Improving natural product research translation: From source to clinical trial.

While great interest in health effects of natural product (NP) including dietary supplements and foods persists, promising preclinical NP research is not consistently translating into actionable clinical trial (CT) outcomes. Generally considered the gold standard for assessing safety and efficacy, CTs, especially phase III CTs, are costly and require rigorous planning to optimize the value of the information obtained. More effective bridging from NP research to CT was the goal of a September, 2018 transdisciplinary workshop. Participants emphasized that replicability and likelihood of successful translation depend on rigor in experimental design, interpretation, and reporting across the continuum of NP research. Discussions spanned good practices for NP characterization and quality control; use and interpretation of models (computational through in vivo) with strong clinical predictive validity; controls for experimental artefacts, especially for in vitro interrogation of bioactivity and mechanisms of action; rigorous assessment and interpretation of prior research; transparency in all reporting; and prioritization of research questions. Natural product clinical trials prioritized based on rigorous, convergent supporting data and current public health needs are most likely to be informative and ultimately affect public health. Thoughtful, coordinated implementation of these practices should enhance the knowledge gained from future NP research.

An Intuitive Approach for Predicting Potential Human Health Risk with the Tox21 10k Library.

In vitro-in vivo extrapolation (IVIVE) analyses translating high-throughput screening (HTS) data to human relevance have been limited. This study represents the first report applying IVIVE approaches and exposure comparisons using the entirety of the Tox21 federal collaboration chemical screening data, incorporating assay response efficacy and quality of concentration-response fits, and providing quantitative anchoring to first address the likelihood of human in vivo interactions with Tox21 compounds. This likelihood was assessed using a maximum blood concentration to in vitro response ratio approach (Cmax/AC50), analogous to decision-making methods for clinical drug-drug interactions. Fraction unbound in plasma (fup) and intrinsic hepatic clearance (CLint) parameters were estimated in silico and incorporated in a three-compartment toxicokinetic (TK) model to first predict Cmax for in vivo corroboration using therapeutic scenarios. Toward lower exposure scenarios, 36 compounds of 3925 unique chemicals with curated activity in the HTS data using high-quality dose-response model fits and ≥40% efficacy gave "possible" human in vivo interaction likelihoods lower than median human exposures predicted in the United States Environmental Protection Agency's ExpoCast program. A publicly available web application has been designed to provide all Tox21-ToxCast dose-likelihood predictions. Overall, this approach provides an intuitive framework to relate in vitro toxicology data rapidly and quantitatively to exposures using either in vitro or in silico derived TK parameters and can be thought of as an important step toward estimating plausible biological interactions in a high-throughput risk-assessment framework.

httk: R Package for High-Throughput Toxicokinetics.

Thousands of chemicals have been profiled by high-throughput screening programs such as ToxCast and Tox21; these chemicals are tested in part because most of them have limited or no data on hazard, exposure, or toxicokinetics. Toxicokinetic models aid in predicting tissue concentrations resulting from chemical exposure, and a "reverse dosimetry" approach can be used to predict exposure doses sufficient to cause tissue concentrations that have been identified as bioactive by high-throughput screening. We have created four toxicokinetic models within the R software package httk. These models are designed to be parameterized using high-throughput in vitro data (plasma protein binding and hepatic clearance), as well as structure-derived physicochemical properties and species-specific physiological data. The package contains tools for Monte Carlo sampling and reverse dosimetry along with functions for the analysis of concentration vs. time simulations. The package can currently use human in vitro data to make predictions for 553 chemicals in humans, rats, mice, dogs, and rabbits, including 94 pharmaceuticals and 415 ToxCast chemicals. For 67 of these chemicals, the package includes rat-specific in vitro data. This package is structured to be augmented with additional chemical data as they become available. Package httk enables the inclusion of toxicokinetics in the statistical analysis of chemicals undergoing high-throughput screening.

Predictive endocrine testing in the 21st century using in vitro assays of estrogen receptor signaling responses.

