The history of the development of the cell transformation assays (CTAs) is described, providing an overview of in vitro cell transformation from its origin to the new transcriptomic-based CTAs. Application of this knowledge is utilized to address how the different types of CTAs, variously addressing initiation and promotion, can be included on a mechanistic basis within the integrated approach to testing and assessment (IATA) for non-genotoxic carcinogens. Building upon assay assessments targeting the key events in the IATA, we identify how the different CTA models can appropriately fit, following preceding steps in the IATA. The preceding steps are the prescreening transcriptomic approaches, and assessment within the earlier key events of inflammation, immune disruption, mitotic signaling and cell injury. The CTA models address the later key events of (sustained) proliferation and change in morphology leading to tumor formation. The complementary key biomarkers with respect to the precursor key events and respective CTAs are mapped, providing a structured mechanistic approach to represent the complexity of the (non-genotoxic) carcinogenesis process, and specifically their capacity to identify non-genotoxic carcinogenic chemicals in a human relevant IATA.

Perfluorinated alkyl substances and polyfluorinated alkyl substances (PFASs) are long-chain compounds, with perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) being the most well-known examples. Both are considered typical non-genotoxic carcinogens (NGTxCs). In this study, we verified whether the Bhas 42 cell transformation assay (Bhas 42 CTA) can be used as an effective in vitro method to predict carcinogenicity of NGTxCs using both PFOA and PFOS as typical representatives. Transcriptome analysis during the PFOA-induced transformation process showed that many factors related to the effects of PFOA on the immune system and cancer hallmarks increased or decreased. Thus, we demonstrated that mechanistic analyses such as transcriptome analyses in combination with the transformation focus formation results from the Bhas 42 CTA may be useful tools when assessing the carcinogenicity and other biological effects of NGTxCs such as PFOA. We propose that the Bhas 42 CTA is a simple in vitro test for the detection of NGTxCs, that it has in vitro oncotransformation as an endpoint, and that it can also detect the activation of factors involved in malignant progression, such as invasion and metastasis. It allows for the comprehensive detection of subtle mechanisms in parallel with focus formation throughout the transformation process, from the early stages to malignancy.