REGULATORY

TOXICOLOGY

AND

PHARMACOLOGY

14,24-40

(199

1)

An Evaluation of the Roles of Mammalian Cell Mutation Assays in the Testing of Chemical Genotoxicity’ A . P* LI*-~ > C.S. AARON,? A.E. AULETTA,# K. L. DEARFIELD,$J.C.RIDDLE,§ R. S. SLESINSKI,’ AND L. F. STANKOWSKI, JR! *Monsanto Company, SZF, 800 N. Lindbergh Blvd., St. Louis, Missouri 63167; tThe Upjohn Company, Kalamazoo, Michigan 49001 $U.S. Environmental Protection Agency, Washington, D.C. 20460; gB. F. Goodrich Company, Brecksville, Ohio 44141; ‘Technical Assessment Systems, Washington, D.C. 20007: and “Pharmakon Research International, Inc., Waverly, Pennsylvania 18471

Received

September

24, 1990

The present status of the applicability of mammalian cell gene mutation assaysin the safety evaluation of industrial chemicals is evaluated from the industrial and regulatory point of view, with emphasis being placed on the CHO/HGPRT and mouse Iymphoma tk+/- assays. The CHO/HGPRT assaywas concluded to be a highly specific assay,but it might be less sensitive to mutagens that mainly induced large deletions. The mouse Iymphoma assaywas concluded to be sensitive, but it might have a lower specificity due to experimental artifacts such as pH and osmolality changes. Mammalian gene mutation assays,when conducted within their limitations, are concluded to be valuable in safety evaluation, providing results complementary to the Ames test and cytogenetic assays. 0 1991 Academic Presr. Inc.

INTRODUCTION Several recent key events have led to the reevaluation of the role of genotoxicity testing in safety assessment of industrial chemicals. These events, in chronological order, include the International Program on Chemical Safety’s (WCS) collaborative study on mutagenesis assays (de Serres and Ashby, 198 1; Ashby et al., 1985); the Phase III Gene-Tox review of selected mutagenesis assays(summarized by Auletta and Ashby ( 1988)); and the National Toxicology Program (NTP) effort in the testing of carcinogens and noncarcinogens in a selected battery of short-term tests (summarized by Tennant et al. ( 1987)). Results of these efforts, especially those of the NTP, led to a series of ’ This paper has been reviewed by the Office of Pesticides and Toxic Substances, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. ’ To whom correspondence should be addressed.

24 0273-2300191

$3.00

Copyright 0 1991 by Academic Press, Inc. AII rights of reproduction in any form reserved.

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25

commentaries and reviews expressing a diversity of opinions (e.g., Brockman and DeMarini ( 1988) and Shelby et al. ( 1988)). The major point of concern: What should be considered the adequate combination of tests for the evaluation of chemical genotoxicity? Because of the known differences between bacteria1 and mammalian ceils, such as membrane permeability, genome organization, and DNA repair, mammalian cell systems have been developed and used in genotoxicity test batteries to augment the SulmonelIu histidine reversion assay developed by Ames et al. (1975). The purpose of this paper is to critically review the role of mammalian mutagenicity assays in the genotoxicity evaluation of industrial chemicals. Based on published information as well as our experience, we present here an upto-date evaluation of the field of mammalian cell mutagenesis, with emphasis on the two widely used assays: the Chinese hamster ovary/hypoxanthine-guanine phosphoribosyltransferase (CHO/HGPRT) and the mouse lymphoma L5 178Y tk+/- mutation assay. INDUSTRIAL

PERSPECTIVES

a. Purposes for Testing Chemicals are tested for genotoxicity in industry for two major reasons: (1) To evaluate potential human health risk for chemicals with anticipated human exposure and (2) to satisfy regulatory requirements. The two reasons are obviously interrelated, as regulatory requirements exist (see below) to evaluate the safety of the chemical products. The extent of testing is generally a function of the known biological activities and the anticipated level of human exposure. b. Influence of Regulatory Agencies Several factors influence the choice of genotoxicity assays, with the requirements or “perceived” requirements of the regulatory agencies being one of the most significant. Industrial scientists, based on their own experience and expertise, may have special preferences for specific tests used for genotoxicity testing. However, for chemicals that require regulatory approval, the tests that are required or considered appropriate by regulatory agencies are usually the ones that are performed. As further described below, industrial chemicals may fall under the jurisdiction of the Toxic Substances Control Act (TSCA) or the Federal Insecticide Fungicide Rodenticide Act (FIFRA) of the U.S. Environmental Protection Agency (USEPA). Pharmaceuticals are mainly under the jurisdiction of the Food and Drug Administration (FDA), which, although it does not have a written requirement for genotoxicity testing, does apply genotoxicity data for the evaluation of human health risk potential. The inclusion of mammalian cell mutation assays in a routine genotoxicity testing battery in industry will be influenced by a perceived or an actual requirement for such assays by regulatory agencies. c. Data Interpretation Carcinogenicity is probably one of the most undesirable chronic toxicological properties of a chemical. Genotoxicity data are often used in industry as initial indicators