Thousands of environmental chemicals are subject to regulatory review for their potential to be endocrine disruptors (ED). In vitro high-throughput screening (HTS) assays have emerged as a potential tool for prioritizing chemicals for ED-related whole-animal tests. In this study, 1814 chemicals including pesticide active and inert ingredients, industrial chemicals, food additives, and pharmaceuticals were evaluated in a panel of 13 in vitro HTS assays. The panel of in vitro assays interrogated multiple end points related to estrogen receptor (ER) signaling, namely binding, agonist, antagonist, and cell growth responses. The results from the in vitro assays were used to create an ER Interaction Score. For 36 reference chemicals, an ER Interaction Score >0 showed 100% sensitivity and 87.5% specificity for classifying potential ER activity. The magnitude of the ER Interaction Score was significantly related to the potency classification of the reference chemicals (p < 0.0001). ERα/ERß selectivity was also evaluated, but relatively few chemicals showed significant selectivity for a specific isoform. When applied to a broader set of chemicals with in vivo uterotrophic data, the ER Interaction Scores showed 91% sensitivity and 65% specificity. Overall, this study provides a novel method for combining in vitro concentration response data from multiple assays and, when applied to a large set of ER data, accurately predicted estrogenic responses and demonstrated its utility for chemical prioritization.

Profiling 976 ToxCast chemicals across 331 enzymatic and receptor signaling assays.

Understanding potential health risks is a significant challenge due to the large numbers of diverse chemicals with poorly characterized exposures and mechanisms of toxicities. The present study analyzes 976 chemicals (including failed pharmaceuticals, alternative plasticizers, food additives, and pesticides) in Phases I and II of the U.S. EPA's ToxCast project across 331 cell-free enzymatic and ligand-binding high-throughput screening (HTS) assays. Half-maximal activity concentrations (AC50) were identified for 729 chemicals in 256 assays (7,135 chemical-assay pairs). Some of the most commonly affected assays were CYPs (CYP2C9 and CYP2C19), transporters (mitochondrial TSPO, norepinephrine, and dopaminergic), and GPCRs (aminergic). Heavy metals, surfactants, and dithiocarbamate fungicides showed promiscuous but distinctly different patterns of activity, whereas many of the pharmaceutical compounds showed promiscuous activity across GPCRs. Literature analysis confirmed >50% of the activities for the most potent chemical-assay pairs (54) but also revealed 10 missed interactions. Twenty-two chemicals with known estrogenic activity were correctly identified for the majority (77%), missing only the weaker interactions. In many cases, novel findings for previously unreported chemical-target combinations clustered with known chemical-target interactions. Results from this large inventory of chemical-biological interactions can inform read-across methods as well as link potential targets to molecular initiating events in adverse outcome pathways for diverse toxicities.

Real-time growth kinetics measuring hormone mimicry for ToxCast chemicals in T-47D human ductal carcinoma cells.

High-throughput screening (HTS) assays capable of profiling thousands of environmentally relevant chemicals for in vitro biological activity provide useful information on the potential for disrupting endocrine pathways. Disruption of the estrogen signaling pathway has been implicated in a variety of adverse health effects including impaired development, reproduction, and carcinogenesis. The estrogen-responsive human mammary ductal carcinoma cell line T-47D was exposed to 1815 ToxCast chemicals comprising pesticides, industrial chemicals, pharmaceuticals, personal care products, cosmetics, food ingredients, and other chemicals with known or suspected human exposure potential. Cell growth kinetics were evaluated using real-time cell electronic sensing. T-47D cells were exposed to eight concentrations (0.006-100 µM), and measurements of cellular impedance were repeatedly recorded for 105 h. Chemical effects were evaluated based on potency (concentration at which response occurs) and efficacy (extent of response). A linear growth response was observed in response to prototypical estrogen receptor agonists (17ß-estradiol, genistein, bisphenol A, nonylphenol, and 4-tert-octylphenol). Several compounds, including bisphenol A and genistein, induced cell growth comparable in efficacy to that of 17ß-estradiol, but with decreased potency. Progestins, androgens, and corticosteroids invoked a biphasic growth response indicative of changes in cell number or cell morphology. Results from this cell growth assay were compared with results from additional estrogen receptor (ER) binding and transactivation assays. Chemicals detected as active in both the cell growth and ER receptor binding assays demonstrated potencies highly correlated with two ER transactivation assays (r = 0.72; r = 0.70). While ER binding assays detected chemicals that were highly potent or efficacious in the T-47D cell growth and transactivation assays, the binding assays lacked sensitivity in detecting weakly active compounds. In conclusion, this cell-based assay rapidly detects chemical effects on T-47D growth and shows potential, in combination with other HTS assays, to detect environmentally relevant chemicals with potential estrogenic activity.

