Environmental Mutagenesis 7263-266 (1985)

Letter to the Editor

Gonadal Genotoxicity Assays as Practical Surrogates for Germ-Cell Mutagenicity Assays

The US Environmental Protection Agency (EPA) recently published an amended criteria document for the assessment of the germ-cell mutagenic hazard posed by environmental chemicals [Anon, 19841. Almost coincident with its publication a review undertaken by an EPA gene-tox committee [Russell et al 19841 of the relative performance of the available rodent germ-cell mutagenicity assays appeared. The latter dealt in detail with the relative performance of a range of resource-consuming rodent germ-cell mutation assays and also approached the question of risk-estimation. On the final page of the gene-tox review, the performance of mouse sperm DNA repair (unscheduled DNA sytheiss [UDS]) assays was considered: of the nine rodent germ-cell mutagens assessed, only one failed to induce UDS in sperm, and both of the nonmutagens were inactive. This class of assay therefore gave the best correlation with heritable germ-cell mutagenicity data of the assays considered by Russell et a1 [1984]. In the EPA document [Anon, 19841 sperm UDS assays were referred to in the section dealing with “evidence that a test chemical had reached and interacted with gonadal DNA,” but their use as surrogates for germ-cell mutation assays was not discussed.

There is general agreement that the expense incurred in mounting definitive germ-cell assays to define agents capable of inducing heritable chromosomal or gene mutations will probably preclude their routine deployment [Anon, 19841. Emphasis therefore switches to the most practical and cost-effective testing scheme for detecting probable mammalian germ-cell mutagens. It may prove profitable to collate the available evidence, and derive new data, to confirm or refute the efficacy of the testing scheme shown below (Fig. 1). Even if successful, this approach would remain essentially empirical; on the other hand, to consider only dcjjtzitive evidence of heritable mutagenic activity may leave the majority of structurally novel germ-cell mutagens undetected (and therefore neglected) over the coming decades owing to financial restraints.

An advantage of the approach proposed (Fig. 1) is that it extends naturally from that generally adopted to define probable mammalian carcinogens. Possible disadvan- tages are that sperm UDS assays are not yet generally practiced, and that they may be complicated by the need to evaluate the several stages of the spermatogenic cycle [Sega, 19841. The stage-specific expression of the myc oncogene in germ cells [Stewart et al, 19841 supports the concept that these cells should not be regarded as a single biological entity; the phrase “sperm UDS assays” used herein is therefore generic.

0 1985 Alan R. Liss, Inc.

264 Ashby

Definition of Genotoxicity in vitro

Salmonella mutation test Chromosomal aberration assay Other genotoxicity data?

0 0 0 I I I I I I I 0 0

+ 1

Assessment of Expression of Genotoxicity in vivo

Assume to be Non-genotoxic

Prediction of Possible Carcinogenicity

Mouse micronucleus test Liver U D S assay Additional ~n VIVO genotoxicity tests’,

Prediction of Possible Germ-cell Mutagenicity

Sperm UDS Additional germ-cell genotoxicity tests?

Fig. 1. Proposed combined scheme for the definition of probable mammalian carcinogens and germ cell mutagens. The definition of genotoxicity in vitro has been suggested to require the conduct of a full Salmonella gene mutation assay and one for the induction of chromosomal aberrations [Ashby et al, 19851. More extensive testing procedures have also been described [Berry and Litchfield, 19851. Use of short-term in vivo genotoxicity assays to discern probable mammalian carcinogens, from among agents defined as genotoxic in vitro, has been discussed earlier [Matter and Schmid, 1971; Stich and Kaiser, 1974; Mirsalis and Butterworth, 1980; de Serres and Ashby, 1981; Ashby, 1983; Beije and Ashby, 1985; Ashby and Beije, 19851. Assays that provide evidence of genotoxic activity in the gonads of rodents have been described by Sega et a1 [1976, 1978, 1981, 19831, and Working and Butterworth [1984] and have been reviewed by Russell et al [1984]. The stage specificity of UDS within the spermatogenic cycle does not always correlate with mutagen sensitivity [Sega, 19841; likewise, the expression of the myc oncogene is dependent upon the stage of the spermatogenic cycle assessed [Stewart et al, 19841. Several related reviews of germ-cell mutagenicity assays have recently appeared [Brusick and Kilbey, 1983; Adler, 1983; Russell and Shelby, 19841.

In order to advance understanding of chemically induced germ-cell mutagen- icity, it is vital that definitive heritable mutagenicity data continue to be generated. However, it may prove expedient to define a class of probable germ-cell mutagens from among the growing number of novel in vitro genotoxins.The implicit proposition is that in vitro genotoxins that elicit somatic cell genotoxicity in acute in vivo studies should be regarded as probable mammalian carcinogens, and that those that elicit acute germ-cell genotoxicity should be regarded as probable inducers of heritable mutations. The recent development of a rat sperm UDS assay Working and Butter- worth, 19841 may increase the potential of this approach. It is suggested that the difference between probable and confirmed germ-cell mutagenicity effects may be too subtle to pursue in the majority of cases. These sentiments echo those of Brusick, as expressed in a recent Litton Newsletter [Brusick, 19841; namely, that a decade of research has emphasized both the value of in vitro genotoxicity assays and the absence or inadequacy of appropriate mammalian carcinogenicity/mutagenicity data. This

Surrogate Germ Cell Assays 265

does not invalidate previous “validation” studies, but it indicates that acute in vivo genotoxicity data may have an important role to play in the future, and that observa- tions of genotoxicity for a new chemical in vitro cannot be dismissed in their absence.

