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The new paradigm of toxicity testing approaches involves rapid screening of thousands of chemicals across hundreds of biological targets through use of in vitro assays. Such assays may lead to false negatives when the complex metabolic processes that render a chemical bioactive in a living system are unable to be replicated in an in vitro environment. In the current study, a workflow is presented for complementing in vitro testing results with in silico and in vitro techniques to identify inactive parents that may produce active metabolites. A case study applying this workflow involved investigating the influence of metabolism for over 1,400 chemicals considered inactive across 18 in vitro assays related to the estrogen receptor (ER) pathway. Over 7,500 first-generation and second-generation metabolites were generated for these in vitro inactive chemicals using an in silico software program. Next, a consensus model comprised of four individual quantitative structure activity relationship (QSAR) models was used to predict ER-binding activity for each of the metabolites. Binding activity was predicted for ∼8–10% of metabolites in each generation, with these metabolites linked to 259 in vitro inactive parent chemicals. Metabolites were enriched in substructures consisting of alcohol, aromatic, and phenol bonds relative to their inactive parent chemicals, suggesting these features are potentially favorable for ER-binding. The workflow presented here can be used to identify parent chemicals that can be potentially bioactive, to aid confidence in high throughput risk screening.

Chemical ionization plays an important role in many aspects of pharmacokinetic (PK) processes such as protein binding, tissue partitioning, and apparent volume of distribution at steady state (Vdss). Here, estimates of ionization equilibrium constants (i.e., pKa) were analyzed for 8,132 pharmaceuticals and 24,281 other compounds to which humans might be exposed in the environment. Results revealed broad differences in the ionization of pharmaceutical chemicals and chemicals with either near-field (in the home) or far-field sources. The utility of these high-throughput ionization predictions was evaluated via a case-study of predicted PK Vdss for 22 compounds monitored in the blood and serum of the U.S. population by the U.S. Centers for Disease Control and Prevention National Health and Nutrition Examination Survey (NHANES). The chemical distribution ratio between water and tissue was estimated using predicted ionization states characterized by pKa. Probability distributions corresponding to ionizable atom types (IATs) were then used to analyze the sensitivity of predicted Vdss on predicted pKa using Monte Carlo methods. 8 of the 22 compounds were predicted to be ionizable. For 5 of the 8 the predictions based upon ionization are significantly different from what would be predicted for a neutral compound. For all but one (foramsulfuron), the probability distribution of predicted Vdss generated by IAT sensitivity analysis spans both the neutral prediction and the prediction using ionization. As new data sets of chemical-specific information on metabolism and excretion for hundreds of chemicals are being made available (e.g., Wetmore et al., 2015), high-throughput methods for calculating Vdss and tissue-specific PK distribution coefficients will allow the rapid construction of PK models to provide context for both biomonitoring data and high-throughput toxicity screening studies such as Tox21 and ToxCast.