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of carcinogenicity for the planning of a product development strategy. In this respect, the NTP findings have a significant impact. The results of the NTP 73-compound study (Tennant et al., 1987) led to the general conclusion that a battery of four shortterm tests (the Ames test, mouse lymphoma gene mutation, in vitro cytogenetics, and in vitro sister-chromatid exchange) did not have a greater concordance with carcinogenicity than any of the individual assays alone. The NTP data therefore provide empirical justification against the use of a large battery of assays. Using a smaller number of tests, lending importance to specificity (ability to give negative results for noncarcinogens) as well as sensitivity (ability to detect carcinogens), and using the weight-of-evidence approach to analyze data (as opposed to placing significance on isolated positive results) are part of the recommendations offered by the American Industrial Health Council Mutagenicity Subcommittee (Kier, 1988). The use of a smaller test battery is also preferred by industry for the submission of data to regulatory agencies. One possible interpretation of the NTP results is that the chances of obtaining a spurious positive result with a nongenotoxic chemical increase with the number of tests performed. To negate a positive finding often is difficult and may require extensive resources. An important consideration for the inclusion of mammalian cell mutation assays in a genotoxicity battery is therefore whether such an inclusion will enhance our understanding of the genotoxic properties of the chemicals in question without significantly increasing the probability of spurious positive responses. Genotoxicity data are also used to gain an understanding of the mechanism of action of a chemical with known carcinogenicity. An awareness of the mechanism of action of a carcinogenic chemical can be useful in making decisions about potential risk to human health. Chemicals that do not act via a genotoxic mechanism, for instance, may act through a mechanism which may not pose a significant risk to human health at expected exposure levels. Mammalian cell mutation assays will be a useful addition to a testing battery if the data obtained will allow a better understanding of the mechanism of action of the chemicals tested.

d. Cost-Efectiveness of Testing Economics also play an important role in the decision to include certain tests in a testing battery. Research using an exhaustive combination of assay systems and experimental conditions may allow the most scientific estimation of human genotoxic risks of the chemicals in question. However, this approach may be cost prohibitive for all but a few chemicals with high production volume or potentially high levels of human exposure. The approach which is most likely to gain widespread acceptance within industry is one which includes tests which are regarded as being predictive of in vivo events and which are acceptable by the regulatory agencies to which data may be submitted. The Ames test, being the test with the most published data as well as the least expensive, definitely is and will continue to be the major genotoxicity assay. The question is whether mammalian cell mutation assays are among the tests accepted by the scientific community as well as the regulatory agencies to complement the Ames test for the best evaluation of mammalian genotoxicity.

MAMMALIAN

CELL

REGULATORY 1. The USEPA’s O&e

GENE

MUTATION

ASSAYS

27

PERSPECTIVES of Pesticide Programs (OPP)

a. Testing Requirements Currently, mutagenicity testing is required for all uses of chemicals when they are registered as pesticides with the EPA’s OPP. Part 158-Data Requirements for Registration-of the Code of Federal Regulations (CFR) (USEPA, 1984) and Subdivision F of the Pesticide Assessment Guidelines (USEPA, 1982) issued by OPP detail the purpose of and recommendations for mutagenicity testing. Subdivision F presents representative tests that may be used to satisfy Part 158 testing requirements. OPP requires minimal testing to be performed in three areas of mutagenicity concern: (1) gene mutation; (2) structural chromosomal aberrations; and (3) other genotoxic effects as appropriate for the test substance. Subdivision F provides a listing of representative tests that may satisfy these three testing areas. Properly conducted cultured mammalian cell gene mutation tests are able to satisfy the requirements for a test in the gene mutation category. The USEPA’s OPP is currently undergoing a review of its Subdivision F Guidelines to decide which mutagenicity tests are necessary to satisfy the Part 158 requirement for mutagenicity testing. Therefore, the main question becomes, which tests are the most useful for producing information that would lead to the best decision(s) for evaluation of risk in light of the three OPP uses (see below) for mutagenicity test data? b. Purposes for Mutagenicity

Testing

Once the chemical has been properly tested in the three mutagenicity areas of concern, OPP evaluates the test results. Further testing may be suggested by the results, for example, to evaluate the potential for heritable risk or to clarify the available test results. Once all appropriate tests are submitted, the data are used to assessa potential heritable risk concern based on the USEPA’s Mutagenicity Risk Assessment Guidelines. To perform a quantitative risk assessment for a heritable mutagenicity risk from a chemical, appropriate data are required. Currently, data from properly conducted specific locus and/or heritable translocation assays are utilized for performing such a quantification. However, these tests are not routinely performed during an initial screen for mutagenicity potential. Other tests are usually performed, which might include a mammalian cell gene mutation assay. Based on the results of these tests, further testing in a specific locus or heritable translocation assay is considered. In addition to heritable risk assessment, mutagenicity data are used in two other roles for mutagenicity testing: (1) as part of the body of information used to make a decision to trigger oncogenicity testing when such testing was not originally required for a chemical, and (2) as part of the weight-of-evidence for determining a carcinogenicity classification for a chemical when a long-term bioassay has been performed. How mutagenicity data are utilized to support a carcinogenic&y classification is based on the USEPA’s Carcinogenicity Risk Assessment Guidelines. Based on these uses of mutagenicity testing, do cultured mammalian cell gene mutation test data fit appropriately into the set of testing requirements for any or all of

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the three purposes for mutagenicity testing? To address this question, it is important for experts in the field of mammalian cell gene mutation assays to decide upon the most appropriate methods and test systems for providing the best information concerning a chemical’s possible genotoxic capability. How mammalian cell gene mutation assays would fit appropriately into the testing requirements could then be rationalized. 2. USEPA TSCA Section 4 Test Schemes a. Present Test Schemes Under Section 4 of TSCA, the USEPA pursues mutagenicity both as an endpoint itself and as an indicator of potential oncogenicity. Testing is required under two separate schemes: one for gene mutation and one for chromosomal effects. The test schemes for mutagenicity were designed during 1979- 1980 and were concerned with the identification of agents which might be potential human mutagens. In 1983, the Agency added triggers to mandate a two-year bioassay as part of its mutagenicity test scheme. As they are now constituted, the schemes are each composed of three tiers. A single positive response in tests in the Iower tiers triggers higher level testing (Figs. 1 and 2). Each endpoint is pursued independently, although a chemical must be tested in both schemes to satisfy the testing requirement.

