The screening of chemicals for the protection of human health and the environment requires the assessment of genetic toxicity. However, existing, internationally‐accepted in vitro mammalian genotoxicity tests have been criticized for their low specificity (i.e. high frequency of “false” or “misleading” positive results for compounds that are negative in vivo). An in vitro transgene mutation assay has been established that uses a metabolically competent cell line derived from MutaMouse lung (i.e. FE1 cells). Mutation scoring employs the well‐characterized lacZ positive selection system, and the assay is proposed as an alternative in vitro assessment tool. In this study, the performance of the FE1 cell assay was evaluated by examining responses to nine non‐DNA‐reactive chemicals that previously elicited misleading positive results in other mammalian cell genotoxicity assays. FE1 cells were exposed to concentrations up to approximately 10 mM and/or concentrations that yielded approximately 80‐90% cytotoxicity (as measured by relative increase in cell count). The assay demonstrated excellent specificity; exposures to the chemicals examined did not yield any positive responses even when tested in the presence of an exogenous metabolic activation system (i.e. S9) or with an extended sampling time. These results indicate that the FE1 cell mutagenicity assay is an effective and practical alternative to traditional mammalian cell gene mutation assays. The development and validation of effective in vitro tools such as the MutaMouse FE1 cell assay will contribute to international efforts to reduce, refine, and replace experimental animals for toxicity assessment. Environ. Mol. Mutagen. 58:582–591, 2017. © 2017 Wiley Periodicals, Inc.