Inter‑laboratory study of human in vitro toxicogenomics‑based tests as alternative methods for evaluating chemical carcinogenicity: a bioinformatics perspective

The assessment of the carcinogenic potential of chemicals with alternative, human-based in vitro systems has become a major goal of toxicogenomics. The central read-out of these assays is the transcriptome and the induced expression changes upon chemical exposure. While many studies exist that describe the transcriptomic responses of such systems, reports on robustness and reproducibility when testing the systems independently in different laboratories are uncommon. Furthermore, there is limited knowledge about variability induced by bioinformatics procedures. We have conducted an inter-laboratory study for testing chemical carcinogenicity with two human in vitro systems: hepatoma-derived cells (HepaRG) and hTERT-immortalized renal proximal tubule epithelial cells (RPTEC/TERT1), representing liver and kidney as major target organs. Cellular systems were initially challenged with thirty compounds from three toxicity classes (ten genotoxic and ten non-genotoxic carcinogens as well as ten non-carcinogens), genome-wide gene expression was measured with microarrays and hazard classifiers were built from this training set. Subsequently, each system was independently established in three different laboratories and gene expression measurements were conducted with three blinded compounds. Data analysis was performed independently by two different groups. Bioinformatic workflows were developed both for the assessment of inter-laboratory reproducibility and for the prediction of carcinogenic hazard. As a result, both workflows came to very similar conclusions with respect to i) identification of non-reproducible experiments, ii) overall assessment of robustness and reproducibility and iii) re-classification of the unknown compounds to the respective toxicity classes. In summary, the developed bioinformatics workflows deliver accurate measures for inter-laboratory comparison studies and can be used as guidance for validation of future carcinogenicity assays in order to implement testing of human in vitro alternatives to animal testing.JRC.I.5-Systems Toxicolog