Positive

Positive

I FIG.

1.

Current

U.S.

EPA

-

Bioassay TIiggW

El

TSCA Section 4 gene mutation test scheme.

MAMMALIAN

IN WV0 MAMMALIAN CYTOCENETICS 2 Metabolic Activation

CELL

Negative

+

GENE

MUTATION

IN VIVO MAMMALIAN CYTOGENETICS

29

ASSAYS

NC?ptiW *

NOFURTHBR TESTING

t Positive

Positive

Bioassay Trigger

Positive

Negative

RODENT HERITABLE TRANSLOCATION

FIG.

2. Current

U.S. EPA TSCA

Section

4

cytogeneticstest scheme.

The first four tests in the scheme, the Salmonella assay, the in vitro assay for gene mutation in mammalian cells in culture, and the in vitro and in vivo cytogenetics assays, are designed to detect intrinsic mutagenic potential. If they are negative, no further mutagenicity testing is required. The next two tests, the sex-linked recessive lethal (SLRL) assay in Drosophila melanogaster and the rodent dominant lethal assay, demonstrate the ability of a test agent to reach the gonad and interact with DNA in the germ cell. If these two assays are negative, no further testing is required. The last two tests, the mouse visible specific locus assay and the rodent heritable translocation assay, measure the ability of an agent to induce heritable gene or chromosomal mutations and can be used to generate data necessary for quantitative risk assessment. At the time the test schemes were finalized, early results were available from the first ICPS large-scale cross laboratory study on the ability of short-term tests to identify carcinogens, (de Serres and Ashby, 1981). These results indicated that agents which were negative in the Salmonella assay were subsequently identified as mutagens by testmg in an in vitro mammalian system. However, the study did not clarify whether gene mutation or cytogenetics assayswere preferred as a complement to the Salmonella assay. It also did not support the preferential use of a specific gene mutation assay for this purpose. In the Section 4 test schemes, a negative result in the Sulmonellu assay must be confirmed by a mammalian cell culture assay for gene mutation. Choice of cell system was left to the investigator unless there were data on a specific chemical which suggested that one system was preferable.

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Using some of the early findings correlating short-term test results and carcinogenicity, particularly those from the Gene-Tox Program (Green and Auletta, 1980, Waters and Auletta, 1981) the USEPA developed a scheme for using short-term tests to trigger in vivo carcinogenicity testing of industrial chemicals under Section 4 of TSCA (Figs. 1 and 2). These tests included a combination of a positive response in the Salmonella assay and the Drosophila SLRL assay; a positive response in an in vitro mammalian cell culture assay for gene mutation; a positive response in an in vitro cytogenetics assay: and a positive response in an in vivo cytogenetics assay. Each of the tests chosen to directly trigger a carcinogen bioassay was shown by the Gene-Tox Program to have a sensitivity greater than 80% toward carcinogens. Each had been designated as routine by the Gene-Tox Panel of the “Developmental Status of Bioassays in Genetic Toxicology” (Brusick and Auletta, 1985). The choice of a mammalian cell culture system to serve as a trigger following a negative Salmonella assay was based on the IPCS findings as described earlier. Emphasis was placed upon both gene mutation and chromosomal effects because both endpoints were used in the IPCS. The in vivo cytogenetics assay was chosen as a trigger because it was an in viva assay, and positive results in this system were considered to be of sufficient concern to warrant long-term testing for oncogenicity. 6. Proposed Revision of the Present Test Schemes Since the publication of the first Section 4 test rule which defined the tests mentioned above as triggers to a two-year bioassay, new information has become available. This new information prompted a review of the mutagenicity tests used as triggers to a bioassay under Section 4. These data were the subject of discussion at an USEPAsponsored “Workshop on the Relationship between Short-term Test Information and Carcinogenicity” held in Williamsburg (Auletta and Ashby, 1988; Kier, 1988). As a result of data presented at the meeting and the data of Tennant et al. (1987), the USEPA is considering the revision of Section 4 test schemes. The proposed revision is shown in Fig. 3. The major change is in the first tier where the test schemes for gene mutation and chromosomal aberrations have been combined. There are now three tests in the first tier: the Salmonella assay, an in vitro cell culture assay for gene mutation, and an in vivo assay for chromosomal effects. The agency is considering a proposal to make the L5 178Y TK+/system the preferred test for gene mutation. If this occurs, data from other systems would continue to be considered if submitted. Investigators who use the L5 178Y system would be encouraged to count both large and small colonies, thus allowing for the detection of chromosomal damage as well as gene mutation in this system. In the proposed revision to the TSCA scheme, there would no longer be a single test trigger to a bioassay. The bioassay trigger would depend on a minimum of two positive responses, at least one of which would be an in vivo assay. The in vitro cytogenetics assay would no longer be part of the test scheme. A positive response in all three first tier tests, a positive response in the Salmonella assay and the in vivo assay for chromosomal effects, or a positive response in the in vitro gene mutation assay and the in vivo assay for chromosomal effects will lead directly to a two-year bioassay.

MAMMALIAN

CELL GENE MUTATION

31

ASSAYS

Positive

Positive

5iihiFJ

pi!jk

FIG. 3. Proposed revision of U.S. EPA TSCA Section 4 mutagenicity test scheme.

