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New approach methodologies (NAMs) that make use of in vitro screening and in silico approaches to inform chemical evaluations rely on in vitro toxicokinetic (TK) data to translate in vitro bioactive concentrations to exposure metrics reflective of administered dose. With 1364 per- and polyfluoroalkyl substances (PFAS) identified as of interest under Section 8 of the U.S. Toxic Substances Control Act (TSCA) and concern over the lack of knowledge regarding environmental persistence, human health, and ecological effects, the utility of NAMs to understand potential toxicities and toxicokinetics across these data-poor compounds is being evaluated. To address the TK data deficiency, 71 PFAS selected to span a wide range of functional groups and physico-chemical properties were evaluated for in vitro human plasma protein binding (PPB) by ultracentrifugation with liquid chromatography-mass spectrometry analysis. For the 67 PFAS successfully evaluated by ultracentrifugation, fraction unbound in plasma ( f up ) ranged from less than 0.0001 (pentadecafluorooctanoyl chloride) to 0.7302 (tetrafluorosuccinic acid), with over half of the PFAS showing PPB exceeding 99.5% ( f up < 0.005). Category-based evaluations revealed that perfluoroalkanoyl chlorides and perfluorinated carboxylates (PFCAs) with 6-10 carbons were the highest bound, with similar median values for alkyl, ether, and polyether PFCAs. Interestingly, binding was lower for the PFCAs with a carbon chain length of ≥11. Lower binding also was noted for fluorotelomer carboxylic acids when compared to their carbon-equivalent perfluoroalkyl acids. Comparisons of the f up value derived using two PPB methods, ultracentrifugation or rapid equilibrium dialysis (RED), revealed RED failure for a subset of PFAS of high mass and/or predicted octanol-water partition coefficients exceeding 4 due to failure to achieve equilibrium. Bayesian modeling was used to provide uncertainty bounds around f up point estimates for incorporation into TK modeling. This PFAS PPB evaluation and grouping exercise across 67 structures greatly expand our current knowledge and will aid in PFAS NAM development.

Per- and polyfluoroalkyl substances (PFAS) are a diverse set of commercial chemicals widely detected in humans and the environment. However, only a limited number of PFAS are associated with epidemiological or experimental data for hazard identification. To provide developmental neurotoxicity (DNT) hazard information, the work herein employed DNT new approach methods (NAMs) to generate in vitro screening data for a set of 160 PFAS. The DNT NAMs battery was comprised of the microelectrode array neuronal network formation assay (NFA) and high-content imaging (HCI) assays to evaluate proliferation, apoptosis, and neurite outgrowth. The majority of PFAS (118/160) were inactive or equivocal in the DNT NAMs, leaving 42 active PFAS that decreased measures of neural network connectivity and neurite length. Analytical quality control indicated 43/118 inactive PFAS samples and 10/42 active PFAS samples were degraded; as such, careful interpretation is required as some negatives may have been due to loss of the parent PFAS, and some actives may have resulted from a mixture of parent and/or degradants of PFAS. PFAS containing a perfluorinated carbon (C) chain length ≥8, a high C:fluorine ratio, or a carboxylic acid moiety were more likely to be bioactive in the DNT NAMs. Of the PFAS positives in DNT NAMs, 85% were also active in other EPA ToxCast assays, whereas 79% of PFAS inactives in the DNT NAMs were active in other assays. These data demonstrate that a subset of PFAS perturb neurodevelopmental processes in vitro and suggest focusing future studies of DNT on PFAS with certain structural feature descriptors.

A structurally diverse set of 147 per- and polyfluoroalkyl substances (PFAS) was screened in a panel of 12 human primary cell systems by measuring 148 biomarkers relevant to (patho)physiological pathways to inform hypotheses about potential mechanistic effects of data-poor PFAS in human model systems. This analysis focused on immunosuppressive activity, which was previously reported as an in vivo effect of perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS), by comparing PFAS responses to four pharmacological immunosuppressants. The PFOS response profile had little correlation with reference immunosuppressants, suggesting in vivo activity does not occur by similar mechanisms. The PFOA response profile did share features with the profile of dexamethasone, although some distinct features were lacking. Other PFAS, including 2,2,3,3-tetrafluoropropyl acrylate, demonstrated more similarity to the reference immunosuppressants but with additional activities not found in the reference immunosuppressive drugs. Correlation of PFAS profiles with a database of environmental chemical responses and pharmacological probes identified potential mechanisms of bioactivity for some PFAS, including responses similar to ubiquitin ligase inhibitors, deubiquitylating enzyme (DUB) inhibitors, and thioredoxin reductase inhibitors. Approximately 21% of the 147 PFAS with confirmed sample quality were bioactive at nominal testing concentrations in the 1-60 micromolar range in these human primary cell systems. These data provide new hypotheses for mechanisms of action for a subset of PFAS and may further aid in development of a PFAS categorization strategy useful in safety assessment.