Evaluation of the herbicide glyphosate, (aminomethyl)phosphonic acid, and glyphosate-based formulations for genotoxic activity using in vitro assays.

Glyphosate, the most heavily used herbicide world-wide, is applied to plants in complex formulations that promote absorption. The National Toxicology Program reported in 1992 that glyphosate, administered to rats and mice at doses up to 50,000 ppm in feed for 13 weeks, showed little evidence of toxicity, and no induction of micronuclei was observed in the mice in this study. Subsequently, mechanistic studies of glyphosate and glyphosate-based formulations (GBFs) that have focused on DNA damage and oxidative stress suggest that glyphosate may have genotoxic potential. However, few of these studies directly compared glyphosate to GBFs, or effects among GBFs. To address these data gaps, we tested glyphosate, glyphosate isopropylamine (IPA), and (aminomethyl)phosphonic acid (AMPA, a microbial metabolite of glyphosate), 9 high-use agricultural GBFs, 4 residential-use GBFs, and additional herbicides (metolachlor, mesotrione, and diquat dibromide) present in some of the GBFs in bacterial mutagenicity tests, and in human TK6 cells using a micronucleus assay and a multiplexed DNA damage assay. Our results showed no genotoxicity or notable cytotoxicity for glyphosate or AMPA at concentrations up to 10 mM, while all GBFs and herbicides other than glyphosate were cytotoxic, and some showed genotoxic activity. An in vitro to in vivo extrapolation of results for glyphosate suggests that it is of low toxicological concern for humans. In conclusion, these results demonstrate a lack of genotoxicity for glyphosate, consistent with observations in the NTP in vivo study, and suggest that toxicity associated with GBFs may be related to other components of these formulations.

Cdc42 regulates extracellular matrix remodeling in three dimensions.

Extracellular matrix (ECM) actively participates in normal cell regulation and in the process of tumor progression. The Rho GTPase Cdc42 has been shown to regulate cell-ECM interaction in conventional two-dimensional culture conditions by using dominant mutants of Cdc42 in immortalized cell lines that may introduce nonspecific effects. Here, we employ three-dimensional culture systems for conditional gene targeted primary mouse embryonic fibroblasts that better simulate the reciprocal and adaptive interactions between cells and surrounding matrix to define the role of Cdc42 signaling pathways in ECM organization. Cdc42 deficiency leads to a defect in global cell-matrix interactions reflected by a decrease in collagen gel contraction. The defect is associated with an altered cell-matrix interaction that is evident by morphologic changes and reduced focal adhesion complex formation. The matrix defect is also associated with a reduction in synthesis and activation of matrix metalloproteinase 9 (MMP9) and altered fibronectin deposition patterning. A Cdc42 mutant rescue experiment found that downstream of Cdc42, p21-activated kinase (PAK), but not Par6 or WASP, may be involved in regulating collagen gel contraction and fibronectin organization. Thus, in addition to the previously implicated roles in intracellular regulation of actin organization, proliferation, and vesicle trafficking, Cdc42 is essential in ECM remodeling in three dimensions.

Microphysiological Systems Evaluation: Experience of TEX-VAL Tissue Chip Testing Consortium.