Any other combination of responses, including a single positive response in any one assay or a positive response in both the Salmonella assay and the in vitro assay for gene mutation would lead to a review of the data in a public program review. As it is now envisioned, the program review would include representatives from all interested parties, including the Agency, the interested public, and the regulated industry. In the proposed scheme, the program review is scheduled to occur before a decision is made to require further testing. It is anticipated that during the program review all available data, including other test results, structure-activity relationships, production volume, and exposure figures, will be considered. It is anticipated that no further testing would be required for the majority of chemicals which are negative in all three of the first tier tests. However, where exposure data, structure-activity relationships, or other factors indicate it is warranted, these agents may also be subject to a program review and subsequent testing in a bioassay. It is also anticipated that a program review will be performed for those agents which may require testing in either the specific locus or the heritable translocation assay. STATUS

OF ASSAYS

1. CHO/HGPR

T

a. General Considerations One outstanding feature of the CHO/HGPRT since its introduction by Hsie, O’Neill, and co-workers (Hsie et al., 1979; O’Neill et al., 1977; Li, 1984, 1985) is the painstaking

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research effort invested in the optimization of the mutagenesis protocol and mutant characterization. The resulting mutagenesis assay is well defined in its procedures and extremely stringent in its mutant selection conditions. A guideline for the performance of the assay has recently been published (Li et al., 1987). Most of the critical steps in the assay performance are well- agreed upon by experts in the field. The CHO/HGPRT is therefore generally considered to be a well-characterized and standardized assay. b. Accuracy in Prediction

of Chemical Carcinogenicity

As the assay was not part of the NTP program, its performance with chemicals of different classes can only be judged based on published information. This has been performed as part of the USEPA Gene-Tox Phase III Program (Li et al., 1988). In the review, 87 chemicals were evaluated positive, 3 negative, and 31 inconclusive. (The inconclusive results are mainly due to testing yielding negative results, but not testing both in the presence and in the absence of activation.) The 87 chemicals span 24 of the 30 chemical classes according to the Gene-Tox classification scheme. The chemicals evaluated include 43 animal carcinogens, out of which 40 were found positive. The three carcinogens found not positive in the assay were formaldehyde, methapyrilene, and benzyl chloride. All three yielded negative results but none were tested both in the presence and in the absence of activation. Only one noncarcinogen was evaluated in the review, caprolactam, yielding a negative response. According to the Gene-Tox review, the assay appears to be responsive to a large variety of chemical classes, with good sensitivity toward carcinogens. The specificity of the assay could not be evaluated from the review as only one noncarcinogen was tested. However, since the review, eight NTP noncarcinogens, ascorbic acid, benzoin, 2-chloroethanol, geranyl acetate, 8-hydroxyquinoline, phenol, propyl gallate, and sulfisoxazole, were tested in the CHO/HGPRT assay (Plowers and Li, 1988; Stankowski et al., 1988b). Of the eight noncarcinogens, six were found negative, one (Zchloroethanol) was found positive, and one (benzoin) was equivocal. In a separate study, glyphosate, a noncarcinogen, was also found negative in the CHO/HGPRT assay (Li and Long, 1988). The Gene-Tox review plus the data on noncarcinogens therefore indicates that the assay has both high sensitivity and specificity. However, in contrast to the Gene-Tox review findings, the assay has been criticized as “insensitive.” This is probably due to two factors: (1) Strong clastogens in general yield positive but weak responses in the assay. Examples are the antibiotics actinomycin D, adriamycin, daunomycin, and bleomycin. (2) The assay in general has only weak responses toward aromatic amines which are strong mutagens in other assays like the Ames test. Examples are 2-aminofluorene, P-naphthylamine, and benzidine. The low response toward clastogens is probably a factor of the presence of “essential” genes adjacent to the hgprt gene on the X chromosome in CHO cells. Clastogens, yielding deletion of both the hgprt gene and the essential genes, are less likely to yield viable hgprtmutants. The reason for the low sensitivity of CHO cells to aromatic amines is not yet clear but probably is a function of the cellular metabolic activities. O-acetylase activity has been proposed to be critical for the formation of mutagenic intermediates from aromatic amines. The enzyme activity is found to be high in Salmonella, which is sensitive to aromatic amines, and low in CHO cells (Heflich et al., 1988). To better

MAMMALIAN

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define low responses, a large sampling size (3-5 X 106) for both mutagen treatment and mutant selection (Li and Shimizu, 1983; Sir@ and Gupta, 1983) as well as the use of multiple S9 levels (Li, 1984, 1985) have been recommended. However, some chemicals which are mutagenic in other systems may not be active in this assay because they may act through a mechanism which will not produce mutations at the HGPRT gene locus. c. Inclusion in Test Battery Because of its well-defined protocol, its sensitivity to a wide number of chemical classes, its high specificity toward noncarcinogens, and a good understanding of its limitations, the CHO/HGPRT assay has merits to be included in a genotoxicity battery. The use of a combination of the Ames test, the CHO/HGPRT assay, and an in vitro or in vivo cytogenetics assay to detect mammalian mutagens also is theoretically satisfying. The CHO/HGPRT assay will complement the Ames test to detect point mutagens that are unique to mammalian cells (see below, under Examples of Industrial Applications). The assay will complement the cytogenetic assay to detect mammalian point mutagens that are not strong clastogens. The Ames test and cytogenetic assay, on the other hand, will detect aromatic amines and strong clastogens, respectively, which yield only low responses in the CHO/HGPRT assay. d. New Development:

