1. Office of Research and Development National Center for Computational Toxicology August 25, 2011 Tox21 & ToxCast Chemical Landscapes: Laying the Foundation for 21 st Century Toxicology 5 th Meeting on U.S. Government Chemical Databases & Open Chemistry, Frederick, MD Ann Richard National Center for Computational Toxicology

2. Office of Research and Development National Center for Computational Toxicology 1 NCCT’s ToxCast Program QSAR

3. Office of Research and Development National Center for Computational Toxicology Generic Chemicals Test Substances NCCT Public Data http://www.epa.gov/ncct/ http://www.epa.gov/ncct/ ToxRefDB ToxCast ACToR DSSTox Tox21

4. Office of Research and Development National Center for Computational Toxicology NCCT Public Data http://www.epa.gov/ncct/ http://www.epa.gov/ncct/ ToxRefDB ToxCast Chemical Structure Annotation Chemical Management Chemical QC DSSTox ACToR Tox21

5. Office of Research and Development National Center for Computational Toxicology NCCT Public Data http://www.epa.gov/ncct/ http://www.epa.gov/ncct/ ToxRefDB Tox21 ToxCast DSSTox Data Integration Chemical – Assay Linkage In vivo data Linkage Analysis tools ACToR

6. Office of Research and Development National Center for Computational Toxicology 20072009 20082010 ToxCast Project ToxCast concept paper ToxSci EPA NCCT launches ToxCast ToxCast Phase I contracts issued ACToR launched NAS Report released Phase I testing begins Phase I data results ToxCast Data Analysis Summit ToxRefDB web access w/in ACToR Full Phase I data publication/release Phase I (309) Phase II: (700)

7. Office of Research and Development National Center for Computational Toxicology Tox21 Project 2005 2007 2009 10K 5.6K 1.4K Tox21 Chemical Library 2.8K 20062008 20102004 2011 EPA Tox21 Plate A NTP HTS Plate A ToxCast Phase I NCGC Drug Plates A,B NCGC Drug Plates A,B Tox21 EPA Plates A’,B,C NTP Tox21 Plates A’,B,C NTP Tox21 Plates A’,B,C NCGC Drug Plates A’,B’,C NCGC Drug Plates A’,B’,C ToxCast Phase II

8. Office of Research and Development National Center for Computational Toxicology 7 Tox21 Chemical x Assay Landscape # Chemicals >500 ~1000~10,000 # Assays 100 ToxCast Phase I & II 960 Chem x >500 assays Tox21 ~10,000 Chem x 50-100 assays 1408 Chem x >100 assays NTP HTS Plate A NTP HTS Plate A Drugs NCCT/EPANIEHS/NTPNIH/NCGC Environmental Industrial Pesticides Food Use Drugs Toxicology 1536 well microplate format (1408 cmpds/plate) x 9 plates 1536 well microplate format (1408 cmpds/plate) x 9 plates FDA CFSAN/CDER

9. Office of Research and Development National Center for Computational Toxicology e1k 820 unique (1000 samples) To be tested in endocrine-related subset of ToxCast assays ToxCast_ 960 Phase I & II(a,b) 111 (+24 PhIIc) failed drugs, CFSAN, pesticides, EPA high interest compounds tested in >500 HTS assays 100 IIc Tox21: EPA Chemical Inventories TOX21_EPA_3726

10. Office of Research and Development National Center for Computational Toxicology Tox21: EPA ToxCast Chemicals ToxCast Phase I (293 unique)  EPA pesticidal actives w/ rich in vivo data  PFOAs, BPA, metabolite/parent pairs ToxCast Phase II a,b (776) +c (100)  EPA pesticides, high interest EPA and stakeholder inventories, data rich chemicals (EDSP, OPPT, Antimicrobials, Inerts, …)  135 Pharma failed drugs w/ pre-clinical & clinical tox data  FDA CFSAN data rich, NCTR LTKB Priority 1 drugs  Toxicity reference chemicals, data-rich chemicals, NTP immunotox Broaden selections from ToxCast Phase II nomination lists to include in Tox21 EPA set

