Regulatory Genotoxicity Testing: A Critical Appraisal

Abstract

This review considers current approaches to regulatory genotoxicity testing, focusing on how the use of animals can be further replaced, reduced and refined. The complementary roles of in vitro and in vivo testing, and the justification for using animals, are discussed in detail. Recommendations are made for improvements and further work, in the light of the considerable current controversy surrounding the composition and deployment of testing strategies, and the interpretation of the data generated, particularly for carcinogenicity prediction. The major problems are the oversensitivity of in vitro tests and the insensitivity of in vivo assays. On the basis of an analysis of some published databases, it is concluded that there is insufficient support for using in vivo genotoxicity assays for screening. Also, it is questionable whether the scientific benefits of using such assays always outweigh the costs to the animals involved. The considerable efforts being made to harmonise in vivo protocols and to develop improved methods for detecting genotoxicity are discussed. It is recommended that more emphasis be placed on characterising genotoxins in vitro, especially for mechanisms of activity, to optimise the benefits of any confirmatory animal tests.. Also, regulatory agencies are urged to require better-designed and more-scientifically sound protocols, in which animal numbers are minimised and data interpretation, particularly that of negative results, is facilitated. Lastly, in the development and validation of transgenic rodent systems, emphasis should be placed on developing protocols in which other acute toxicity and metabolism endpoints can be measured simultaneously with in vivo mutagenesis, while minimising animal numbers.

Cell Transformation Assays: Are we Barking up the Wrong Tree?

“…What is also of interest from Tables 1 and 2 is that only two known or probable human carcinogens were uniquely positive in one or more Tier 2 (in vivo) tests, necessitating further investigation to explain the pattern of activity (18), whereas 11 chemicals were uniquely positive in supplementary genotoxicity tests, many of which were in vitro assays. This supports efforts designed to reduce the number of false positives generated by in vitro Tier 1 genotoxicity tests (19) and the incorporation of supplementary in vitro tests in any integrated testing strategy, as opposed to extending the range of in vivo tests involved in Tier 2 (16,20,21). Others have commented on the inability of standard genotoxicity tests to detect carcinogens (22).…”

Proposed Integrated Decision-tree Testing Strategies for Mutagenicity and Carcinogenicity in Relation to the EU REACH Legislation

“…If the in vitro micronucleus test were to become an acceptable substitute for the chromosomal aberration test, it would provide a way of detecting aneugens (by the induction of spindle disruption resulting in micronuclei containing centromeres) and clastogens (by the induction of micronuclei lacking centromeres), in one test. This would be less labour-intensive than performing metaphase analysis (6), and cheaper than conducting an in vivo bone-marrow micronucleus test (see later).…”

“…Many in vitro methods are available for detecting genotoxicity (6)(7)(8)(9). As described in more detail below, these are divided into tier 1 and tier 2, core and ancillary, assays (the former usually comprising bacterial mutagenicity and cytogenetics tests, although gene mutation testing in cultured mammalian cells is sometimes also undertaken).…”

“…It is also important to score numerical aberrations (polyploidy and hypodiploidy/hyperdiploidy), indicative of aneuploidy (14,15). Ancillary in vitro genotoxicity assays (6,7) include in silico modelling, gene mutation in cultured mammalian cells, differential DNA repair, the in vitro micronucleus assay, unscheduled DNA synthesis (UDS), mitotic recombination, gene conversion/ mutation in yeast (Saccharomyces cerevisiae), the COMET assay, the use of genetically engineered cell lines, and toxicogenomics. Some of the more widely used and potentially available methods for mutagenicity testing are listed in Table 2 and summarised below.…”

Chapter 9: Genotoxicity and Carcinogenicity

“…Genotoxicity testing is an important part of the hazard assessment of chemicals for regulatory purposes (2,3). It is undertaken for two main reasons (4,5): a) to detect chemicals that might cause genetic damage, including point mutations, in germ cells, and thus increase the burden of genetic disease in the human population; and b) to detect chemicals that might be carcinogenic (based on the assumption that mutagenesis is a key event in the process of carcinogenesis). A scheme for genotoxicity testing is presented in Figure 9.1.…”