In vitro transcriptomic analyses reveal pathway perturbations, estrogenic activities, and potencies of data-poor BPA alternative chemicals.

Since initial regulatory action in 2010 in Canada, bisphenol A (BPA) has been progressively replaced by structurally related alternative chemicals. Unfortunately, many of these chemicals are data-poor, limiting toxicological risk assessment. We used high-throughput transcriptomics to evaluate potential hazards and compare potencies of BPA and 15 BPA alternative chemicals in cultured breast cancer cells. MCF-7 cells were exposed to BPA and 15 alternative chemicals (0.0005-100 µM) for 48 h. TempO-Seq (BioSpyder Inc) was used to examine global transcriptomic changes and estrogen receptor alpha (ERα)-associated transcriptional changes. Benchmark concentration (BMC) analysis was conducted to identify 2 global transcriptomic points of departure: (1) the lowest pathway median gene BMC and (2) the 25th lowest rank-ordered gene BMC. ERα activation was evaluated using a published transcriptomic biomarker and an ERα-specific transcriptomic point of departure was derived. Genes fitting BMC models were subjected to upstream regulator and canonical pathway analysis in Ingenuity Pathway Analysis. Biomarker analysis identified BPA and 8 alternative chemicals as ERα active. Global and ERα transcriptomic points of departure produced highly similar potency rankings with bisphenol AF as the most potent chemical tested, followed by BPA and bisphenol C. Further, BPA and transcriptionally active alternative chemicals enriched similar gene sets associated with increased cell division and cancer-related processes. These data provide support for future read-across applications of transcriptomic profiling for risk assessment of data-poor chemicals and suggest that several BPA alternative chemicals may cause hazards at similar concentrations to BPA.

Quantitative in vitro to in vivo extrapolation of genotoxicity data provides protective estimates of in vivo dose.

Genotoxicity assessment is a critical component in the development and evaluation of chemicals. Traditional genotoxicity assays (i.e., mutagenicity, clastogenicity, and aneugenicity) have been limited to dichotomous hazard classification, while other toxicity endpoints are assessed through quantitative determination of points-of-departures (PODs) for setting exposure limits. The more recent higher-throughput in vitro genotoxicity assays, many of which also provide mechanistic information, offer a powerful approach for determining defined PODs for potency ranking and risk assessment. In order to obtain relevant human dose context from the in vitro assays, in vitro to in vivo extrapolation (IVIVE) models are required to determine what dose would elicit a concentration in the body demonstrated to be genotoxic using in vitro assays. Previous work has demonstrated that application of IVIVE models to in vitro bioactivity data can provide PODs that are protective of human health, but there has been no evaluation of how these models perform with in vitro genotoxicity data. Thus, the Genetic Toxicology Technical Committee, under the Health and Environmental Sciences Institute, conducted a case study on 31 reference chemicals to evaluate the performance of IVIVE application to genotoxicity data. The results demonstrate that for most chemicals considered here (20/31), the PODs derived from in vitro data and IVIVE are health protective relative to in vivo PODs from animal studies. PODs were also protective by assay target: mutations (8/13 chemicals), micronuclei (9/12), and aneugenicity markers (4/4). It is envisioned that this novel testing strategy could enhance prioritization, rapid screening, and risk assessment of genotoxic chemicals.