11. Office of Research and Development National Center for Computational Toxicology 211 volatile/ insufficient sample COA/MSDS review Unable to procure Too expensive 4371 compounds Able to purchase 3726 unique substances EPA Tox21 Inventory e.g., EPA Tox21 Chemical Procurement ~7,000 compounds Candidates for procurement Complex mixtures Ill-defined substances No structure Insoluble (est. LogP) Volatiles (est. VP) Inorganics Explosive Reactive Polymers … 435 Insoluble in DMSO (approx 10%) Solubilize in DMSO Solubilize in DMSO HPV, MPV Substances Antimicrobials Pesticide activesPesticide inerts Water contaminants Industrial chemicals NCCT Programs EU, FDA In vivo data availability (EPA, NTP, FDA) Endocrine disruptors ~19,000 compounds EPA Tox21/ToxCast Phase II Chemical Nominations (over 100 lists) Green Chemistry 10

12. Office of Research and Development National Center for Computational Toxicology Tox21 Chemicals: EPA Selection Strategy Nominations & tracking of inventory overlaps by CAS Prioritize for procurement Cost & availability Filter by MW, physchem, volatility, MSDS cautions DMSO solubility Nominations & tracking of inventory overlaps by CAS Prioritize for procurement Cost & availability Filter by MW, physchem, volatility, MSDS cautions DMSO solubility

13. Office of Research and Development National Center for Computational Toxicology 12 ToxCast PhI&PhII 960: # Compounds per Inventory (960 x 16) 960 Total chemicals  2740 total overlaps across 16 diverse inventories Excellent coverage of multiple high-interest inventories Broad diversity of chemical- use and types Large overlap with data-rich inventories 960 Total chemicals  2740 total overlaps across 16 diverse inventories Excellent coverage of multiple high-interest inventories Broad diversity of chemical- use and types Large overlap with data-rich inventories

14. Office of Research and Development National Center for Computational Toxicology 13 ToxCast_960 Pharmaceuticals: Multipurposing (255) 111 Failed pharmaceuticals – no overlaps 144 additional cmpds classified as drugs; on other lists with other uses 137 of these appear on 2 or more lists Caffeine appears on 18 lists 111 Failed pharmaceuticals – no overlaps 144 additional cmpds classified as drugs; on other lists with other uses 137 of these appear on 2 or more lists Caffeine appears on 18 lists

15. Office of Research and Development National Center for Computational Toxicology 14 Chemical Sample Registration Workflow Place chemical orders Procure 2x100mg compound in tared, barcoded vials Store neat Solubilize in DMSO + Partition to Matrix racks Solution Plates Insoluble Review COA QC structure Register in DSSTox Inventory Register in Sample Tracking Database X Bottle IDs Remove from DSSTox Update Solution IDs Update Assay Providers ACToR/ PubChem NCGC Plate Address Plate ID Assay Results Analytical QC

16. Office of Research and Development National Center for Computational Toxicology Publish QC summary results in association with assay data 15 Tox21 Analytical QC LC-MS GC-MS PASS = Confirm parent ion peak and >90% purity Fail, inconclusive or analytical method inappropriate Retest at later time point under assay conditions for stability A copy of each parent Tox21 assay plate (352 cmpds/plate) will be subjected to analytical QC for assessing purity, identity, stability

17. Office of Research and Development National Center for Computational Toxicology Analytical QC Summary PDF results will be available for each sample NCGC Analytical Chemist (W. Leister) will review all preliminary LC results and QC “FAIL” compounds, and supervise follow-up testing Prime objective of QC is to inform analysis & interpretation of assay results: high confidence  low confidence  fail Analytical QC Summary PDF results will be available for each sample NCGC Analytical Chemist (W. Leister) will review all preliminary LC results and QC “FAIL” compounds, and supervise follow-up testing Prime objective of QC is to inform analysis & interpretation of assay results: high confidence  low confidence  fail