CHO/AS52

Cells

Recently, a new CHO cell line, CHO-AS52, has been developed via genetic-engineering. The CHO-AS52 cells were constructed from an X-ray-induced hgprt deletion CHO mutant (X3/5 cells) by transformation with the plasmid pSV2gpt and they contain a single, functional copy of the bacterial gene encoding the purine salvage pathway enzyme XPRT (Tindall, et al., 1984, 1986). Thus, the hgprt gene normally found in CHO-Kl-BH4 cells is replaced by the functionally analogous gpt in AS52 cells. In fact, the assay protocols for measuring mutation to HGPRT- in CHO-Kl-BH4 cells and to XPRT- in AS52 cells are virtually identical. During the development of the assay, it was observed that XPRT mutant frequencies induced by X rays were much higher than those previously observed in the CHO/ HGPRT assay (Tindall, et al., 1984, 1986). Subsequent studies demonstrated that HPRT and XPRT mutant frequencies induced by ethylmethane sulfonate (EMS), uv light, and ICR 19 1 were very similar and that these agents induced primarily presumptive point mutations at hprt and gpt (Stankowski and Hsie, 1986; Stankowski et al., 1986). In contrast, X rays induced primarily (or exclusively) deletion mutations and induced much higher XPRT than HPRT mutant frequencies (Stankowski and Hsie, 1986). Based on these results, it was proposed that the relatively lower mutability of hprt by clastogens was due to an inability to recover viable HPRT mutants with deletions extending into adjacent essential sequences of the monosomic Xchromosome. Due to the presumed location of gpt on an autosome (or other site able to withstand extremely large deletions), the AS52/XPRT assay was therefore predicted not to have similar limitations.

34

Ll ET AL.

More recent studies have supported this hypothesis. During concurrent comparisons with the CHO/HPRT assay, the AS52/XPRT assay was found to be much more sensitive to a host of clastogenic agents, including actinomycin D, adriamycin, bleomycin, cis-dichlorodiamine platinum, cyclophosphamide, daunomycin, ethanol, formaldehyde, hexamethylphosphoramide, methanol, and mitomycin C (Stankowski et al., 1988a,b). Southern blot analyses of mutants induced by several of these agents revealed that each primarily (or exclusively) induced deletion mutations at gpt (HPRT mutants not analyzed) (Tindall et al., 1986). However, similar induced HPRT and XPRT mutant frequencies were observed with classical point or frameshift mutagens such as dimethylnitrosamine (DMN) or ICR 19 1, and negative results were observed in both cell lines for cytotoxic concentrations of acetone, dimethyl sulfoxide (DMSO), hydrochloric acid (HCl), and sucrose (Stankowski, personal communication). The AS52/XPRT assay may be considered an improvement upon the more familiar CHO/HPRT assay. Results of concurrent evaluations of 36 agents in the two assays are available from the laboratory of Stankowski. Although admittedly few in number, the agents were carefully selected. Based on the demonstrated (or presumed) carcinogenic properties of these agents, the assay exhibits a sensitivity of 100% (20/20) with a specificity of 87.5% (14/16). While experts in the field of mammalian mutagenesis are hopeful that the AS52/ XPRT system will provide high quality mammalian mutagenicity data, it needs to be emphasized that most of the data on specificity and sensitivity came from the laboratories of the founders (Hsie and co-workers in Oak Ridge and Galveston; Stankowski and co-workers in Waverly). It is essential to demonstrate the same performance in other laboratories. Taking the present information at face value, it appears that the test system is a more sensitive version of the CHO/HPRT assay with a similarly high specificity. e. Examples

of Industrial

Applications

Testing results for several compounds from the Upjohn Co. (Kalamazoo, MI) are used here to illustrate the advantages of using CHO gene mutation assays to identify compounds that are capable of causing mutations. One involves materials which form formaldehyde (Aaron et al., 1989). Almost all N-methyl compounds (such as morphine) produce formaldehyde via N-demethylase activity. One such compound was U48,753E, a potential drug with an N-methyl substituent. The genetic toxicology profile with this drug was that it was negative in the Ames test, the micronucleus test, the rat hepatocyte UDS assay, and the sex-linked recessive lethal test and V-79/HGPRT. However, the compound was weakly mutagenic in the CHO/HGPRT and was clearly and reproducibly mutagenic in the AS52/XPT system. By adding formaldehyde dehydrogenase to the incubation mixture in the AS52/XPT system, the mutagenicity could be eliminated. Another case (Aaron et al., 1988) involved U-73,9755, a potential anticancer agent with potent DNA binding characteristics. Because of the high specificity of the drug for AT-rich regions of the DNA, the compound is negative in the Ames test using the standard strains. TA- 102 and mammalian cells (CHO/HGPRT and AS52/XPT) detect this class of compounds very efficiently. Furthermore, the electrophilic portion of the

MAMMALIAN

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35

molecules is not from one of the common functional groups but is a cyclopropane ring. This example illustrates the insufficiency of applying only the routine Ames test and structure alerts to identify mutagens. When six drug candidates were evaluated with both the CHO/HGPRT and the AS52/XPT assays, similar results were found (Aaron and Stankowski, 1989). 2. Mouse Lymphoma

tk+/-

Gene Mutation

Assay

a. General Considerations The mouse lymphoma assay is used to quantify genetic alterations affecting the expression of the thymidine kinase (TK) locus (Clive and Spector, 1975; Clive et al., 1979). Extensive research has resulted in an optimization of the mutagenesis protoco1 (Moore and Clive, 1982; Moore and Howard, 1982; Moore, 1984; Meyer et al., 1986; Majeska and Matheson, 1990). The genetic analysis of mutants using a combination of molecular and banded karyotype analyses indicates that both single-gene mutations and chromosomal mutations can be recovered (Hozier et al., 1985; Moore et al., 1985, 1987; Blazak et al., 1986). The assay is well characterized, but not particularly well standardized, for optimal performance. Recently a series of meetings have been held by some of the major users of the assay. These discussions indicate the need for a definitive document detailing the performance, presentation, and interpretation of test data for the assay. It is clear from the literature that the quality of published data for this assay is variable. 6. Accuracy in Prediction

