How accurate is in vitro prediction of carcinogenicity?

Positive genetic toxicity data suggest carcinogenic hazard, and this can stop a candidate pharmaceutical reaching the clinic. However, during the last decade, it has become clear that many non-carcinogens produce misleading positive results in one or other of the regulatory genotoxicity assays. These doubtful conclusions cost a lot of time and money, as they trigger additional testing of apparently genotoxic candidates, both in vitro and in animals, to discover whether the suggested hazard is genuine. This in turn means that clinical trials can be put on hold. This review describes the current approaches to the ‘misleading positive’ problem as well as efforts to reduce the use of animals in genotoxicity assessment. The following issues are then addressed: the application of genotoxicity testing screens earlier in development; the search for new or improved in vitro genotoxicity tests; proposed changes to the International Committee on Harmonisation guidance on genotoxicity testing [S2(R1)]. Together, developments in all these areas offer good prospects of a more rapid and cost-effective way to understand genetic toxicity concerns

Nongenotoxic apoptosis inducers do not produce misleading positive results in the TK6 cell-based GADD45a-GFP genotoxicity assay

The in vitro mammalian genotoxicity tests identify some carcinogens not identified by the bacterial Ames test. However, historically they have produced rather more misleading predictions of carcinogenicity than the Ames test. This liability has been reduced in pharmaceutical testing by lowering the top-testing dose and rejecting data from excessively toxic doses. It also stimulated the development of new assays with inherently higher specificity. Among these, the GADD45a-GFP assay has been recognized as a maturing technology by th

Development of a High-Throughput Gaussia

Exposure to genotoxic carcinogens leads to increased expression of the GADD45a gene in mammalian cells. This signature of genotoxic hazard has previously been exploited in the GreenScreen HC assay, in which GADD45a expression is linked to green fluorescent protein (GFP) expression in the human TK6 lymphoblastoid cell line. This article describes the development and validation of an alternative assay ("BlueScreen HC"), in which expression is linked to Gaussia luciferase (GLuc) expression, yielding a luminescent reporter, the preferred optical output in high-throughput screening. The coelentrazine substrate of GLuc is relatively unstable, and a new buffer is reported that improves its stability. A more sensitive method is demonstrated for the measurement of cell densities in the assay, using the fluorescent cyanine dye thiazole orange. A protocol amendment also allows the assessment of pro-genotoxicity using S9 liver extracts. Compounds from the European Centre for the Validation of Alternative Methods (ECVAM) recommended list for the assessment of new or improved genotoxicity assays were evaluated with and without S9 in the new assay. The new GLuc assay was as effective as the GFP assay in producing positive results for all classes of genotoxic carcinogen and negative results for all nongenotoxins tested. © 2012 Society for Laboratory Automation and Screening