Investigating the dependency of in vitro benchmark concentrations on exposure time in transcriptomics experiments

There is increasing interest to employ in vitro transcriptomics experiments in toxicological testing, for example to determine a point-of-departure (PoD) for chemical safety assessment. However current practices to derive PoD tend to utilise a single exposure time despite the importance of exposure time on the manifestation of toxicity caused by a chemical. Therefore it is important to investigate both concentration and exposure time to determine how these factors affect biological responses, and as a consequence, the derivation of PoDs. In this study, metabolically competent HepaRG cells were exposed to five known toxicants over a range of concentrations and time points for subsequent gene expression analysis, using a targeted RNA expression assay (TempO-Seq). A non-parametric factor-modelling approach was used to model the collective response of all significant genes, which exploited the interdependence of differentially expressed gene responses. This in turn allowed the determination of an isobenchmark response (isoBMR) curve for each chemical in a reproducible manner. For 2 of the 5 chemicals tested, the PoD was observed to vary by 0.5-1 log-order within the 48-h timeframe of the experiment. The approach and findings presented here clearly demonstrate the need to take both concentration and exposure time into account when designing in vitro toxicogenomics experiments to determine PoD. Doing so also provides a means to use concentration-time-response modelling as a basis to extrapolate a PoD from shorter to longer exposure durations, and to identify chemicals of concern that can cause cumulative effects over time.