Incorporating High-Throughput Exposure Predictions With Dosimetry-Adjusted In Vitro Bioactivity to Inform Chemical Toxicity Testing

TOXICOLOGICAL SCIENCES, 148(1), 2015, 121–136 doi: 10.1093/toxsci/kfv171 Advance Access Publication Date: August 6, 2015 Research Article Incorporating High-Throughput Exposure Predictions With Dosimetry-Adjusted In Vitro Bioactivity to Inform Chemical Toxicity Testing *The Hamner Institutes for Health Sciences, Institute for Chemical Safety Sciences, Research Triangle Park, North Carolina 27709-2137; †United States Environmental Protection Agency, Office of Research and Development, National Center for Computational Toxicology, Research Triangle Park, North Carolina 27711; and ‡Life Technologies, ADME/ Tox Division of the Primary and Stem Cell Systems Business Unit, Durham, North Carolina 27703 1 To whom correspondence should be addressed at The Hamner Institutes for Health Sciences, Institute for Chemical Safety Sciences, PO Box 12137, 6 Davis Drive, Research Triangle Park, NC 27709 Fax: (919) 558-1300. E-mail: . Present address: National Institute of Environmental Health Sciences, National Toxicology Program, Research Triangle Park, NC 27711. 3 Present address: United States Environmental Protection Agency, Office of Research and Development, National Center for Computational Toxicology, Research Triangle Park, NC 27711. 2 Disclaimer: The United States Environmental Protection Agency through its Office of Research and Development reviewed and approved this publication. However, it may not necessarily reflect official Agency policy, and reference to commercial products or services does not constitute endorsement. ABSTRACT We previously integrated dosimetry and exposure with high-throughput screening (HTS) to enhance the utility of ToxCast HTS data by translating in vitro bioactivity concentrations to oral equivalent doses (OEDs) required to achieve these levels internally. These OEDs were compared against regulatory exposure estimates, providing an activity-to-exposure ratio (AER) useful for a risk-based ranking strategy. As ToxCast efforts expand (ie, Phase II) beyond food-use pesticides toward a wider chemical domain that lacks exposure and toxicity information, prediction tools become increasingly important. In this study, in vitro hepatic clearance and plasma protein binding were measured to estimate OEDs for a subset of Phase II chemicals. OEDs were compared against high-throughput (HT) exposure predictions generated using probabilistic modeling and Bayesian approaches generated by the U.S. Environmental Protection Agency (EPA) ExpoCast program. This approach incorporated chemical-specific use and national production volume data with biomonitoring data to inform the exposure predictions. This HT exposure modeling approach provided predictions for all Phase II chemicals assessed in this study whereas estimates from regulatory sources were available for only 7% of chemicals. Of the 163 chemicals assessed in this study, 3 or 13 chemicals possessed AERs < 1 or < 100, respectively. Diverse bioactivities across a range of assays and concentrations were also noted across the wider chemical space surveyed. The availability of HT exposure estimation and bioactivity screening tools provides an opportunity to incorporate a risk-based strategy for use in testing prioritization. Key words: predictive toxicology; ToxCast; in vitro-in vivo extrapolation; dosimetry; exposure assessment C The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. V This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact 121 Barbara A. Wetmore,*,1 John F. Wambaugh,† Brittany Allen,* Stephen S. Ferguson,‡,2 Mark A. Sochaski,* R. Woodrow Setzer,† Keith A. Houck,† Cory L. Strope,* Katherine Cantwell,* Richard S. Judson,† Edward LeCluyse,* Harvey J. Clewell,* Russell S. Thomas,*,†,3 and Melvin E. Andersen* 122 | TOXICOLOGICAL SCIENCES, 2015, Vol. 148, No. 1 expressed in mg/kg/day, provides a useful first-order approximation of activity-to-exposure ratios (AERs)—in essence a margin of exposure (MOE)—that can help shift from a hazardcentric approach toward a more risk-based strategy that can inform prioritization strategies (Thomas et al., 2013). MATERIALS AND METHODS Chemical selection and stock preparation. The 178 ToxCast Phase II chemicals (http://www.epa.gov/ncct/toxcast/chemicals.html) [last accessed August 20, 2015] analyzed in this study were selected based on the existence of an analytical chemistry detection method and the availability of human exposure data. Compounds for the plasma protein binding and metabolic stability assays were obtained from Compound Focus, Inc (Evotec, South San Francisco, California) in neat form. Dimethyl sulfoxide (DMSO) stock solutions were prepared from the neat chemicals to generate the analytical calibration curves and for use in the assays. All stock solutions were stored at < 70 C. Specific vendor and vendor-supplied purity information for each chemical is provided as Supplementary material (Supplementary Table S1). Plasma protein binding assay. Plasma protein binding was measured for each chemical using either the rapid equilibrium dialysis (RED) method as described previously (Rotroff et al., 2010b; Waters et al., 2008; Wetmore et al., 2012) or ultrafiltration as described later. The human plasma used in the assay was obtained from healthy, consented, paid donors at a U.S. Food and Drug Administration-licensed and inspected donor center (#HMPLEDTA2; Bioreclamation, Inc, Westbury, New York). The plasma was pooled from 5 male (37, 22, 27, 36, and 21 years old) and 5 female (30, 40, 47, 55, and 54 years old) adults and stored at < 70 C until use. Determination of plasma protein binding by ultrafiltration was conducted on a subset of chemicals for which equilibrium dialysis resulted in unbound values  100%. This phenomenon has been observed with a subset of ToxCast industrial chemicals (eg, plasticizers, phthalates) and is believed to occur due to binding and/or interactions with dialysis plate components (data not shown). Briefly, plasma was thawed to room temperature and, if necessary, pH adjusted to 7.4. DMSO stocks of chemicals (200X) were added to plasma to achieve a final concentration of 10 mM. Samples were vortexed and incubated at 37 C in a water bath in polypropylene tubes prior to centrifugation in a Centrifree ultrafiltration device (Millipore Cat No. 4104, Billerica, Massachusetts) at 2000  g for 20 min at 37 C. Ultrafiltrates were collected for analysis. This procedure ensured that the ultrafiltrate did not exceed 40% of the initial volume and minimized dissociation of bound compound due to removal of free compound (Whitlam and Brown, 1981). Nonspecific binding (NSB) was measured in a similar manner, with chemic (...truncated)