute increase in mutant frequency required to establish a positive response should be estab- lished in each laboratory based on analysis of variation of that laboratory's negative control data. The following frequencies of induced (net over concurrent background) mutants ( x 10 6) seems widely accepted as a cutoff for concluding that an observed mutation frequency represents a positive response: CHO, V79 (HPRT) > 20; L5178Y TK +/- > 100 and A S 5 2 / X P R T > 30. However, the cutoff frequency for inferring a positive response using a particular test system should be established in each laboratory (con- sensus). Positive results may be corroborated by statistical analysis, concentrat ion/response rela- tionships a n d / o r repeat of the experiments (thus, repeat of clearly positive experiments is optional). Specific recommendations with regard to the im- portance of dose dependence, or arbitrary 'fold increase' as criteria for positive responses were not developed by the working group. Further- more, no specific statistical tests were uniformly endorsed by the working group for use in the evaluation of mammalian cell gene mutation as- says.

C.S. Aaron et al. / Mutation Research 312 (1994) 235-239 239

7. Summary

The working group on mammal ian cell gene mutat ion assays discussed a wide range of proto- col issues related to study design. Agreement was reached on several issues as follows: • The upper limit of concentration for testing

non-toxic substances should be 10 mM or 5 m g / m l , whichever is lower.

• For testing toxic substances the criteria of an acceptable upper limit of concentration should yield 10-20% survival.

• Any of several established mammal ian cell mu- tation assays (L5178Y TK ÷/- , C H O / H P R T , A S 5 2 / X P R T , V 7 9 / H P R T ) can be used to evaluate mutagenesis in mammalian cells; the ouabain ( N a / K - A T P a s e ) system is not an ac- ceptable mutat ion assay for routine evaluation of mutagenesis in mammalian ceils.

• Ability to recover small colonies must be con- vincingly demonstra ted when using the L5178Y TK +/- mouse lymphoma assay.

• In the mouse lymphoma assay (L5178Y T K + / - ) , colonies in positive controls and at least two (if available) representative positive doses of the test compound should be sized if a positive response is seen; in the event of a negative response due to the test compound, colony sizing of the positive control is neces- sary to validate the conduct of the assay.

• Testing both in the presence and absence of $9 metabolic activation is necessary (consensus).

• It was not possible to come to a firm conclu- sion about the length of t reatment. There was a general agreement that extended t reatment times ( > 2 cell cycles) often has more disad- vantages than advantages and should only be used with adequate justification.

• It is not necessary to repeat clearly positive or clearly negative tests when the assay has been adequately performed.

• If t rea tment groups are not replicated, the number of doses tested should be increased.

• Each laboratory should establish a historical database for the performance of a given assay in that laboratory.

References

Cole, J., D.B. McGregor, M. Fox, J. Thacker and R.C. Garner (1990) Gene mutations in cultured mammalian cells, in: D.J. Kirkland (Ed.), Basic Mutagenicity Test: UKEMS Recommended Procedures, Cambridge University Press, Cambridge.

Kirkland, D.J. (1994a) Report of the International Workshop on Standardization of Genotoxicity Test Procedures: Pref- ace, Mutation Res., this issue.

Kirkland, D.J. (1994b) Report of in vitro subgroup, Mutation Res., this issue.

Li, A.P., C.S. Aaron, A.E. Auletta, K.L. Dearfield, J.C. Rid- dle, R.S. Slesinski and L.F. Stankowski Jr. (1991) An evaluation of the role of mammalian cell mutation assays in the testing of chemical genotoxicity, Reg. Toxicol. Phar- macol., 14, 24-40.