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Integration of Dosimetry, Human Exposure and High-Throughput Screening Data in the Toxicity Assessment of Environmental

Integration of Dosimetry, Human Exposure and High-Throughput Screening Data in the Toxicity Assessment of Environmental Chemicals. Barbara A. Wetmore The Hamner Institutes for Health Sciences. June 7, 2011 DRSG Teleseminar Series.

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Integration of Dosimetry, Human Exposure and High-Throughput Screening Data in the Toxicity Assessment of Environmental

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  1. Integration of Dosimetry, Human Exposure and High-Throughput Screening Data in the Toxicity Assessment of Environmental Chemicals Barbara A. Wetmore The Hamner Institutes for Health Sciences June 7, 2011 DRSG Teleseminar Series

  2. Broad-Based Movement in Toxicology Towards In Vitro Testing and Hazard Prediction

  3. ToxCast: Forecasting the Toxicity of Environmental Chemicals Convergence of diverse fields and technologies to predict toxicity of chemicals and to aid in prioritization efforts -- high-throughput screening (HTS) -- bioinformatics -- computational toxicology Collins et al., Science 319:906, 2008

  4. ToxCast Assays ~500 HTS Assays: Multiple Cell Types, Pathways Assessed Judson et al. Env Health Perspect 118:485-92, 2010.

  5. Difficulty Translating Nominal Testing Concentrations into In Vivo Doses Knudsen et al. Toxicology 282:1-15, 2011

  6. Question Is there a way to incorporate human dosimetry and exposure information with AC50 or LEC data to better understand the impact of a chemical on human health?

  7. Defining Dosimetry and Exposure in High Throughput Toxicity Screens Toxicokinetic Parameters ~500 In Vitro ToxCast Assays Plasma Protein Binding Hepatic Clearance ToxCast AC50 or LEC Values In Vitro-to-In Vivo Extrapolation Estimated Target Tissue Bioactivity Concentration Predicted Assay Oral Equivalent Doses Human Exposure Estimates Provided by ToxCast Data Generated In Vitro Data Obtained from Registration Documents Chemicals with Potential to Perturb Cellular Pathways at Relevant Human Exposure Levels Computational Modeling

  8. High-Throughput PharmacokineticsPerformed on ToxCast Phase I Chemicals Human Hepatocytes (10 donor pool) Hepatic Clearance Population-Based In Vitro-to-In Vivo Extrapolation Plasma Concentration at Steady State for 100 Healthy Individuals of Both Sexes from 20 to 50 Yrs Old Human Plasma (6 donor pool) Plasma Protein Binding Rotroff et al., Toxicol Sci 117:348, 2010 Wetmore et al., In preparation.

  9. Distribution of In Vitro Pharmacokinetic Data for ToxCast Phase I Chemicals Distribution Summary Statistics Median 2.44 Upper Quartile 18.41 Lower Quartile 0.50 Distribution Summary Statistics Median 6.21 Upper Quartile 17.96 Lower Quartile 0.00 Percent Unbound Hepatic Clearance (µl/min/106 cells)

  10. Estimating Steady State Plasma Concentrations Using the In Vitro Assay Results Hepatic Clearance Plasma Protein Binding Population-Based In Vitro to In Vivo Extrapolation Software Plasma Concentration at Steady State for 100 Healthy Individuals of Both Sexes from 20 to 50 Yrs Old Estimated Renal Clearance

  11. Estimating Steady State Plasma Concentrations Using the In Vitro Assay Results Dose Rate * Body Weight [Conc]SS = CLWholeBody + CLR CLH where HPGL ≈ 137 million cells/g, VL ≈ 1820 g CLInt = HPGL * VL * Clinvitro CLR = fU * GFR where GFR ≈ 6.7 L/hr fU * QL * ClInt QL + fU * ClInt where QL ≈ 90 L/hr CLH = 100% oral bioavailability assumed for both CLR and CLH Kinetics are assumed to be linear CSS = DR * BW / (CLR + CLH) where DR = 0.042 mg/kg/hr and BW = 70 kg

  12. Assessment of IVIVE-derived values using Published PK and PBPK Models Non-restrictive CL Restrictive CL Restrictive CL: 6/13 agree; Css for 5 others are overpredicted Non-restrictive CL: 8/13 agree; Css for 4 others are underpredicted Css overprediction afforded by restrictive clearance model generates lower and more conservative estimates for oral equivalent values, which would be more protective of human health. Bioavailability an issue in 2 of 13 compounds a Css, concentration at steady state for 1 mg/kg/day dose;predicted using the 1 mM metabolic clearance rate. c IVIVE performed incorporating Caco-2 data into the simulation. d Css value in (Volkel et al., 2002) represented total bisphenol A, of which 99% is glucuronidated. The published value was divided by 100 to estimate the free concentration for this table. e Caco-2 assay to be verified for these chemicals. fPFOS and PFOA undergo active renal resorption (Andersen et al., 2006) and may explain the discrepancy in the listed values.

