Does the 2-test in vitro genotoxicity battery of Ames plus in vitro micronucleus tests detect relevant substances that induce gene mutations in vivo?

The in vitro genotoxicity testing battery comprising a bacterial gene mutation test (Ames test) and an in vitro micronucleus (MNvit) test is widely recommended by regulatory authorities since it detects the 3 key modes of genotoxic action (gene mutations, clastogenicity and aneuploidy). The recommendations that this battery is acceptable for regulatory use are based on its ability to effectively detect in vivo genotoxins and carcinogens. However, substances inducing gene mutations are a particular concern (representing stable genetic change and a key step in the carcinogenic process), and more in vivo gene mutation data have been published in recent years due to the wider use of transgenic rodent gene mutation (TGR) and Pig-a assays. It is appropriate to question whether the Ames + MNvit battery is efficient at detecting substances that induce gene mutations in vivo. A review of several compilations of genotoxicity studies revealed 26 substances that were reported positive in TGR and/or Pig-a but negative in the Ames test. A detailed analysis showed that 25 of these results were questionable for various reasons, and not robust examples of the failure of the 2-test in vitro battery. Only procarbazine was convincingly positive in vivo but predominantly negative in vitro, and this is mainly due to its complex metabolism which is probably not optimal with standard in vitro metabolic activation systems. The analysis shows no evidence that adding a mammalian cell gene mutation test to the in vitro battery would improve the detection of in vivo gene mutagens.