Abstract
As demonstrated in Part I, cultured MutaMouse primary hepatocytes (PHs) are suitable cells for use in an in vitro gene mutation assay due to their metabolic competence, their “normal” phenotype, and the presence of the MutaMouse transgene for reliable mutation scoring. The performance of these cells in an in vitro gene mutation assay is evaluated in this study, Part II. A panel of 13 mutagenic and nonmutagenic compounds was selected to investigate the performance of the MutaMouse PH in vitro gene mutation assay. The nine mutagens represent a range of classes of chemicals and include mutagens that are both direct‐acting and requiring metabolic activation. All the mutagens tested, except for ICR 191, elicited significant, concentration‐dependent increases in mutant frequency (MF) ranging from 2.6‐ to 14.4‐fold over the control. None of the four nonmutagens, including two misleading, or “false,” positives (i.e., tertiary butylhydroquinone [TBHQ] and eugenol), yielded any significant increases in MF. The benchmark dose covariate approach facilitated ranking of the positive chemicals from most (i.e., 3‐nitrobenzanthrone [3‐NBA], benzo[a]pyrene [BaP], and aflatoxin B1 [AFB1]) to least (i.e., N‐ethyl‐N‐nitrosourea [ENU]) potent. Overall, the results of this preliminary validation study suggest that this assay may serve as a complimentary tool alongside the standard genotoxicity test battery. This study, alongside Part I, illustrates the promise of MutaMouse PHs for use in an in vitro gene mutation assay, particularly for chemicals requiring metabolic activation. Environ. Mol. Mutagen. 60:348–360, 2019. © 2019 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.
Highlights
Regulatory evaluations of new and existing substances always require genetic toxicity assessment, and this generally includes in vitro assessments of mutagenic activity
PhIP yielded significant mutant frequency (MF) increases at the top three concentrations tested with a 3.7-fold increase at 10 μg/mL (Fig. 2F). 1,8-DNP yielded a significant MF increase of 3.6-fold at 10 μg/mL (Fig. 2G). 3-NBA elicited a significant MF fold-increase of 8.2 at 0.5 μg/mL, and this was accompanied by a sharp increase in cytotoxicity (Fig. 2H)
This study aimed for an initial characterization of the performance and applicability domain of the in vitro gene mutation assay in MutaMouse primary hepatocytes (PHs)
Summary
Regulatory evaluations of new and existing substances always require genetic toxicity assessment, and this generally includes in vitro assessments of mutagenic activity. The United States Federal Insecticide, Fungicide, and Rodenticide Act (US FIFRA) Pesticide Assessment Guidelines require that in vitro mammalian cell mutagenicity tests are performed (Jaeger, 1984). The Canadian Environmental Protection Act (CEPA) requires an in vitro test for gene mutation, with and without metabolic activation, for certain substances not on the Domestic Substances List (DSL) (Minister of Justice, 2018). Conference on Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidance on genotoxicity testing and data interpretation for pharmaceuticals intended for human use S2 (R1), and Japan’s Chemical Substance Control Law all require, under certain conditions, an in vitro assessment of either chromosomal damage or mutagenesis (USFDA, 2007; METI, 2009; ICH, 2011; ECHA, 2017).
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