  13. Reverse Dosimetry Modeling for Interpreting In Vitro Assay Results Assay X (e.g., ACE inhibition) 1 mg/kg/day Oral Exposure Metabolic and binding parameters ToxCast AC50 Value Oral Dose Required to Achieve Steady State Plasma Concentrations Equivalent to AC50 Oral Exposure Plasma Concentration Upper 95th Percentile Css Among 100 Healthy Individuals of Both Sexes from 20 to 50 Yrs Old Reverse Dosimetry Oral Equivalent (mg/kg/day) 1 mg/kg/day = ToxCast AC50 (uM) Upper 95th Percentile Css (uM)

  14. Reverse Dosimetry Modeling for Interpreting In Vitro Assay Results 500 In Vitro ToxCast Assays In Vitro Bioactivity ToxCast AC50 Value Oral Equivalent Dose (mg/kg/day) Represented as a Box Plot ? Oral Exposure Plasma Concentration What are humans exposed to? ? Upper 95th Percentile Css Among 100 Healthy Individuals of Both Sexes from 20 to 50 Yrs Old Oral Dose Required to Achieve Steady State Plasma Concentrations Equivalent to In Vitro Bioactivity ? Reverse Dosimetry Chemical

  15. Assessment of ToxCast Phase I Chemicals Approximately 12% of ToxCast Phase I chemicals have in vitro bioactivity at oral equivalent doses that overlap with estimated human exposures.

  16. Assessment of ToxCast Phase I Chemicals Isoxaben Fenbuconazole Difenoconazole Acifluorfen Buprofezin Fludioxonil Triflumizole Dichloran Quinclorac Pyriproxyfen Spiroxamine Pyraclostrobin Cyprodinil PFOS Prometon

  17. Assessment of ToxCast Phase I Chemicals Piperonyl butoxide Chlorpropham Tetraconazole

  18. Assessment of ToxCast Phase I Chemicals 2-phenylphenol Triclosan

  19. Assessment of ToxCast Phase I Chemicals Fluroxypyr-meptyl Dicamba Fenhexamid

  20. Incorporation of Dosimetry Provides Greater Context to AC50 Data Pyraclostrobin Dichloran Isoxaben Cyprodinil Pyriproxyfen Fenbuconazole Spiroxamine Triflumizole Fludioxonil Acifluorfen Difenoconazole Buprofezin Quinclorac PFOS Prometon

  21. ToxCast Assays with Oral Equivalent Values Overlapping Human Exposures Are these related to adverse effects in vivo? Is the AC50 value the right basis for calculating the oral equivalent dose?

  22. Summary of use patterns, in vivo effects and assay hits for flagged Phase I Chemicals

  23. Distribution of Ad hoc Margin of Exposure Values for the ToxCast Phase I Chemicals Distribution Summary Statistics Median 1.59 (38.90) Upper Quartile 2.67 (467.74) Lower Quartile 0.70 (5.01) Distribution Summary Statistics Median 2.07 (117.49) Upper Quartile 2.90 (794.33) Lower Quartile 0.97 (9.33) Log10 Transformed Margin-of-Exposure Most Highly Exposed Subpopulation Log10 Transformed Margin-of-Exposure General US Population Of the 24 chemicals that overlapped using the upper bound of exposure estimates 16 also overlapped with oral equivalent values when general U.S. population exposure estimates were employed.

  24. Reverse Dosimetry Project has Generated Two Manuscripts Thus Far

  25. In progress…Analysis of 350 Phase II Compounds Underway http://www.epa.gov/pesticides/ppdc/testing/feb09/toxcast-presentation.pdf

  26. Conclusions • Integration of in vitro pharmacokinetic assays with computational modeling allows estimation of oral equivalent doses required to produce steady state in vivo concentrations equivalent to AC50 or LEC values in HTS assays. • Of the 239 chemicals tested thus far, only 23 had overlapping human exposure estimates and oral equivalent doses. • For 16 of these 23 chemicals, exposure estimates for the general U.S. population – and not just the most highly exposed subpopulation – displayed overlap with the oral equivalent values. • Incorporation of dosimetry and exposure information with AC50/LEC values provides a necessary context for interpretation of in vitro toxicity screening data. • The pharmacokinetic approaches presented in this study have the potential to move beyond a hazard identification paradigm towards the use of in vitro data in a risk assessment context.

  27. Acknowledgements Thomas Lab External Collaborators Rusty Thomas Frank Boellmann Eric Healy Reetu Singh Ling-Chieh Tsai David Dix (EPA) John Wambaugh (EPA) Daniel Rotroff (EPA) Richard Judson (EPA) Keith Houck (EPA) Bob Kavlock (EPA) Matt Martin (EPA) David Reif (EPA) Stephen Ferguson (CellzDirect) Kimberly Freeman (CellzDirect) Cornelia Smith (CellzDirect) Institute Collaborators Harvey Clewell Mel Andersen Mark Sochaski Brittany Allen Katherine Cantwell Ed LeCluyse Funding American Chemistry Council

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