Much has been written and said about the promise and excitement of microphysiological systems, miniature devices that aim to recreate aspects of human physiology on a chip. The rapid explosion of the offerings and persistent publicity placed high expectations on both product manufacturers and regulatory agencies to adopt the data. Inevitably, discussions of where this technology fits in chemical testing paradigms are ongoing. Some end-users became early adopters, whereas others have taken a more cautious approach because of the high cost and uncertainties of their utility. Here, we detail the experience of a public-private collaboration established for testing of diverse microphysiological systems. Collectively, we present a number of considerations on practical aspects of using microphysiological systems in the context of their applications in decision-making. Specifically, future end-users need to be prepared for extensive on-site optimization and have access to a wide range of imaging and other equipment. We reason that cells, related reagents, and the technical skills of the research staff, not the devices themselves, are the most critical determinants of success. Extrapolation from concentration-response effects in microphysiological systems to human blood or oral exposures, difficulties with replicating the whole organ, and long-term functionality remain as critical challenges. Overall, we conclude that it is unlikely that a rodent- or human-equivalent model is achievable through a finite number of microphysiological systems in the near future; therefore, building consensus and promoting the gradual incorporation of these models into tiered approaches for safety assessment and decision-making is the sensible path to wide adoption.

Zebrafish: as an integrative model for twenty-first century toxicity testing.

The zebrafish embryo is a useful small model for investigating vertebrate development because of its transparency, low cost, transgenic and morpholino capabilities, conservation of cell signaling, and concordance with mammalian developmental phenotypes. From these advantages, the zebrafish embryo has been considered as an alternative model for traditional in vivo developmental toxicity screening. The use of this organism in conjunction with traditional in vivo developmental toxicity testing has the potential to reduce cost and increase throughput of testing the chemical universe, prioritize chemicals for targeted toxicity testing, generate predictive models of developmental toxicants, and elucidate mechanisms and adverse outcome pathways for abnormal development. This review gives an overview of the zebrafish embryo for pre dictive toxicology and 21st century toxicity testing. Developmental eye defects were selected as an example to evaluate data from the U.S. Environmental Protection Agency's ToxCast program comparing responses in zebrafish embryos with those from pregnant rats and rabbits for a subset of 24 environmental chemicals across >600 in vitro assay targets. Cross-species comparisons implied a common basis for biological pathways associated with neuronal defects, extracellular matrix remodeling, and mitotic arrest.

Benchmark Concentrations for Untargeted Metabolomics Versus Transcriptomics for Liver Injury Compounds in In Vitro Liver Models.

Interpretation of untargeted metabolomics data from both in vivo and physiologically relevant in vitro model systems continues to be a significant challenge for toxicology research. Potency-based modeling of toxicological responses has served as a pillar of interpretive context and translation of testing data. In this study, we leverage the resolving power of concentration-response modeling through benchmark concentration (BMC) analysis to interpret untargeted metabolomics data from differentiated cultures of HepaRG cells exposed to a panel of reference compounds and integrate data in a potency-aligned framework with matched transcriptomic data. For this work, we characterized biological responses to classical human liver injury compounds and comparator compounds, known to not cause liver injury in humans, at 10 exposure concentrations in spent culture media by untargeted liquid chromatography-mass spectrometry analysis. The analyte features observed (with limited metabolites identified) were analyzed using BMC modeling to derive compound-induced points of departure. The results revealed liver injury compounds produced concentration-related increases in metabolomic response compared to those rarely associated with liver injury (ie, sucrose, potassium chloride). Moreover, the distributions of altered metabolomic features were largely comparable with those observed using high throughput transcriptomics, which were further extended to investigate the potential for in vitro observed biological responses to be observed in humans with exposures at therapeutic doses. These results demonstrate the utility of BMC modeling of untargeted metabolomics data as a sensitive and quantitative indicator of human liver injury potential.

Predictive modeling of biological responses in the rat liver using in vitro Tox21 bioactivity: Benefits from high-throughput toxicokinetics.

