Analysis of published data for top concentration considerations in mammalian cell genotoxicity testing

Abstract

The ability of the in vitro mammalian cell tests currently used to identify genotoxins has been shown to be limited by a high rate of false-positive results, triggering further unnecessary testing in vivo. During an European Centre for the Validation of Alternative Methods workshop on how to improve the specificity of these assays, testing at high concentrations was identified as one possible source of false positives. Thus far, Organisation for Economic Co-operation and Development genotoxicity test guidelines have required testing of chemicals using mammalian cells in vitro should be undertaken to concentrations as high as 10 mM (5000 μg/ml). Recently, a draft revision of the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use genotoxicity test guidelines has recommended that testing concentrations should be reduced to 1 mM (500 μg/ml). To assess the impact that this lowering would have on the outcome of in vitro genotoxicity testing, we established a database of 384 chemicals classified as rodent carcinogens and reported Ames test results and the test concentrations that produced positive results in the mouse lymphoma assay (MLA), in vitro chromosome aberration (CA) assay and in vitro micronucleus test. Genotoxicity testing results were illustrated for 229 and 338 compounds in the MLA and in vitro CA assay, respectively. Of these test compounds, 62.5% produced positive results in the MLA, of which 20.3% required testing between 1 and 10 mM. A total of 58.0% produced positive results in in vitro CA assays, of which 25.0% required testing between 1 and 10 mM. If the testing concentration limit for mammalian cell assays was reduced to 1 mM, 24 (6.25%) potential carcinogens would not be detected in any part of the standard in vitro genotoxicity test battery (Ames test, MLA and in vitro CA assay). Further re-evaluation and/or retest of these compounds by Kirkland and Fowler [Kirkland, D. and Fowler, P. (2010) Further analysis of Ames-negative rodent carcinogens that are only genotoxic in mammalian cells in vitro at concentrations exceeding 1 mM, including retesting of compounds of concern. Mutagenesis 25, 539-553] suggest that the current 10 mM top concentration can be reduced without any loss of sensitivity in detecting rodent carcinogens.