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An Intuitive Approach for Predicting Potential Human Health Risk with the Tox21 10k Library Nisha S Sipes, John F. Wambaugh, Robert Pearce, Scott S. Auerbach, Barbara Anne Wetmore, Jui-Hua Hsieh, Andrew J. Shapiro, Daniel Svoboda, Michael J DeVito, and Stephen S. Ferguson Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b00650 • Publication Date (Web): 15 Aug 2017 Downloaded from http://pubs.acs.org on August 17, 2017
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An Intuitive Approach for Predicting Potential Human Health Risk with the Tox21 10k Library
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Nisha S. Sipes1*, John F. Wambaugh2, Robert Pearce2, Scott S. Auerbach1, Barbara A. Wetmore4, Jui-Hua
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Hsieh1, Andrew J. Shapiro1, Daniel Svoboda3, Michael J. DeVito1, Stephen S. Ferguson1
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1NTP/NIEHS/NIH, RTP, NC
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2NCCT/ORD/EPA, RTP, NC
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3Sciome, RTP, NC
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4NERL/ORD/EPA, RTP, NC
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*Corresponding Author: Nisha S. Sipes, 111 T.W. Alexander Drive, PO Box 12233, MD: K2-17, Research
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Triangle Park, NC 27709, Telephone: 919-316-4603, Email:
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ABSTRACT
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In vitro-in vivo extrapolation (IVIVE) analyses translating high-throughput screening (HTS) data to human
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relevance have been limited. This study represents the first report applying IVIVE approaches and
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exposure comparisons using the entirety of the Tox21 federal collaboration chemical screening data,
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incorporating assay response efficacy and quality of concentration-response fits, and providing
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quantitative anchoring to first address the likelihood of human in vivo interactions with Tox21
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compounds. This likelihood was assessed using a maximum blood concentration to in vitro response
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ratio approach (Cmax/AC50), analogous to decision-making methods for clinical drug-drug interactions.
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Fraction unbound in plasma (fup) and intrinsic hepatic clearance (CLint) parameters were estimated in
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silico and incorporated in a 3-compartment toxicokinetic (TK) model to first predict Cmax for in vivo
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corroboration using therapeutic scenarios. Toward lower exposure scenarios, 36 compounds of 3,925
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with curated activity in the HTS data using high quality dose-response model fits and ≥40% efficacy gave
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‘possible’ human in vivo interaction likelihoods lower than median human exposures predicted in EPA’s
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ExpoCast program. A publicly available web application has been designed to provide all Tox21/ToxCast
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dose likelihood predictions. Overall, this approach provides an intuitive framework to relate in vitro
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toxicology data rapidly and quantitatively to exposures using either in vitro or in silico derived TK
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parameters, and can be thought of as an important step towards estimating plausible biological
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interactions in a high throughput risk assessment framework.
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INTRODUCTION
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Likelihood of an adverse human response to environmental chemical exposure is not adequately
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characterized for most of the thousands of chemicals with potential human exposure due to the
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limitations of traditional toxicity testing methods.1 To address the needs for higher throughput
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approaches reflective of human molecular pathways, the Toxicology in the 21st Century(Tox21) federal
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collaboration was formed with an initial focus of employing in vitro high-throughput screening (HTS)
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assays with a 10k chemical library (e.g., environmental, pharmaceutical, consumer- and industrial-use) in
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>60 HTS assays (e.g., cytotoxicity, cell stress, mitochondrial, nuclear receptors).2 Of these chemicals,
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>1,000 have been evaluated in the EPA’s ToxCast program (>800 HTS assays) to broaden the biological
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coverage.3 Hazard-based chemical assessments, using HTS data, have related chemicals to biological
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pathways responsible for adverse in vivo effects, but have typically not incorporated concentrations or
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estimated required doses needed to achieve these effects.4-9 While hypothesized pathways of concern
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are uncovered, doses at which these effects may manifest remain generally unknown and unaddressed
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for Tox21 data.
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Recent efforts are building quantitative approaches to translate in vitro toxicity potencies to equivalent
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in vivo doses using in vitro-in vivo extrapolation (IVIVE) techniques.10-13 These approaches utilize
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pharmacokinetic equations to estimate chemical steady-state concentrations (Css) in plasma and to
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dosimetrically adjust in vitro HTS potencies to estimate external dose equivalents (intake rates).10-13
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Additionally the High-Throughput Toxicokinetic (HTTK) open-source R-package14, 15 can quickly calculate
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equivalent external doses from HTS data using multiple IVIVE models, and conversely, calculate plasma
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concentrations given certain dosing scenarios. The current R-package utilizes in silico estimated
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physicochemical parameters (e.g., hydrophobicity, acid/base dissociation constants) and two widely
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applied in vitro measured parameters, the chemical fraction unbound in plasma (fup) and the intrinsic
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hepatic clearance (CLint), to predict TK, plasma concentrations and equivalent doses. While these models
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have made enormous strides in translating in vitro assay data to human relevance, the analyses are
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limited to 1)495 chemicals with human in vitro fup and CLint data, 2)a steady-state concentration vs. a
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dynamic peak plasma concentration (Cmax) approach, 3)HTS without consideration of assay response
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efficacies or quality of HTS concentration-response data, and, most importantly, 4)methods
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inadequately evaluating in vitro responses with respect to clinical therapeutic outcomes to estimate the
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likelihood of potential human interactions with Tox21 compounds.
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Pharmaceutical researchers have addressed these types of challenges using predictive in vitro models to
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forecast chemical-induced clinical effects. For example, the FDA has provided draft guidance documents
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to predict drug interaction potential including the likelihood of interaction with drug metabolizing
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enzymes and transporters in vivo.16 The only types of “interactions” considered here are chemical-
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biological target interactions. This guidance describes calculating the ratio of peak plasma
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concentrations (Cmax) of the chemical, at the exposure level of interest, divided by observed in vitro
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inhibition constant (Ki) reflective of the half-maximal in vitro activity. In this approach, a Cmax/Ki ratio ≥1
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(plasma concentrations equivalent or higher than in vitro half-maximal applied concentrations) indicates
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a ‘likely’ in vivo interaction. ‘Remote’ possibility exists if the ratio is