of Chemical Carcinogenicity

The mouse lymphoma assay was used by NTP in its testing program, and the qualitative results were published by Tennant et al. ( 1987). In this study, the sensitivity of the assay for the prediction of rodent carcinogenicity was reported to be 0.70, with a specificity of 0.45, and the concordance was 0.60. One criticism of the NTP study was that the tests were “blindly” performed under a “standard” protocol. There was no attempt to determine the suitability of the test conditions or assay system for the particular compounds. Upon close evaluation, a number of the test compounds (approx. 17 out of 73) were not appropriate for testing due to problems of solubility, pH, etc. The Gene-Tox committee is currently reevaluating all of the published mouse lymphoma data to further examine the performance of the assay. It is clear from literature that the tk locus in cultured mammalian cells can be used to detect compounds that are clastogenic (e.g., Moore et al., (I 987)). This is in contrast with the hgprt locus (see above) which appears to be less sensitive to clastogens. c. Inclusion in Test Battery The assay appears to detect a broad spectrum of genetic events, including a series of chromosomal events not readily detected by either the Ames test or the CHO/ HGPRT assay. In contrast with aberration analysis, it detects only those genetic al-

36

LI ET AL.

terations compatible with cell survival. Furthermore, the molecular and cytogenetic analysis of mutations suggests that this assay registers the range of genetic lesions recently found in a wide variety of human tumors. It should be noted that a goal of genetic toxicology is to predict which chemicals will have significant human risks. Test systems capable of detecting genetic events critical to human cancer, such as the mouse lymphoma assay, should be a valuable component of a test battery. GENERAL

CONCLUSIONS

ON MAMMALIAN

CELL MUTATION

ASSAYS

a. Advantages A genotoxicity battery that includes mammalian cell mutation assays is scientifically valid. Consider the battery of the Ames test, mammalian cell mutation, and in vivo cytogenetics (or micronucleus): It includes the two major endpoints of genotoxicity (mutation and chromosomal aberration), in vitro correlation between prokaryotes and eukaryotes, and in vitro: in vivo correlation. Further, while in vivo mammalian gene mutation assays such as the specific locus assay of Russell et al. (1981) have been developed, they are not yet available for general use. The only practical assays to study mammalian gene mutation today are the mammalian cell assays. Mechanistic studies on mammalian cells have furthered our understanding of the assays. We now have evidence that the CHO/HGPRT assay measures mainly point mutations rather than deletions (Tindall et al., 1986); the small colonies of the mouse lymphoma assay represent mainly deletion mutations (Blazak et al., 1986). And, through genetic engineering, new assay systems such as CHO-AS52 can be developed to enhance our genotoxicity testing capability (Tindall et al., 1984). State-of-the-art endpoints, such as gene sequencing and oncogene activation, are applicable to mammalian cell systems and have potential to further enhance our ability to detect mammalian mutagens. Compared with another widely used mammalian cell assay, the in vitro cytogenetics assay, mammalian gene mutation assays in general have a better-defined protocol. The simplicity of the mutant selection and counting procedures allows a more efficient and objective quantitation of mutagenicity. Because of the standardized procedures and the ease of quantitation, the assays can be performed entirely by well-trained technicians rather than experienced cytogeneticists, therefore saving further on cost. In fact, the cost for a routine mammalian cell mutation assay (five test doses with and without activation or three test doses with multiple levels of S9) with an independent repeat experiment is approximately the same as that for an in vitro cytogenetics experiment (three test doses, with and without S9; three harvest times) without an independent repeat. b. Areas of Improvement For any other genotoxicity assays, it is important to know the limitations of the assays and to apply the assays within their range of limitations. It wiI1 not be fruitful to claim more significance than the assays can truly deliver. As an example, knowing the low sensitivity of CHO cells to aromatic amines, it will not be scientifically valid

MAMMALIAN

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to argue for a lower mammalian genotoxicity for an aromatic amine because it only elicits a strong response in prokaryotes. This argument, however, will have more scientific validity for a chemical belonging to a chemical class that the CHO/HGPRT assay is known to detect. Similarly, for the mouse lymphoma assay, where factors such as low pH and high osmolality are known to induce a mutagenic response, a unique positive response needs to be interpreted with care, as the results may not be a factor of the genotoxicity of the chemicals tested. Scientific judgment based on our understanding of the test systems is necessary for the interpretation of test data. Judgment on health risk based on test data reduced to “+” and “-” is a practice that needs to be avoided. It is only through our acceptance of the assay limitations that improvements can be made. For the CHO/HGPRT assay, research should be performed on the definition of experimental conditions with which aromatic amines and strong clastogens can be detected. This may involve the introduction of the O-acetylase gene through genetic engineering as well as the placement of the HGPRT gene on an autosome (as presumably occurred for the gpt gene in the CHO-AS52 cells) to increase its sensitivity toward clastogens. The CHO-AS52 system has the potential to be a mammalian gene mutation assay with both high sensitivity and specificity. The assay should be evaluated in multiple laboratories to test for its general applicability in genotoxicity testing. For the mouse lymphoma assay, research should be performed to determine the scientific validity of the reported positive findings of noncarcinogenic agents, especially those yielding negative responses in other genotoxicity assays. If they are due to a common misuse of the test protocol or data interpretation, the “correct” protocol and data interpretation criteria should be communicated by the experts. If the results are believed to reflect genuine mammalian genotoxicity, their validity should be carefully studied and documented. Data should continue to be generated with chemicals of known in vivo genotoxicity and carcinogenicity with respect to mammalian gene mutation assays in order to enhance our data base on specificity and sensitivity. Studies to demonstrate complementarity of the mammalian gene mutation assays with the Ames test and/or cytogenetics assays similar to that of Aaron et al. (1988, 1989) should be continued. c. CHO versus Mouse Lymphoma

