A novel method to derive a human safety limit for PFOA by gene expression profiling and modelling

Abstract

Perfluorooctanoic acid (PFOA) is a persistent environmental contaminant that can accumulate in the human body due to its long half-life. This substance has been associated with liver, pancreatic, testicular and breast cancers, liver steatosis and endocrine disruption. PFOA is a member of a large group of substances also known as "forever chemicals" and the vast majority of substances of this group lack toxicological data that would enable their effective risk assessment in terms of human health hazards. This study aimed to derive a health-based guidance value for PFOA intake (ng/kg BW/day) from in vitro transcriptomics data. To this end, we developed an in silico workflow comprising five components: (i) sourcing in vitro hepatic transcriptomics concentration-response data; (ii) deriving molecular points of departure using BMDExpress3 and performing pathway analysis using gene set enrichment analysis (GSEA) to identify the most sensitive molecular pathways to PFOA exposure; (iii) estimating freely-dissolved PFOA concentrations in vitro using a mass balance model; (iv) estimating in vivo doses by reverse dosimetry using a PBK model for PFOA as part of a quantitative in vitro to in vivo extrapolation (QIVIVE) algorithm; and (v) calculating a tolerable daily intake (TDI) for PFOA. Fourteen percent of interrogated genes exhibited in vitro concentration-response relationships. GSEA pathway enrichment analysis revealed that "fatty acid metabolism" was the most sensitive pathway to PFOA exposure. In vitro free PFOA concentrations were calculated to be 2.9% of the nominal applied concentrations, and these free concentrations were input into the QIVIVE workflow. Exposure doses for a virtual population of 3,000 individuals were estimated, from which a TDI of 0.15 ng/kg BW/day for PFOA was calculated using the benchmark dose modelling software, PROAST. This TDI is comparable to previously published values of 1.16, 0.69, and 0.86 ng/kg BW/day by the European Food Safety Authority. In conclusion, this study demonstrates the combined utility of an "omics"-derived molecular point of departure and in silico QIVIVE workflow for setting health-based guidance values in anticipation of the acceptance of in vitro concentration-response molecular measurements in chemical risk assessment.

Keywords: Bayesian; Markov chain Monte Carlo; NAMs; PBK; PFOA; in silico; omics; reverse dosimetry.

FIGURE 1

Workflow for the integration of in vitro and in silico new approach methodologies to derive health-based guidance values for the risk assessment of a chemical.

FIGURE 2

Gene accumulation plots for the enriched pathways derived using BMDExpress3 concentration-response modelling of transcriptomics data derived from the exposures of human primary liver cell spheroids to PFOA. (A) Fatty acid metabolism pathway, which was identified as the most sensitive pathway using gene set enrichment analysis; (B) MYC Targets V1 and V2; and (C) unfolded protein response. Colours indicate the duration of exposure to PFOA, highlighting the consistent effects on fatty acid metabolism after 4, 10 or 14 days of exposure.