Computational methods are needed to more efficiently leverage data from in vitro cell-based models to predict what occurs within whole body systems after chemical insults. This study set out to test the hypothesis that in vitro high-throughput screening (HTS) data can more effectively predict in vivo biological responses when chemical disposition and toxicokinetic (TK) modeling are employed. In vitro HTS data from the Tox21 consortium were analyzed in concert with chemical disposition modeling to derive nominal, aqueous, and intracellular estimates of concentrations eliciting 50% maximal activity. In vivo biological responses were captured using rat liver transcriptomic data from the DrugMatrix and TG-Gates databases and evaluated for pathway enrichment. In vivo dosing data were translated to equivalent body concentrations using HTTK modeling. Random forest models were then trained and tested to predict in vivo pathway-level activity across 221 chemicals using in vitro bioactivity data and physicochemical properties as predictor variables, incorporating methods to address imbalanced training data resulting from high instances of inactivity. Model performance was quantified using the area under the receiver operator characteristic curve (AUC-ROC) and compared across pathways for different combinations of predictor variables. All models that included toxicokinetics were found to outperform those that excluded toxicokinetics. Biological interpretation of the model features revealed that rather than a direct mapping of in vitro assays to in vivo pathways, unexpected combinations of multiple in vitro assays predicted in vivo pathway-level activities. To demonstrate the utility of these findings, the highest-performing model was leveraged to make new predictions of in vivo biological responses across all biological pathways for remaining chemicals tested in Tox21 with adequate data coverage (n = 6617). These results demonstrate that, when chemical disposition and toxicokinetics are carefully considered, in vitro HT screening data can be used to effectively predict in vivo biological responses to chemicals.

Profiling the Tox21 Chemical Collection for Acetylcholinesterase Inhibition.

BACKGROUND: Inhibition of acetylcholinesterase (AChE), a biomarker of organophosphorous and carbamate exposure in environmental and occupational human health, has been commonly used to identify potential safety liabilities. So far, many environmental chemicals, including drug candidates, food additives, and industrial chemicals, have not been thoroughly evaluated for their inhibitory effects on AChE activity. AChE inhibitors can have therapeutic applications (e.g., tacrine and donepezil) or neurotoxic consequences (e.g., insecticides and nerve agents). OBJECTIVES: The objective of the current study was to identify environmental chemicals that inhibit AChE activity using in vitro and in silico models. METHODS: To identify AChE inhibitors rapidly and efficiently, we have screened the Toxicology in the 21st Century (Tox21) 10K compound library in a quantitative high-throughput screening (qHTS) platform by using the homogenous cell-based AChE inhibition assay and enzyme-based AChE inhibition assays (with or without microsomes). AChE inhibitors identified from the primary screening were further tested in monolayer or spheroid formed by SH-SY5Y and neural stem cell models. The inhibition and binding modes of these identified compounds were studied with time-dependent enzyme-based AChE inhibition assay and molecular docking, respectively. RESULTS: A group of known AChE inhibitors, such as donepezil, ambenonium dichloride, and tacrine hydrochloride, as well as many previously unreported AChE inhibitors, such as chelerythrine chloride and cilostazol, were identified in this study. Many of these compounds, such as pyrazophos, phosalone, and triazophos, needed metabolic activation. This study identified both reversible (e.g., donepezil and tacrine) and irreversible inhibitors (e.g., chlorpyrifos and bromophos-ethyl). Molecular docking analyses were performed to explain the relative inhibitory potency of selected compounds. CONCLUSIONS: Our tiered qHTS approach allowed us to generate a robust and reliable data set to evaluate large sets of environmental compounds for their AChE inhibitory activity. https://doi.org/10.1289/EHP6993.

Characterizing cleft palate toxicants using ToxCast data, chemical structure, and the biomedical literature.

Cleft palate has been linked to both genetic and environmental factors that perturb key events during palatal morphogenesis. As a developmental outcome, it presents a challenging, mechanistically complex endpoint for predictive modeling. A data set of 500 chemicals evaluated for their ability to induce cleft palate in animal prenatal developmental studies was compiled from Toxicity Reference Database and the biomedical literature, which included 63 cleft palate active and 437 inactive chemicals. To characterize the potential molecular targets for chemical-induced cleft palate, we mined the ToxCast high-throughput screening database for patterns and linkages in bioactivity profiles and chemical structural descriptors. ToxCast assay results were filtered for cytotoxicity and grouped by target gene activity to produce a "gene score." Following unsuccessful attempts to derive a global prediction model using structural and gene score descriptors, hierarchical clustering was applied to the set of 63 cleft palate positives to extract local structure-bioactivity clusters for follow-up study. Patterns of enrichment were confirmed on the complete data set, that is, including cleft palate inactives, and putative molecular initiating events identified. The clusters corresponded to ToxCast assays for cytochrome P450s, G-protein coupled receptors, retinoic acid receptors, the glucocorticoid receptor, and tyrosine kinases/phosphatases. These patterns and linkages were organized into preliminary decision trees and the resulting inferences were mapped to a putative adverse outcome pathway framework for cleft palate supported by literature evidence of current mechanistic understanding. This general data-driven approach offers a promising avenue for mining chemical-bioassay drivers of complex developmental endpoints where data are often limited.