Among bacterial mutagenesis assays, the Ames test is almost universally accepted as the standard test. For mammalian mutation assays, the CHO and mouse lymphoma assays are the two most commonly used. Experts in mammalian mutation assays cannot reach a consensus on which assay is the more appropriate for genotoxicity testing. The investigators with expertise in the CHO assay see it as an assay of high specificity and acceptable sensitivity for correlation with rodent carcinogenicity. The new advancement, using the CHO-AS52 cells, definitely improves the sensitivity of the assay for clastogens. Investigators with expertise in the mouse lymphoma assay view that assay as one of high sensitivity and acceptable specificity. The general view of “low” specificity for the mouse lymphoma assay, as that reported by Tennant et al. ( 1987) for the NTP results, can be overcome by setting a more stringent requirement for positive results. In general, however, the following is basically agreed upon:

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1. The CHO assay using the wild-type CHO cells is appropriate for genotoxicity testing if accompanied by an assay for clastogenicity or if the CHO-AS52 cell line is used. 2. The mouse lymphoma assay is appropriate for genotoxicity testing if controls for osmolality and pH are present, if more stringent criteria for positive results are adopted, and if both large and small colonies are considered. FINAL

REMARKS

AND RECOMMENDATIONS

Mammalian cell mutagenesis represents an important field in genetic toxicology, backed by significant research and an extensive data base. An essential key for successful application of assays in genotoxicity testing is to understand their limitations and to subsequently utilize the assays within these limits. Working within the known limitations of the assaysand paying attention to both specificity and sensitivity will further enhance the utility of the assays in genotoxicity testing. REFERENCES AARON, C. S., AND STANKOWSKI, L. F., JR. (1989). Comparison of the ASSZ/XPT and the CHO/HPRT assays:Evaluation of six drug candidates. Mutat. Rex 223, 12 1- 128. AARON, C. S.. MAZUREK, J., HARBACH, P., WISER, S. K., SMITH, A., STANKOWSKI, L., AND SORG, R. (1988). Comparative mutagenicity testing of a drug candidate: U-73,975, a potent AT-binding agent, is also extremely potent in inducing genetic changes in a variety of systems.Environ. Mol. Mutagen. 11, 2 (abstract). AARON, C. S., STANKOWSKI,L. F., JR.,HARBACH, P. R., VALENCIA, R., MAYO, J. K., MIRSALIS,J., MAZUREK, J. H., STEIMETZ, K. L., WISER, S. K., ZIMMER, D. M.. AND T~zos, R. J. (1989). Comparative mutagenicity testing of a drug candidate, U-48753E: Mechanism of induction of gene mutations in mammalian cells and quantitation of potential hazard. Mutat. Rex 223, 1 11- 120. AMES, 9. N., MCCANN, J., AND YAMASAKI, E. (1975). Methods for detecting carcinogens and mutagens with the .Sulmonella/mammalian microsome mutagenicity test. Mutat. Rex 31, 347-364. ASHBY, J., DE SERRES,F. J., DRAPER, M., ISHIDATE, M., MARCOLIN, 9. H., MATTER, 9. E., AND SHELBY, M. D. (1985). Progress in Mutation Research, Vol. 5. Evaluation of Short-term Tests for Carcinogens: Report of the International Programme on Chemical Safety’s Collaborative Study on In Vitro Assays. Elsevier, Amesterdam. AULETTA, A., AND ASHBY, J. (1988). Workshop on the relationship between short-term test information and carcinogenicity. Williamsburg, Virginia, January 20-23, 1987. Environ. Mol. Mutagen. 11, 135-146. BLAZAK, W. F., STEWART, 9. E., GALPERIN, 1..ALLEN, K. L., RUDD. C. J., MITCHELL, A. D., AND CASPARY. W. J. ( 1986). Chromosome analysis of trifuorothymidine-resistant L5 178 mouse lymphoma cell colonies. Environ. Mutagen. 8, 229-240. BROCKMAN, H. E., AND DEMARINI, D. M. (1988). Commentary: Utility of short-term tests for genetic toxicity in the aftermath of the NTP’s analysis of 73 chemicals. Environ. Mol. Mutagen. 11, 42 l-435. BRUSICK, D., AND AULETTA, A. (1985). Developmental status of bioassays in genetic toxicology: A report of Phase II of the U.S. Environmental Protection Agency Gene-Tox Program. Mutat. Res. 153, l-10. CLIVE, D., JOHNSON.K. O., SPECTOR,J. F. S., BATON, A. G., AND BROWN, M. M. M. (1979). Validation and characterization of the L5 178Y Tk+/- mouse lymphoma mutagen assaysystem. Mutnt. Res. 59, 61-108. CLIVE, D., AND SPECTOR,J. F. S. (1975). Laboratory procedures for assessing specific locus mutations at the Tk locus in cultured L5 178Y mouse lymphoma cells. Mutat. Rex 31, 17-29. DE SERRES.F. J., AND ASHBY, J. (1981). Progress in Mutation Research, Vol. 1, Evaluation of Short-Term Tests for Carcinogens: Report of the International Collaborative Program. Elsevier, Amsterdam. FLOWERS,L. J., AND LI, A. P. (1988). Evaluation of eight noncarcinogens in the CHO/HGPRT gene mutation system. Environ. Mol. Mutagen. 11, 34-35 (abstract).

