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High-throughput in vitro Data To Inform Prioritization of Ambient Water Monitoring and Testing for Endocrine Active Chemicals Wendy J. Heiger-Bernays, Susanna Wegner, and David Dix Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b00014 • Publication Date (Web): 07 Dec 2017 Downloaded from http://pubs.acs.org on December 14, 2017
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Environmental Science & Technology
High-throughput in vitro Data To Inform Prioritization of Ambient Water Monitoring and Testing for Endocrine Active Chemicals a
Wendy J. Heiger-Bernaysa*, Susanna Wegnerb and David Dixc Boston University School of Public Health, 715 Albany St. Boston, MA 02118, USA b Oak Ridge Institute of Science and Education, Oak Ridge, TN, USA c DJD Consulting, PO Box 4518, Paso Robles, CA 93447, USA
*Department of Environmental Health, (617) 358-2431;
[email protected] ACS Paragon Plus Environment
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ABSTRACT
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The presence of industrial chemicals, consumer product chemicals, and pharmaceuticals is well
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documented in waters in the US and globally. Most of these chemicals lack health-protective
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guidelines and many have been shown to have endocrine bioactivity. There is currently no
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systematic or national prioritization for monitoring waters for chemicals with endocrine
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disrupting activity. We propose Ambient Water Bioactivity Concentrations (AWBCs) generated
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from high throughput data as a health-based screen for endocrine bioactivity of chemicals in
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water. The US EPA ToxCast program has screened over 1800 chemicals for estrogen receptor
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(ER) and androgen receptor (AR) pathway bioactivity. AWBCs are were calculated for 110 ER
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and 212 AR bioactive chemicals using high throughput ToxCast data from in vitro screening
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assays and predictive pathway models, high-throughput toxicokinetic data, and data-driven
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assumptions about consumption of water. Chemical-specific AWBCs are compared with
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measured water concentrations in datasets from the greater Denver area, Minnesota lakes, and
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Oregon waters, demonstrating a framework for identifying endocrine bioactive chemicals. This
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approach can be used to screen potential cumulative endocrine activity in drinking water and to
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inform prioritization of future monitoring, chemical testing and pollution prevention efforts.
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INTRODUCTION
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Since the 1990s, contaminants of emerging concern (CECs) have been measured in US surface
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waters,1,2 groundwaters3 and drinking water4,5 and waters globally.6,7 CECs include agents that
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are unregulated in water supplies or those which lack sufficient toxicological data for decision-
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making. Unregulated CECs such as pharmaceuticals, personal care products, and agricultural2 or
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industrial2 chemicals enter into water supplies from municipal wastewater treatment plants8,9
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croplands,10 industrial and commercial facilities,11 septic systems,12 landfills13 and concentrated
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animal feeding operations.14 The growing need for recycled water coupled with increasing
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drought conditions15 heighten the need for new tools to aid in assessment of potential human and
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ecological risks associated with CECs in water. Many of the CECs detected in water are of
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particular concern due to their potential to alter hormone signaling pathways in living systems at
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relatively low concentrations.16 The biological signaling pathways that regulate brain
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development, reproductive capabilities, growth and behavior are exquisitely sensitive to very
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small changes in concentration and temporal presence of chemicals with endocrine activity,16
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including endocrine signaling molecules. While there are some state and regional water
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monitoring efforts, the US lacks a systematic national monitoring program that prioritizes CECs
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that have endocrine bioactivity.