High-throughput (HT) in vitro to in vivo extrapolation (IVIVE) is an integral component in new approach method (NAM)-based risk assessment paradigms, for rapidly translating in vitro toxicity assay results into the context of in vivo exposure. When coupled with rapid exposure predictions, HT-IVIVE supports the use of HT in vitro assays for risk-based chemical prioritization. However, the reliability of prioritization based on HT bioactivity data and HT-IVIVE can be limited as the domain of applicability of current HT-IVIVE is generally restricted to intrinsic clearance measured primarily in pharmaceutical compounds. Further, current approaches only consider parent chemical toxicity. These limitations occur because current state-of-the-art HT prediction tools for clearance and metabolite kinetics do not provide reliable data to support HT-IVIVE. This paper discusses current challenges in implementation of IVIVE for prioritization and risk assessment and recommends a path forward for addressing the most pressing needs and expanding the utility of IVIVE., Competing Interests: Authors MM, PM, MS, SH, CN, SP, PM and MY were employed by the company ScitoVation, LLC, author RC was employed by the company. 21st Century Tox Consulting and authors ML and HC were employed by the company Ramboll US Corp. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Moreau, Mallick, Smeltz, Haider, Nicolas, Pendse, Leonard, Linakis, McMullen, Clewell, Clewell and Yoon.)

Per- and polyfluoroalkyl substances (PFAS) are a broad class of hundreds of fluorinated chemicals with environmental health concerns due to their widespread presence and persistence in the environment. Several of these chemicals have been comprehensively studied for experimental toxicity, environmental fate and exposure, and human epidemiology; however, most chemicals have limited or no data available. To inform methods for prioritizing these data-poor chemicals for detailed toxicity studies, we evaluated 142 PFAS using an in vitro screening platform consisting of two multiplexed transactivation assays encompassing 81 diverse transcription factor activities and tested in concentration-response format ranging from 137 nM to 300 μM. Results showed activity for various nuclear receptors, including three known PFAS targets--specifically estrogen receptor alpha and peroxisome proliferator receptors alpha and gamma. We also report activity against the retinoid X receptor beta, the key heterodimeric partner of type II, non-steroidal nuclear receptors. Additional activities were found against the pregnane X receptor, nuclear receptor related-1 protein, and nuclear factor erythroid 2-related factor 2, a sensor of oxidative stress. Using orthogonal assay approaches, we confirmed activity of representative PFAS against several of these targets. Finally, we identified key PFAS structural features associated with nuclear receptor activity that can inform future predictive models for use in prioritizing chemicals for risk assessment and in the design of new structures devoid of biological activity., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Chemical risk assessment relies on toxicity tests that require significant numbers of animals, time and costs. For the >30,000 chemicals in commerce, the current scale of animal testing is insufficient to address chemical safety concerns as regulatory and product stewardship considerations evolve to require more comprehensive understanding of potential biological effects, conditions of use, and associated exposures. We demonstrate the use of a multi-level new approach methodology (NAMs) strategy for hazard- and risk-based prioritization to reduce animal testing. A Level 1/2 chemical prioritization based on estrogen receptor (ER) activity and metabolic activation using ToxCast data was used to select 112 chemicals for testing in a Level 3 human uterine cell estrogen response assay (IKA assay). The Level 3 data were coupled with quantitative in vitro to in vivo extrapolation (Q-IVIVE) to support bioactivity determination (as a surrogate for hazard) in a tissue-specific context. Assay AC 50 s and Q-IVIVE were used to estimate human equivalent doses (HEDs), and HEDs were compared to rodent uterotrophic assay in vivo-derived points of departure (PODs). For substances active both in vitro and in vivo, IKA assay-derived HEDs were lower or equivalent to in vivo PODs for 19/23 compounds (83%). Activity exposure relationships were calculated, and the IKA assay was as or more protective of human health than the rodent uterotrophic assay for all IKA-positive compounds. This study demonstrates the utility of biologically relevant fit-for-purpose assays and supports the use of a multi-level strategy for chemical risk assessment., Competing Interests: Declaration of competing interest None., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Following the 2010 Gulf of Mexico oil spill, concerns were raised regarding exposure of fish to crude oil components, particularly polycyclic aromatic hydrocarbons (PAHs). This three year study examined hepatic enzymes in post-mitochondrial supernatant fractions from red snapper (Lutjanus campechanus) and gray triggerfish (Balistes capriscus) collected in the north central Gulf of Mexico between 2011 and 2014. Biomarker activities evaluated included benzo(a)pyrene hydroxylase (AHH), ethoxyresorufin O-deethylase (EROD), glutathione transferase (GST), and glutathione peroxidase (GPx). Mean EROD activity was higher in gray triggerfish (12.97 ± 7.15 pmol/min/mg protein [mean ± SD], n = 115) than red snapper (2.75 ± 1.92 pmol/min/mg protein, n = 194), p < 0.0001. In both species, EROD declined over time between 2011 and 2014. Declines in GST and GPx activities were also noted over this time period for both species. Gray triggerfish liver was fatty, and heptane extracts of the liver fat contained fluorescent substances with properties similar to known PAHs, however the origin of these PAHs is unknown., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Glutathione transferase zeta 1 (GSTZ1-1) is a homodimeric enzyme found in the cytosol and mitochondrial matrix of the liver and other tissues. It catalyzes the glutathione-dependent isomerization of maleylacetoacetate to fumarylacetoacetate in the tyrosine catabolic pathway and can metabolize small halogenated carboxylic acids. GSTZ1a-1a crystals diffracted to a resolution of 3.1 Å and belonged to space group P1, with unit-cell parameters a = 42.0, b = 49.6, c = 54.6 Å, α = 82.9, β = 69.9, γ = 73.4°, with a calculated Matthews coefficient of 2.1 Å(3) Da(-1) assuming a dimer in the crystallographic asymmetric unit. Refinement of the structure is currently in progress.