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GREEN, S., AND AULETTA, A. (1980). Editorial introduction to the reports of the Gene-Tox Program: An evaluation of bioassays in genetic toxicology. Mutat. Res. 76, 165-168. HEFLICH, R. H., DJLJRIC,Z., ZHIJO, Z., FULLERTON, N. F., CASCIANO,D. A., AND BELAND, F. A. (1988). Metabolism of 2-acetylaminofluorene in the Chinese hamster ovary cell mutation assay.Mol. Environ. Mutug. 11, 167-182. HOZIER, J., SAWYER, J., CLIVE, D., AND MOORE, M. M. (1985). Chromosome 11 aberrations in small colony L5 178 YK-/- mutants early in their clonal history. Mutat. Res. 147, 237-242. HSIE, A. W., Q’NEILL, J. P., COUCH, D. B., SAN~EBASTIAN,J. R., BRIMER, P. A., MACHANOFF, R., RIDDLE, J. C., LI, A. P., FUSCOE, J. C., FORBES, N. L., AND HSIE, M. H. (1979). Utilization of a quantitative mammalian cell mutation system in experimental mutagenesis and genetic toxicology. In Strategy for Short-Term Testingfor Mutagens/Carcinogens. (B. Butterworth, Ed.), pp. 39-54. R. C. Press, West Palm Beach, FL. KIER, L. D. (1988). Commentary: Comments and perspective on the EPA workshop on “The Relationship between Short-term Test Information and Carcinogenicity.” Environ. Mol. Mutagen. 11, 147-I 57. LI, A. P. (1984). Use of Aroclor 1254-induced rat liver homogenate in the assaying of promutagens in Chinese hamster ovary cells. Environ. Mutagen. 6, 539-544. Lr. A. P. (1985). A testing strategy to evaluate the mutagenic activity of industrial chemicals. Regul. To.uicol. Pharmacol. 5,207-2 1I, LI, A. P. AND LONG, T. J. (1988). An evaluation of the genotoxic potential of glyphosate. Fundum. Appl. Toxicol. 11, 21-28. LI, A. P., AND SHIMIZU, R. W. (1983). A modified agar assay for the quantitation of mutation at the hypoxanthine guanine phosphoribosyl transferase gene locus in Chinese hamster ovary cells. Mutat. Res. 111,365-370.

LI, A. P., CARVER, J. H., CHOY, W. N., HSIE, A. W., CURA, R. S., LOVEDAY, K. S., O’NEILL, J. P., RIDDLE, J. C.. STANKOWSKI, L. F., JR., AND YANG, L. L. (1987). A guide for the performance of the Chinese hamster ovary cell/hypoxanthine guanine phosphoribosyl transferase gene mutation assay.Mutat. Rex 189, 135-141. LI, A. P., GUPTA, R. S., HEF’LICH, R. H., AND WASSOM, J. S. (1988). A review and analysis of the Chinese hamster ovary/hypoxanthine guanine transferase assayto determine the mutagenicity of chemical agents: A report of Phase III of the U.S. Environmental Protection Agency Gene-Tox Program. Mutat. Res. 196, 17-36. MAJESKA, J. B.. AND MATHESON, D. W. (1990). Development of an optimal S9 activation mixture for the L5 178 TKk mouse lymphoma mutation assay.Environ. Mol. Mutagen. 16, 31 l-3 19. MEYER, M., BROCK, K., LAWRENCE, K., CASTO, B., AND MOORE. M. M. (1986). Evaluation of the effect of agar on the results obtained in the L5 178 mouse lymphoma assay.Environ. Mutagen. 8, 727-740. MOORE, M. M. (I 984). The evaluation of the use of 10% rather than 20% horse serum in the cloning and selection of TK-/mutants of L5 178/TK+/- mouse lymphoma cells. Mutat. Rex 140, 2 15-2 18. MOORE, M. M., AND CLIVE, D. (1982). The quantitation of TK-/- and HGPRT- mutants of L5 178/TK+/ - mouse lymphoma cells at varying times post-treatment. Environ. Mutagen. 4,499-5 19. MOORE, M. M., AND HOWARD, B. E. (1982). Quantitation of small colony trifluorothymidine-resistant mutants of L5 178/TK+/mouse lymphoma cells in RPMI-1640 medium. Mutat. Res. 104, 287-294. MOORE, M. M., CLIVE, D., HOWARD, B. E., BATSON, A. G., AND TURNER, N. T. (1985). In situ analysis of trifluorothymidine-resistant (TFT’) mutants of L5 178Y/TK+/- mouse lymphoma cells. Mutat. Res. 151, 147-159. MOORE, M. M., BROCK, K. H., DEMARINI, D. M., AND DOERR. C. L. (1987). Differential recovery of induced mutants at the TK+/- and hgprt loci in mammalian cells. In Banbwy Report 28: Mammalian Cell Mutagenesis, pp. 93-108. Cold Spring Harbor, Cold Spring Harbor, NY. O’NEILL, J. P., BRIMER, P. A., MACHANOFF, R., HIRSCH, G. P., AND HSIE, A. W. (1977). A quantitative assay of mutation induction at the hypoxanthine guanine phosphirobosyl transferase locus in Chinese hamster ovary cells (CHO/HGPRT system): Development and definition of the system. Mutat. Rex 45, 91-101. RUSSELL, L. B., SELBY, P. B., VON HALLE, E., SHERIDAN, W., AND VALCOVIC, L. (198 I). Use of the mouse spot test in chemical mutagenesis: Interpretation of past data and recommendations for future work. Mutat. Res. 86, 355-379. SHELBY, M. D., ZEIGER, E., AND TENNANT, R. W. (1988). Commentary on the status of short-term tests for chemical carcinogens. Environ. Mol. Mutagen. 11, 437-442.

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An evaluation of the roles of mammalian cell mutation assays in the testing of chemical genotoxicity.

The present status of the applicability of mammalian cell gene mutation assays in the safety evaluation of industrial chemicals is evaluated from the ...
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