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The U.S. EPA’s Endocrine Disrupting Screening Program (EDSP) is responsible for evaluating
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potential endocrine effects of all pesticide active and inert ingredients, and chemicals found in
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drinking water sources with the potential for significant human exposures that conceivably could
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include many chemicals in commerce.17 In the 20 years since the EDSP legislative mandate, only
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52 pesticides have been evaluated in the current screening battery of tests, and no chemicals have
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been conclusively designated by U.S. EPA for regulation as endocrine disruptors. However,
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there are an estimated 10,000 unique chemicals in the EDSP universe of chemicals that are
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candidates for testing for potential endocrine activity.18 Understanding the prevalence and
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bioactivity of these chemicals in current and potential drinking water sources is only now
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possible with newly validated high throughput tools developed in the EDSP to identify chemicals
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with potential endocrine activity.19
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In the past five years, the U.S. EPA has made an effort to replace existing testing requirements
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for endocrine activity with high throughput in vitro screening assays. The most progress has been
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made on the estrogen receptor and androgen receptor pathways. U.S. EPA's Toxicity Forecaster
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(ToxCast) program has developed a panel of high throughput in vitro screening assays and
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computational toxicology methods to identify chemicals with estrogen receptor (ER) bioactivity
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and/or androgen receptor (AR) bioactivity. A computational network model is used to integrate
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in vitro assay responses for chemicals into the ER or AR pathway based on the molecular events
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that typically occur in a steroid hormone receptor-mediated response. To identify ER agonist and
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antagonist bioactivity,20,21 the ToxCast ER pathway model integrates concentration:response data
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from 18 in vitro high throughput assays. To identify AR agonist and antagonist bioactivity,22 the
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ToxCast AR pathway model integrates concentration:response data from nine in vitro high
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throughput assays. Assays included in these pathway models target a range of molecular events,
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including receptor binding, dimerization, co-factor recruitment, DNA binding, and protein
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production. The output of these models provides a score of potential ER or AR agonist and
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antagonist activity, chemical potency, and a measure of assay-specific false positive activity of
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each chemical run in ToxCast.21,22 These models have been shown in multiple reviews and
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analyses to perform at least as well as validated toxicological assays in identifying endocrine
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bioactivity. 18, 21,22
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The U.S. EPA develops Ambient Water Quality Criteria (AWQC) for the protection of
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human health and the environment under the US Clean Water Act.24 AWQCs are designed to
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protect and maintain quality of surface waters for potential use as drinking water, not only those
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waters that are currently used for drinking water. Promulgated AWQCs are derived from
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traditional in vivo toxicological data from which points of departure are identified. They provide
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a point of reference for interpretation of potential risks associated with chemical concentrations
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detected in the environment. However, AWQCs are not available for the large number of
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contaminants frequently detected in water, presenting a challenge for interpretation of
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monitoring data on data-poor chemicals, including potential endocrine disrupting chemicals.
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In the absence of AWQCs, high-throughput in vitro data and computational models such
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as EPA’s ToxCast program23 provide endocrine bioactivity data that can put monitoring data on
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otherwise data-poor contaminants into context. To inform monitoring efforts for the large
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number of data-poor chemicals, we propose the use of Ambient Water Bioactivity
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Concentrations (AWBCs) derived from high-throughput ToxCast data. We present a
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methodology to develop AWBCs as analogs to AWQCs, using an approach analogous to
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promulgated AWQCs. An AWBC is a chemical-specific reference concentration in water that is
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based on endocrine bioactivity of the chemical in the high-throughput assays and is protective of
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human health. By combining AWBCs, cumulative potential for bioactivity can be assessed for
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mixtures of chemicals identified in individual water samples. This is the first use of high
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throughput in vitro toxicological data for deriving human health screening level concentrations
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for comparison to measured concentrations of CECs in US waters. The aims of this manuscript
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are: 1) to present a generalizable framework for deriving screening level human health AWBCs
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using ToxCast data; 2) to demonstrate this approach for estrogen receptor (ER) and androgen
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receptor (AR) bioactive chemicals; 3) to illustrate how measured water concentrations of ER and
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AR bioactive chemicals can be compared with chemical-specific AWBCs; and 4) to extend the
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single chemical methodology to estimating bioactivity in chemical mixtures for multiple ER and
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AR bioactive chemicals, showing how AWBCs can be used to evaluate potential for cumulative
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endocrine bioactivity.
