Using Zebrafish to Screen Developmental Toxicity of Per- and Polyfluoroalkyl Substances (PFAS).

Per- and polyfluoroalkyl substances (PFAS) are found in many consumer and industrial products. While some PFAS, notably perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS), are developmentally toxic in mammals, the vast majority of PFAS have not been evaluated for developmental toxicity potential. A concentration-response study of 182 unique PFAS chemicals using the zebrafish medium-throughput, developmental vertebrate toxicity assay was conducted to investigate chemical structural identifiers for toxicity. Embryos were exposed to each PFAS compound (≤100 µM) beginning on the day of fertilization. At 6 days post-fertilization (dpf), two independent observers graded developmental landmarks for each larva (e.g., mortality, hatching, swim bladder inflation, edema, abnormal spine/tail, or craniofacial structure). Thirty percent of the PFAS were developmentally toxic, but there was no enrichment of any OECD structural category. PFOS was developmentally toxic (benchmark concentration [BMC] = 7.48 µM); however, other chemicals were more potent: perfluorooctanesulfonamide (PFOSA), N-methylperfluorooctane sulfonamide (N-MeFOSA), ((perfluorooctyl)ethyl)phosphonic acid, perfluoro-3,6,9-trioxatridecanoic acid, and perfluorohexane sulfonamide. The developmental toxicity profile for these more potent PFAS is largely unexplored in mammals and other species. Based on these zebrafish developmental toxicity results, additional screening may be warranted to understand the toxicity profile of these chemicals in other species.

The association between histopathologic effects and liver weight changes induced in mice and rats by chemical exposures: an analysis of the data from Toxicity Reference Database (ToxRefDB).

Absolute (ALW) and relative (RLW) liver weight changes are sensitive endpoints in repeat-dose rodent toxicity studies, and their changes are often used for quantitative assessment of health effects induced by hepatotoxic chemicals using the benchmark dose-response modeling (BMD) approach. To find biologically relevant liver weight changes to chemical exposures, we evaluated all data available for liver weight changes and associated liver histopathologic findings from the Toxicity Reference Database (ToxRefDB). Our analysis of 389 subchronic mouse and rat studies for 273 chemicals found significant differences in treatment-related ALW and RLW changes between dose groups with and without liver histopathologic changes. In addition, we demonstrate that chemical treatment-induced ALW and RLW changes can predict the presence of histopathologic findings and inform the selection of biologically relevant liver weight changes for BMD modeling and derivation of toxicity values.

Reproducibility of organ-level effects in repeat dose animal studies.

This work estimates benchmarks for new approach method (NAM) performance in predicting organ-level effects in repeat dose studies of adult animals based on variability in replicate animal studies. Treatment-related effect values from the Toxicity Reference database (v2.1) for weight, gross, or histopathological changes in the adrenal gland, liver, kidney, spleen, stomach, and thyroid were used. Rates of chemical concordance among organ-level findings in replicate studies, defined by repeated chemical only, chemical and species, or chemical and study type, were calculated. Concordance was 39 - 88%, depending on organ, and was highest within species. Variance in treatment-related effect values, including lowest effect level (LEL) values and benchmark dose (BMD) values when available, was calculated by organ. Multilinear regression modeling, using study descriptors of organ-level effect values as covariates, was used to estimate total variance, mean square error (MSE), and root residual mean square error (RMSE). MSE values, interpreted as estimates of unexplained variance, suggest study descriptors accounted for 52-69% of total variance in organ-level LELs. RMSE ranged from 0.41 - 0.68 log10-mg/kg/day. Differences between organ-level effects from chronic (CHR) and subchronic (SUB) dosing regimens were also quantified. Odds ratios indicated CHR organ effects were unlikely if the SUB study was negative. Mean differences of CHR - SUB organ-level LELs ranged from -0.38 to -0.19 log10 mg/kg/day; the magnitudes of these mean differences were less than RMSE for replicate studies. Finally, in vitro to in vivo extrapolation (IVIVE) was employed to compare bioactive concentrations from in vitro NAMs for kidney and liver to LELs. The observed mean difference between LELs and mean IVIVE dose predictions approached 0.5 log10-mg/kg/day, but differences by chemical ranged widely. Overall, variability in repeat dose organ-level effects suggests expectations for quantitative accuracy of NAM prediction of LELs should be at least ± 1 log10-mg/kg/day, with qualitative accuracy not exceeding 70%.

ToxRefDB v2.1: update to curated in vivo study data in the Toxicity Reference Database.

The Toxicity Reference Database (ToxRefDB) contains in vivo study data from over 5,900 guideline or guideline-like studies for over 1,100 chemicals. The database includes information regarding study design, chemical treatment, dosing, treatment group parameters, treatment-related (significantly different from control) and critical (adverse) effects, guided by a controlled effect vocabulary, as well as endpoint testing status according to health effects guideline requirements. ToxRefDB v2.1 is an update to address a compilation error found in ToxRefDB v2.0 that resulted in some effects being inadvertently omitted from the database. Though effect data has been recovered, no new studies were added. The recovered data improves the utility of ToxRefDB as a resource for curated legacy in vivo information, which enhances scientific confidence in vitro high-throughput screening of chemicals and supports retrospective and predictive toxicology applications for which outcomes in traditional regulatory toxicology studies serve as reference information.

Evaluation of a high-throughput H295R homogenous time resolved fluorescence assay for androgen and estrogen steroidogenesis screening.

The H295R test guideline assay evaluates the effect of test substances on synthesis of 17ß-estradiol (E2) and testosterone (T). The objective of this study was to leverage commercial immunoassay technology to develop a more efficient H295R assay to measure E2 and T levels in 384-well format. The resulting Homogenous Time Resolved Fluorescence assay platform (H295R-HTRF) was evaluated against a training set of 36 chemicals derived from the OECD inter-laboratory validation study, EPA guideline 890.1200 aromatase assay, and azole fungicides active in the HT-H295R assay. Quality control performance criteria were met for all conditions except E2 synthesis inhibition where low basal hormone synthesis was observed. Five proficiency chemicals were active for both the E2 and T endpoints, consistent with guideline classifications. Of the nine OECD core reference chemicals, 9/9 were concordant with outcomes for E2 and 7/9 for T. Likewise, 9/13 and 11/13 OECD supplemental chemicals were concordant with anticipated effects for E2 and T, respectively. Of the 10 azole fungicides screened, 7/10 for E2 and 8/10 for T exhibited concordant outcomes for inhibition. Generally, all active chemicals in the training set demonstrated equivalent or greater potency in the H295R-HTRF assay, supporting the sensitivity of the platform. The adaptation of HTRF technology to the H295R model provides an efficient way to evaluate E2 and T modulators in accordance with guideline specifications.

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.