It is appreciated that this letter will not lead to the mass adoption of sperm UDS assays, but the principle of practical surrogate assays may prove worthy of debate in this journal. The fact that all germ cell mutagens are also active as mutagens in the bone marrow [Holden, 19821 is also highly pertinent.

REFERENCES

Adler ID (1983): Comparison of types of chemically induced genetic changes in mammals. Mutation Res 115:293-321.

Anon (1984): Proposed guidelines for mutagenicity risk assessment. Federal Register 49(227):463 14- 4632 I .

Ashby J (1983): The unique role of rodents in the detection of possible human mutagens and carcinogens, Mutation Res 115: 177-213.

Ashby J, Beije B (1985): Concomitant observations of UDS in the liver and micronuclei in the bone marrow of rats exposed to cyclophosphamide or 2-acetylaminofluorene. Mutation Res 150 (in press).

Ashby J, de Serres FJ, Draper M, Ishidate M, Margolin B Jr, Matter B, Shelby MD (eds) (1985): Collaborative study of short-term tests for carcinogens. “Progress in Mutation Research, Vol 5. ” Amsterdam: Elsevier.

Beije B, Ashby J (1985): Use of an in vivolin vitro rat liver DNA repair assay to predict the relative rodent hepatocarcinogenic potency of 3 new azo mutagens. Carcinogenesis (in press).

Berry DJ, Litchfield MH (1985): A review of the current regulatory requirements for mutagenicity testing. In Ashby J et al (eds): Collaborative Study of short-term tests for carcinogens. “Progress in Mutation Research, Vol 5.” Amsterdam: Elsevier.

Brusick DJ (1984): Molecular Toxicology Newsletter (lV)2; Fall 1984. Litton Bionetics Inc, Kensington, MD.

Brusick D, Kilbey BJ (1983): Screening strategy for chemicals that are potential germ-cell mutagens in mammals. ICPEMC Committee 1, Final Report. Mutation Res 114: 117-177.

de Serres FJ, Ashby J (1981): Evaluation of short-term tests for carcinogens: Results of the International Collaborative Study. Amsterdam: Elsevier/North Holland.

Holden HE (1982) Comparison of somatic and germ cell models for cytogenetic screening. J Appl Toxicol 2: 196-200.

Matter B, Schmid N (1971): Trenimon-induced chromosomal damage in bone marrow cells of six mammalian species, evaluated by the micronucleus test. Mutation Res 12:417-425.

Mirsalis JC, Butterworth BE (1980): Detection of unscheduled DNA synthesis in hepatocytes isolated from rats treated with genotoxic agents. Carcinogenesis 1 :62 1-625.

Russell LB, Aaron CS, de Serres F, Generoso WM, Kannan KL, Shelby M, Springer J , Voytek P (1984): Evaluation of mutagenicity assays for purposes of genetic risk assessment. A report of the USA EPA Gene-Tox Program. Mutation Res 134:143-157.

Russell LB, Shelby MD (1984): Tests for heritable genetic damage and for evidence of gonadal exposure in mammals. Mutation Res (in press).

Sega GA (1984): A review of the genetic effects of ethyl methanesulphonate. Mutation Res 134:113- 142.

Sega GA, Owens JG and Cumming RO (1976): Studies on DNA repair in early spermatid stages of male mice after in vivo treatment with methyl, ethyl, propyl and isopropyl methanesulphonate. Muta- tion Res 36:193-213.

Sega GA, Sotomayer RE, Owens JG (1978): A study of unscheduled DNA synthesis by X rays in the germ cells of male mice. Mutation Res 49:239-257.

Sega GA, Wolfe KW, Owens JG (1981): A comparison of the molecular action of an SN-I type methylating agent, methylnitrosourea and an SN-2 type methylating agent, methyl methanesul- phonate, in the germ cells of male mice. Chem Biol Interact 33:253-269.

Sega GA, Kelley MR, Owens JG, Carricarte VC (1983): Caffeine pre-treatment enhances unscheduled DNA synthesis in spermatids of mice exposed to methyl methanesulphonate. Mutation Res 108:345-358.

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Stewart TA, Bellve AR, Leder P (1984): Transcription and promoter usage of the myc gene in normal

Stich HF, Kaiser D (1974): Use of DNA repair synthesis in detecting organotropic actions of chemical

Working PK, Butterworth BE (1984): An assay to detect chemically induced DNA repair in rat

somatic and spermatogenic cells. Science 226:707-710.

carcinogens. Proc SOC Exp Biol Med 145: 1339-1342.

spermatocytes. Environ Mutagen 6:273-286.

John Ashby Central Toxicology Laboratory Imperial Chemical Industries PLC Cheshire, United Kingdom