Per- and polyfluoroalkyl substances (PFAS) in mixtures show additive effects on transcriptomic points of departure in human liver spheroids.

Per- and polyfluoroalkyl substances (PFAS) are a wide range of chemicals that are used in a variety of consumer and industrial products leading to direct human exposure. Many PFAS are chemically nonreactive and persistent in the environment, resulting in additional exposure from water, soil, and dietary intake. While some PFAS have documented negative health effects, data on simultaneous exposures to multiple PFAS (PFAS mixtures) are inadequate for making informed decisions for risk assessment. The current study leverages data from previous work in our group using Templated Oligo-Sequencing (TempO-Seq) for high-throughput transcriptomic analysis of PFAS-exposed primary human liver cell spheroids; herein, we determine the transcriptomic potency of PFAS in mixtures. Gene expression data from single PFAS and mixture exposures of liver cell spheroids were subject to benchmark concentration (BMC) analysis. We used the 25th lowest gene BMC as the point of departure to compare the potencies of single PFAS to PFAS mixtures of varying complexity and composition. Specifically, the empirical potency of 8 PFAS mixtures were compared to predicted mixture potencies calculated using the principal of concentration addition (ie, dose addition) in which mixture component potencies are summed by proportion to predict mixture potency. In this study, for most mixtures, empirical mixture potencies were comparable to potencies calculated through concentration addition. This work supports that the effects of PFAS mixtures on gene expression largely follow the concentration addition predicted response and suggests that effects of these individual PFAS in mixtures are not strongly synergistic or antagonistic.

Potency Ranking of Per- and Polyfluoroalkyl Substances Using High-Throughput Transcriptomic Analysis of Human Liver Spheroids.

Per- and polyfluoroalkyl substances (PFAS) are some of the most prominent organic contaminants in human blood. Although the toxicological implications of human exposure to perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) are well established, data on lesser-understood PFAS are limited. New approach methodologies (NAMs) that apply bioinformatic tools to high-throughput data are being increasingly considered to inform risk assessment for data-poor chemicals. The aim of this study was to compare the potencies (ie, benchmark concentrations: BMCs) of PFAS in primary human liver microtissues (3D spheroids) using high-throughput transcriptional profiling. Gene expression changes were measured using TempO-seq, a templated, multiplexed RNA-sequencing platform. Spheroids were exposed for 1 or 10 days to increasing concentrations of 23 PFAS in 3 subgroups: carboxylates (PFCAs), sulfonates (PFSAs), and fluorotelomers and sulfonamides. PFCAs and PFSAs exhibited trends toward increased transcriptional potency with carbon chain-length. Specifically, longer-chain compounds (7-10 carbons) were more likely to induce changes in gene expression and have lower transcriptional BMCs. The combined high-throughput transcriptomic and bioinformatic analyses support the capability of NAMs to efficiently assess the effects of PFAS in liver microtissues. The data enable potency ranking of PFAS for human liver cell spheroid cytotoxicity and transcriptional changes, and assessment of in vitro transcriptomic points of departure. These data improve our understanding of the possible health effects of PFAS and will be used to inform read-across for human health risk assessment.

High-Throughput Transcriptomic Analysis of Human Primary Hepatocyte Spheroids Exposed to Per- and Polyfluoroalkyl Substances as a Platform for Relative Potency Characterization.

Per- and poly-fluoroalkyl substances (PFAS) are widely found in the environment because of their extensive use and persistence. Although several PFAS are well studied, most lack toxicity data to inform human health hazard and risk assessment. This study focused on 4 model PFAS: perfluorooctanoic acid (PFOA; 8 carbon), perfluorobutane sulfonate (PFBS; 4 carbon), perfluorooctane sulfonate (PFOS; 8 carbon), and perfluorodecane sulfonate (PFDS; 10 carbon). Human primary liver cell spheroids (pooled from 10 donors) were exposed to 10 concentrations of each PFAS and analyzed at 4 time points. The approach aimed to: (1) identify gene expression changes mediated by the PFAS, (2) identify similarities in biological responses, (3) compare PFAS potency through benchmark concentration analysis, and (4) derive bioactivity exposure ratios (ratio of the concentration at which biological responses occur, relative to daily human exposure). All PFAS induced transcriptional changes in cholesterol biosynthesis and lipid metabolism pathways, and predicted PPARα activation. PFOS exhibited the most transcriptional activity and had a highly similar gene expression profile to PFDS. PFBS induced the least transcriptional changes and the highest benchmark concentration (ie, was the least potent). The data indicate that these PFAS may have common molecular targets and toxicities, but that PFOS and PFDS are the most similar. The transcriptomic bioactivity exposure ratios derived here for PFOA and PFOS were comparable to those derived using rodent apical endpoints in risk assessments. These data provide a baseline level of toxicity for comparison with other known PFAS using this testing strategy.

A decision tree approach to screen drinking water contaminants for multiroute exposure potential in developing guideline values.

The consideration of inhalation and dermal routes of exposures in developing guideline values for drinking water contaminants is important. However, there is no guidance for determining the eligibility of a drinking water contaminant for its multiroute exposure potential. The objective of the present study was to develop a 4-step framework to screen chemicals for their dermal and inhalation exposure potential in the process of developing guideline values. The proposed framework emphasizes the importance of considering basic physicochemical properties prior to detailed assessment of dermal and inhalation routes of exposure to drinking water contaminants in setting guideline values.