Correction: Development and evaluation of a high throughput inhalation model for organic chemicals.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Development and evaluation of a high throughput inhalation model for organic chemicals.

Currently it is difficult to prospectively estimate human toxicokinetics (particularly for novel chemicals) in a high-throughput manner. The R software package httk has been developed, in part, to address this deficiency, and the aim of this investigation was to develop a generalized inhalation model for httk. The structure of the inhalation model was developed from two previously published physiologically based models from Jongeneelen and Berge (Ann Occup Hyg 55:841-864, 2011) and Clewell et al. (Toxicol Sci 63:160-172, 2001), while calculated physicochemical data was obtained from EPA's CompTox Chemicals Dashboard. In total, 142 exposure scenarios across 41 volatile organic chemicals were modeled and compared to published data. The slope of the regression line of best fit between log-transformed simulated and observed blood and exhaled breath concentrations was 0.46 with an r2 = 0.45 and a root mean square error (RMSE) of direct comparison between the log-transformed simulated and observed values of 1.11. Approximately 5.1% (n = 108) of the data points analyzed were >2 orders of magnitude different than expected. The volatile organic chemicals examined in this investigation represent small, generally lipophilic molecules. Ultimately this paper details a generalized inhalation component that integrates with the httk physiologically based toxicokinetic model to provide high-throughput estimates of inhalation chemical exposures.

Screening for neurotoxic potential of 15 flame retardants using freshwater planarians.

Asexual freshwater planarians are an attractive invertebrate model for high-throughput neurotoxicity screening, because they possess multiple quantifiable behaviors to assess distinct neuronal functions. Planarians uniquely allow direct comparisons between developing and adult animals to distinguish developmentally selective effects from general neurotoxicity. In this study, we used our automated planarian screening platform to compare the neurotoxicity of 15 flame retardants (FRs), consisting of representative phased-out brominated (BFRs) and replacement organophosphorus FRs (OPFRs). OPFRs have emerged as a proposed safer alternative to BFRs; however, limited information is available on their health effects. We found 11 of the 15 FRs (3/6 BFRs, 7/8 OPFRs, and Firemaster 550) caused adverse effects in both adult and developing planarians with similar nominal lowest-effect-levels for BFRs and OPFRs. This suggests that replacement OPFRs are comparably neurotoxic to the phased-out compounds. BFRs were primarily systemically toxic, whereas OPFRs, except Tris(2-chloroethyl) phosphate, shared a behavioral phenotype in response to noxious heat at sublethal concentrations, indicating specific neurotoxic effects. We found this behavioral phenotype was correlated with cholinesterase inhibition, thus linking behavioral outcomes to molecular targets. By directly comparing effects on adult and developing planarians, we further found that one BFR (3,3',5,5'-Tetrabromobisphenol A) caused a developmental selective defect. Together, these results demonstrate that our planarian screening platform yields high content data from various behavioral and morphological endpoints, allowing us to distinguish selective neurotoxic effects and effects specific to the developing nervous system. Ten of these 11 bioactive FRs were previously found to be bioactive in other models, including cell culture and alternative animal models (nematodes and zebrafish). This level of concordance across different platforms emphasizes the urgent need for further evaluation of OPFRs in mammalian systems.

The Carcinogenome Project: In Vitro Gene Expression Profiling of Chemical Perturbations to Predict Long-Term Carcinogenicity.