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EXPERIMENTAL (MATERIALS and METHODS)
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Selection of ER and AR Bioactive Chemicals
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ToxCast pathway models were developed for estrogen and androgen (ER and AR) and used to
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produce an overall activity score for each chemical tested on a scale of 0-1.20,21,22 For this
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analysis, chemicals with overall ER or AR bioactivity rankings equal to or greater than 0.1 are
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considered bioactive. The ER and AR models were validated based on high balanced accuracy in
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identification of sets of reference chemicals.20,22 Assay-specific concentration:response curves
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modeled for each chemical were used to identify median concentrations producing 50% of
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maximum (AC50) responses across all active assays, providing an estimate of activity in a
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biologically relevant model.21,22 The AC50s were selected because they represent the average
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responses across the 18 ER and nine AR assays, decreasing the influence of outlier responses
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from individual assays. Proprietary pharmaceuticals included in ToxCast (largely candidate
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compounds donated by pharmaceutical companies for ToxCast testing that are not currently
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marketed) were excluded from the current analysis as they are unlikely to be present in water and
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they cannot be identified for monitoring in US waters.
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Conversion between In Vitro Concentrations and Exposure Rates
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U.S EPA has developed high throughput toxicokinetic models (HTTK) that predict human doses
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of chemicals based on the associated bioactivity in vitro concentrations. Reverse dosimetry
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assuming first-order metabolism using toxicokinetic (TK) modeling was used to predict the oral
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equivalent dose (OED) of a chemical needed to produce an internal (plasma) concentration equal
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to a bioactive in vitro concentration. 25, 26, 27 Monte Carlo simulations informed by experimental
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data on protein binding and metabolism have been used to estimate a distribution of steady state
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blood concentrations across a hypothetical population (based on exposure assumptions for a 35
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year old Caucasian male) with 1 mg/kg/day of exposure to a given chemical. 25, 26, 27, 28 We use
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the population 95th percentile of these previously simulated blood concentrations as a
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conservative set of conversion factors (to convert concentrations to doses) to estimate the oral
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equivalent dose (OED) in mg/kg/day required to achieve blood steady state (Css) levels identical
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to the bioactive concentrations in the ToxCast assays. For the 150 chemicals lacking
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experimentally derived HTTK data, the geometric mean of the HTTK conversion factors
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available for the other 95 ER and AR active chemicals were used to estimate exposure rates.
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Calculation of the Ambient Water Bioactivity Concentrations (AWBC)
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An Ambient Water Bioactivity Concentration (AWBC) was calculated for each ER and AR
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bioactive chemical and its respective OED using an approach similar to that used by the US EPA
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to develop Ambient Water Quality Criteria29 as follows:
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Equation 1: =
!"
# 1,000,000
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where OED (mg/kg-day) = Oral Equivalent Dose = ToxCast Bioactivity(µM) x ((1 mg/kg)-day)/HTTKCss
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vivo extrapolation already accounts for variability and sensitivity in the population. The
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AWBCAdult is based on assumptions describing US adult population characteristics for body
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weight and daily water consumption. The Daily Intake (DI) of 2.4 L/day and Body Weight (BW)
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of 80 kg represent the per capita estimate of combined direct and indirect community water
(µM)
RSC = Relative Source Contribution, unitless (EPA, 2000) BW = Body Weight (kg) DI = Drinking Water Intake (liters/day) The OED is not modified by application of traditional uncertainty factors since the in vitro to in
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ingestion at the 90th percentile for adults ages 21 and older and associated body weights30. The
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AWBCInfant is based on assumptions describing infants for whom reconstituted infant formula
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and water are the major sources of nutrition and liquids for the first six months of life. Daily
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Intake (DI) and Body Weights (BW) for infants 0-1 month, 1-3 months and 3-6 months were
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used to calculate 95th percentile population estimates for the most vulnerable population: the 0-1
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month infant30. The Relative Source Contribution (RSC) is a factor used to account for the
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percentage of a person’s exposure to chemicals that may come from drinking water. The EPA
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default factor of 0.2, indicating that 80% of a person’s exposure comes from a non-drinking
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water source, is assigned, although this factor should be modified based on exposure data
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specific for each chemical29. The Daily Intake (DI) of 0.184 L/day and Body Weight (BW) of 4.8
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kg represent the per capita estimate of water ingestion at the 95th percentile for infants under 3
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months and mean body weight31. Inputs and calculated AWBCs are included in Table S-1. For
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bioactivity-based prioritization of chemicals for monitoring, we identify priority ER and AR
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active chemicals, those with an AWBCInfant
