1. The Tox21 Program Christopher P. Austin, M.D. Director, NIH Chemical Genomics Center The Future of Chemical Toxicity Testing in the U.S.: Creating a Roadmap to Implement the NRC’s Vision and Strategy June 21, 2010

2. 2 The Tox21 Community Tox21 has become….

3. 3 The Tox21 Community Tox21

4. 4 Scientific collaboration began 2005 (NTP-NCGC) – 2006 (NTP-NCGC-EPA) Memorandum of Understanding on “High-Throughput Screening, Toxicity Pathway Profiling and Biological Interpretation of Findings” (http://ntp.niehs.nih.gov/go/28213) –Signed February 14, 2008 by: NIH/NIEHS/NTP by Dr. S. Wilson NIH/NHGRI by Dr. F. Collins EPA/ORD by Dr. G. Gray –Points of Contact EPA - Bob Kavlock (Director, National Center for Computational Toxicology) NCGC - Chris Austin (Director) NTP - Ray Tice (Chief, NTP Biomolecular Screening Branch) –Public status made available through monthly meetings of the EPA CPCP (Chemical Prioritization Community of Practice) Leverage –Pools resources for common goal –Overcomes the resource limitations of a single agency –Builds on existing expertise –Avoids the need to create a new administrative and support structure Interagency Coordination on Biomolecular Screening

5. 5 ActivitiesNTPNCGCEPA Historical Toxicology Data Experimental Toxicology Ultra High-Throughput Testing Mid- to High Throughput Systems Lower Organism Model System C. elegans Zebrafish In Vitro 3-D Model Systems Effect of Human/Rodent Genetic Background on Toxic Effects Computational Toxicology Validation Experience (NICEATM-ICCVAM ) The Tox21 Community

6. Tox21 activity matrix Rodent in vitro Human in vitro Rodent in vivo Human in vivo Approved drug data Historical EPA and NTP data Tox21

7. 7 Leadership: meets every 2 wks –B. Kavlock (EPA), R. Tice (NTP), C. Austin (NCGC) Working Groups: chairs meet together every 4 wks –Compounds, Assays, Informatics, Targeted Testing –Co-leads from each agency Community: meets every 3 months –Larger group of interested parties from 3 agencies Oversight: component Scientific Advisory Boards –Reports at least once/yr Tox21: Organization

8. 8 All have been evaluated in one or more toxicological tests NTP 1408 (1353 unique compounds) –1206 with NTP test data, 147 ICCVAM reference substances –MW = 32-1168, calculated log p = -3 to 13.2 EPA: 1462 (1384 unique compounds) –MW = 58-516, calculated log p = -2.8 to 8.2 ~400 compound overlap NTP compounds The Current Tox21 Compound Library

9. Proof of Principle Toxicology qHTS

10. Unsupervised clustering in combination with Dunn’s cluster validity index is a robust method for identifying mechanisms of action without requiring a priori knowledge about mechanisms of toxicity.

11. Universe13,247 With structures 8,277 Plausible P-chem (logP) 7,116 CurrentAdditional NTP1353~1400 EPA1330~2800 NCGC~3000 drugs - Library, Fall 2010 ~10,000 Sources include NTP, EPA HPV, CCL, OPPIN, OW, Inerts, ToxCast, DSSTox, EU Carcinogenomics, Pharmaceuticals, others Full Tox21 Chemical Library: Fall 2010

12. Office of Research and Development National Center for Computational Toxicology Tox21 IDs # Chemicals 309 >500 ToxCast Phase I ~1000 >50 ~10,000 # Assays ToxCast Phase II NCCT/EPANIEHS/NTPNIH/NCGC Tox21 Drugs Environmental Industrial/Tox Environmental Industrial/Tox Drugs Food Use NCCT/EPA NIEHS/NTP NIH/NCGC Tox21_3_###### Tox21_2_###### Tox21_1_######

13. NIH Chemical Genomics Center 75 scientists: biology, chemistry, informatics, robotics >100 collaborations with investigators worldwide Chemical genomics: biological profiles of chemical activity Chemical probes of novel targets, rare/neglected diseases