BACKGROUND: Most chemicals in commerce have not been evaluated for their carcinogenic potential. The de facto gold-standard approach to carcinogen testing adopts the 2-y rodent bioassay, a time-consuming and costly procedure. High-throughput in vitro assays are a promising alternative for addressing the limitations in carcinogen screening. OBJECTIVES: We developed a screening process for predicting chemical carcinogenicity and genotoxicity and characterizing modes of actions (MoAs) using in vitro gene expression assays. METHODS: We generated a large toxicogenomics resource comprising [Formula: see text] expression profiles corresponding to 330 chemicals profiled in HepG2 (human hepatocellular carcinoma cell line) at multiple doses and replicates. Predictive models of carcinogenicity and genotoxicity were built using a random forest classifier. Differential pathway enrichment analysis was performed to identify pathways associated with carcinogen exposure. Signatures of carcinogenicity and genotoxicity were compared with external sources, including Drugmatrix and the Connectivity Map. RESULTS: Among profiles with sufficient bioactivity, our classifiers achieved 72.2% Area Under the ROC Curve (AUC) for predicting carcinogenicity and 82.3% AUC for predicting genotoxicity. Chemical bioactivity, as measured by the strength and reproducibility of the transcriptional response, was not significantly associated with long-term carcinogenicity in doses up to [Formula: see text]. However, sufficient bioactivity was necessary for a chemical to be used for prediction of carcinogenicity. Pathway enrichment analysis revealed pathways consistent with known pathways that drive cancer, including DNA damage and repair. The data is available at https://clue.io/CRCGN_ABC , and a portal for query and visualization of the results is accessible at https://carcinogenome.org . DISCUSSION: We demonstrated an in vitro screening approach using gene expression profiling to predict carcinogenicity and infer MoAs of chemical perturbations. https://doi.org/10.1289/EHP3986.

Using Tox21 High-Throughput Screening Assays for the Evaluation of Botanical and Dietary Supplements.

Introduction: Recent nationwide surveys found that natural products, including botanical dietary supplements, are used by ∼18% of adults. In many cases, there is a paucity of toxicological data available for these substances to allow for confident evaluations of product safety. The National Toxicology Program (NTP) has received numerous nominations from the public and federal agencies to study the toxicological effects of botanical dietary supplements. The NTP sought to evaluate the utility of in vitro quantitative high-throughput screening (qHTS) assays for toxicological assessment of botanical and dietary supplements. Materials and Methods: In brief, concentration-response assessments of 90 test substances, including 13 distinct botanical species, and individual purported active constituents were evaluated using a subset of the Tox21 qHTS testing panel. The screen included 20 different endpoints that covered a broad range of biologically relevant signaling pathways to detect test article effects upon endocrine activity, nuclear receptor signaling, stress response signaling, genotoxicity, and cell death signaling. Results and Discussion: Botanical dietary supplement extracts induced measurable and diverse activity. Elevated biological activity profiles were observed following treatments with individual chemical constituents relative to their associated botanical extract. The overall distribution of activity was comparable to activities exhibited by compounds present in the Tox21 10K chemical library. Conclusion: Botanical supplements did not exhibit minimal or idiosyncratic activities that would preclude the use of qHTS platforms as a feasible method to screen this class of compounds. However, there are still many considerations and further development required when attempting to use in vitro qHTS methods to characterize the safety profile of botanical/dietary supplements.

Organophosphate Ester Flame Retardants: Are They a Regrettable Substitution for Polybrominated Diphenyl Ethers?

As the use of polybrominated diphenyl ethers (PBDEs), and the entire class of organohalogen flame retardants, is declining, the use of organophosphate esters flame retardants (OPFRs) is increasing. In this paper, we ask whether OPFRs are a better choice than PBDEs. To address this question, we compared OPFRs with PBDEs for a wide range of properties. OPFRs exposure is ubiquitous in people and in outdoor and indoor environments, and are now often found at higher levels compared to PBDE peak exposure levels. Furthermore, data from toxicity testing, epidemiological studies, and risk assessments all suggest that there are health concerns at current exposure levels for both halogenated and non-halogenated OPFRs. Obtaining the scientific evidence needed for regulation of OPFRs can take many years. Given the large number of OPFRs in use, manufacturers can move towards healthier and safer products by developing innovative ways to reduce fire hazard for electronics enclosures, upholstered furniture, building materials and other consumer products without adding flame retardant chemicals.