Targeted Per- and Polyfluoroalkyl substances (PFAS) assessments for high throughput screening: Analytical and testing considerations to inform a PFAS stock quality evaluation framework.

Per- and polyfluoroalkyl substances (PFAS) represent a large chemical class lacking hazard, toxicokinetic, and exposure information. To accelerate PFAS hazard evaluation, new approach methodologies (NAMs) comprised of in vitro high-throughput toxicity screening, toxicokinetic data, and computational modeling are being employed in read across strategies to evaluate the larger PFAS landscape. A critical consideration to ensure robust evaluations is a parallel assessment of the quality of the screening stock solutions, where dimethyl sulfoxide (DMSO) is often the diluent of choice. Challenged by the lack of commercially available reference standards for many of the selected PFAS and reliance on mass spectrometry approaches for such an evaluation, we developed a high-throughput framework to evaluate the quality of screening stocks for 205 PFAS selected for these NAM efforts. Using mass spectrometry coupled with either liquid or gas chromatography, a quality scoring system was developed that incorporated observations during mass spectral examination to provide a simple pass or fail notation. Informational flags were used to further describe findings regarding parent analyte presence through accurate mass identification, evidence of contaminants and/or degradation, or further describe characteristics such as isomer presence. Across the PFAS-DMSO stocks tested, 148 unique PFAS received passing quality scores to allow for further in vitro testing whereas 57 received a failing score primarily due to detection issues or confounding effects of DMSO. Principle component analysis indicated vapor pressure and Henry's Law Constant as top indicators for a failed quality score for those analyzed by gas chromatography. Three PFAS in the hexafluoropropylene oxide family failed due to degradation in DMSO. As the PFAS evaluated spanned over 20 different structural categories, additional commentary describes analytical observations across specific groups related to PFAS stock composition, detection, stability, and methodologic considerations that will be useful for informing future analytical assessment and downstream HTS efforts. The high-throughput stock quality scoring workflow presented holds value as a tool to evaluate chemical presence and quality efficiently and for informing data inclusion in PFAS or other NAM screening efforts.

Evaluation of 147 perfluoroalkyl substances for immunotoxic and other (patho)physiological activities through phenotypic screening of human primary cells.

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.

Bioactivity profiling of per- and polyfluoroalkyl substances (PFAS) identifies potential toxicity pathways related to molecular structure.

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.