14. NCGC Screening System 1: BSL1/Kalypsys Capacity:3.0MM Assay Wells 5.0MM Compound Wells Throughput:1400 plates/day Readers:ViewLux (2) Acumen (2) Envision (2) New Capabilities: Automatic Loading and Unloading stations using commercially available plate stackers Dispense Inspection Systems using integrated CCD cameras

15. HTS Throughput Molecular mechanism Immediate organismal relevance 10’s/day 1000’s/day 10,000’s/day 100,000’s/day “MTS”“LTS”

16. HTS as done for drug discovery is not suitable for toxicity testing High false positive rate: up to 90% High false negative rate: up to 70% Prior probability of activity low Cannot reliably “bank” or computationally synthesize results Purpose is to generate a few leads for subsequent chemical optimization

17. Quantitative High-Throughput Screening (qHTS) Conventional HTS done at single concentration –typically 10 uM qHTS tests all compounds at 15 concentrations –Range = 5nM – 92uM –Assay volumes 2-6 uL in 1536- well plate format –Concentration-response curve generated for each compound from primary screen Produces robust activity profiles of all compounds –Dramatically reduced FP and FN Throughput >200,000 concentration-response profiles (2M wells) per week –Entire Tox21 collection is tested at 15 concs in single day

18. Tox21 assays screened at NCGC to date General toxicity –Cytotoxicity assays Cell viability assay (measures ATP) –Apoptosis assays Caspase assays (measure activity of Caspase 3/7, 8, 9) –Membrane integrity assay LDH release Protease release –Mitochondrial Toxicity assay Mitochondrial membrane potential –Gene tox assays Micronucleus DNA repair “Tox Pathways” –CREB –ER stress –HRE/Hypoxia –NFkB –P53 –ARE –HSE Targets –Nuclear receptor assays: AR, AhR, ER , FXR, GR, LXR, PPARδ, PPARγ, PXR, RXR, TRβ, VDR, ROR  –hERG channel Inter-individual variation –87 HapMap lines

19. Rapidly access (inhouse) biological profiles of chemical series Browse via interactive heatmap that provides details of assay response Cluster heatmap by assay response and chemical similarity

20. 20 Approaches for Identifying Key Toxicity Pathways - Toxicogenomic data - Human disease - genetic associations - The Pathway Universe - Contract Research Organizations

21. Not all in vitro assays are suitable for HTS and not all substances can be tested in vitro (volatiles, solvent requirement, practical concentration limitation) Responses are for the most part limited to cell- autonomous effects of parent compound Exposure (route, extent), metabolism Genetic heterogeneity relating to differences in sensitivity (A gene is not a pathway, a pathway is not a cell, a cell is not an organ, an organ is not an animal……) Limitations

22. View all results with skepticism“All results are artifacts until proven otherwise” Confirm results in other assays of same phenomenon and/or lower-throughput more physiological systems Test same pathway/readout in multiple different assays/approaches Move from cell lines to primary cells Incorporate metabolism (e.g., co-culture, S9) Determine responses in cells from differing genetic backgrounds Ways to address these limitations

23. Determine which cell types (human vs rodent cell lines, primary cells, stem cell-derived cells, etc.) are most useful for HTS Develop comprehensive battery of pathway and phenotypic assays for testing Incorporate metabolism and genetic heterogeneity Secondary assays to evaluate compounds ID’ed in HTS (e.g., in vitro 3D organ models, C. elegans, zebrafish?) Obtain existing in vitro, experimental animal, and human data on compounds from industry Developing cross-assay meta-analysis informatics algorithms/browsers that enable identification of correlations among the many in vitro assays and in vivo readouts Validate resulting testing strategies for reliability and relevance, develop strategies for incorporation into regulatory decision-making Needs

24. Prediction is very difficult, especially if it's about the future. - Niels Bohr

25. Contact Information Chris Austin, NCGC: austinc@mail.nih.gov Ray Tice, NTP: tice@niehs.nih.gov Bob Kavlock, EPA: kavlock.robert@epamail.epa.gov