The use of exposure source allocation factor in the risk assessment of drinking-water contaminants.

In the risk assessment process, the reference dose, tolerable intake, or acceptable daily intake (RfD, TDI, ADI) is apportioned to specific exposure sources on the basis of a source allocation factor (AF) or relative source contribution (RSC). The U.S. Environmental Protection Agency (EPA) published an exposure decision tree framework in 2000 to guide the determination of AF (or RSC) of drinking-water contaminants (DWC). Besides that, there has not been any systematic analysis of the basis of the use of AF in DWC risk assessments. This article therefore critically reviews and integrates current knowledge and approaches for the development of AF, while focusing on its consistent use in DWC risk assessments based on consideration of (i) risk assessment endpoint, (ii) existing guidelines, (iii) exposure estimates, (iv) usage pattern and environmental fate information, (v) physicochemical properties, (vi) bounds of AF, (vii) multiroute exposures, and (viii) target population characteristics. Accordingly, for a DWC for which drinking water is not a major source of exposure and for which there is documented evidence of widespread presence in one or more of the other media (i.e., air, food, soil, or consumer products), the use of an AF value of 0.2 is suggested. For DWC for which drinking water represents nearly the single major source of exposure, a ceiling AF value of 0.8 is suggested. For other situations, chemical- and context-specific AF values can be developed based on exposure data or models, which should in turn be bounded by the floor and ceiling AF values as originally described by the U.S. EPA (i.e., 0.2-0.8). Future studies need to focus on improvements in methods for deriving AF, by basing it on the consideration of bioavailability, target tissue dose, and extent of route-specific absorption, as well as improvement in the modeling of dose received via direct/voluntary exposure through consumer products and at workplaces.

Approaches for evaluating the relevance of multiroute exposures in establishing guideline values for drinking water contaminants.

In establishing the guideline values for chemical contaminants in drinking water, the contribution of inhalation and dermal routes associated with showering/bathing needs to be evaluated. The present article reviews the current approaches available for evaluating the importance of inhalation and dermal routes of exposure to drinking water contaminants (DWCs) and integrates them within a 2-tier approach. Accordingly, tier 1 would evaluate whether the dermal or inhalation route is likely to contribute to at least 10% of the dose received from ingestion of drinking water (i.e., 0.15 L-equivalent per day based on the daily water intake rate of 1.5 L/day typically used in Health Canada assessments). Based on the route-specific exposure parameters (i.e., area of skin exposed, effective skin permeability coefficient [K(p)], and air to water concentration ratio during use conditions [F(air-water)], breathing rate, duration of contact, and fraction absorbed), it was determined that for DWCs with K(p) less than 0.024 cm/hr and F(air - water) less than 0.0063, the dermal and inhalation routes during showering or bathing are unlikely to contribute significantly to the total dose. For DWCs with K(p) value equal to or greater than 0.025 cm/hr, dermal notation is implied, and as such, tier 2 calculation of L-equivalent associated with dermal exposure needs to be performed. Similarly, for DWCs with F(air-water) greater than 0.00063, inhalation notation is implied, and detailed evaluation of the L-equivalent associated with inhalation exposure (i.e., tier 2) is suggested. In general, data from human volunteer studies, observational measurements, and targeted modeling studies are useful for deriving L-equivalents, reflective of the magnitude of dose received via dermal and inhalation routes relative to the oral route. However, in resource-limited situations, these approaches can be integrated within a 2-tier approach for prioritizing and providing quantitative evaluations of the relevance of dermal and inhalation routes for developing exposure guidelines for DWCs.

Deriving uncertainty factors for threshold chemical contaminants in drinking water.

Uncertainty factors are used in the development of drinking-water guidelines to account for uncertainties in the database, including extrapolations of toxicity from animal studies and variability within humans, which result in some uncertainty about risk. The application of uncertainty factors is entrenched in toxicological risk assessment worldwide, but is not applied consistently. This report, prepared in collaboration with Health Canada, provides an assessment of the derivation of the uncertainty factor assumptions used in developing drinking-water quality guidelines for chemical contaminants. Assumptions used by Health Canada in the development of guidelines were compared to several other major regulatory jurisdictions. This assessment has revealed that uncertainty factor assumptions have been substantially influenced by historical practice. While the application of specific uncertainty factors appears to be well entrenched in regulatory practice, a well-documented and disciplined basis for the selection of these factors was not apparent in any of the literature supporting the default assumptions of Canada, the United States, Australia, or the World Health Organization. While there is a basic scheme used in most cases in developing drinking-water quality guidelines for nonthreshold contaminants by the jurisdictions included in this report, additional factors are sometimes included to account for other areas of uncertainty. These factors may include extrapolating subchronic data to anticipated chronic exposure, or use of a LOAEL instead of a NOAEL. The default value attributed to each uncertainty factor is generally a factor of 3 or 10; however, again, no comprehensive guidance to develop and apply these additional uncertainty factors was evident from the literature reviewed. A decision tree has been developed to provide guidance for selection of appropriate uncertainty factors, to account for the range of uncertainty encountered in the risk assessment process. Recent development of a series of "decision trees" by WHO to derive chemical specific adjustment factors for inter- and intraspecies variability may present an opportunity for a more systematic approach for the identification of evidence-based uncertainty factors.