18. Office of Research and Development National Center for Computational Toxicology Structure-based Data Mining PubChem ChemSpider EPA ACToR Analog Searching AIM DSSTox External Resources S t r u c t u r e Chemical structure - CID Substance details - SID Project inventory record - RID DSSTox Test Sample Assay Results Structures ToxCast/Tox21 Chemical Registry SAR Modeling CID Table RID Table SID Table Tox21ToxCastToxRefDB 7

19. Office of Research and Development National Center for Computational Toxicology Tox21 Sample Tracking Database DSSTox RID Bottle ID (  COA ID) Solution ID (  QC ID) Chemical structure - CID Substance details - SID Project inventory record - RID DSSTox Test Sample Assay Results Structures ToxCast/Tox21 Chemical Registry Bottle_ID Soln_ID QC_ID DSSTox_RID COA_ID 8

20. Office of Research and Development National Center for Computational Toxicology ACToR/ToxMiner Solution ID Plate ID Plate Address ID DSSTox RID Assay name Assay details Assay outcome Assay Results DSSTox RID Bottle ID (  COA ID) Solution ID (  QC ID) Tox21 Sample Tracking Database Chemical structure - CID Substance details - SID Project inventory record - RID DSSTox Test Sample Structures ToxCast/Tox21 Chemical Registry 9 PubChem_CID S t r u c t u r e PubChem_SID Tox21_ID

21. Chemical Information & Sample QC “Whoever is careless with the truth in small matters cannot be trusted with important matters.” – Albert Einstein

22. Gene-expression Chemical Supplier Lot/Batch Certificate of Analysis Solubility Purity & Stability Chemical Sample Details Chemical Name Toxicology literature Public databases Cheminformatics resources CAS SMILES InChI / Structure Substance details Chemical Annotation Layer

23. Name is misspelled or incorrect CAS is invalid or retired CAS and name do not agree Name and structure do not agree Name is insufficient for structure assignment Insufficient description of substance Chemical Annotation of Public resources Generic Chemical of Toxicological Interest Chemical Information & Sample QC Sample Annotation & QC Not same as generic chemical Salt, isomer, … Name does not match info on COA hydrate, stereo Wrong MW COA expired Purity <90% Active impurities Sample degrades Rxn with solvent Hydrolysis Procure from Chemical Supplier Name, CAS, purity (COA) MW, dose IC 50 No CAS or wrong Name wrong Structure wrong

24. Office of Research and Development National Center for Computational Toxicology 23 Tox21 Cheminformatics Plate IDs, Plate Addresses Solution IDs Bottle IDs Assay Results Analytical QC Tox21/ToxCast Features Classes Descriptors Properties Mixture IDs Chemical Substance: SID Structure: CID Parent: PID 2D, 3D Conformers Tautomers Salts Complexes (Metabolites?) CAS/Name Groups: MapID Analogs, classifiers, stereo family, etc. Creating SAR-ready files: How to process salts, complexes, charged species How to aggregate results for resulting structure “duplicates” How to deal with enriched features & “families” of structures within dataset Creating SAR-ready files: How to process salts, complexes, charged species How to aggregate results for resulting structure “duplicates” How to deal with enriched features & “families” of structures within dataset

25. Office of Research and Development National Center for Computational Toxicology 24 DSSTox TOX21E1_3619 NCGC Pharmaceuticals NTP compounds FDA compounds EPA compounds NCGC Pharmaceuticals NTP compounds FDA compounds EPA compounds 13 macromolecule 155 mixture or formulation 1 unspecified or multiple forms 45 inorganics 100 organometallics 173 complexes 219 salts 12 no structures 13 macromolecule 155 mixture or formulation 1 unspecified or multiple forms 45 inorganics 100 organometallics 173 complexes 219 salts 12 no structures 91 “parent” groupings (2-3) MW ranges from 30 (formalin) to 1700 (tannic acid) Alkylbenzenes Chlorobenzenes Methyl phenols Nitrobenzenes Aromatic amines Phthalates Perfluorinates… Alkylbenzenes Chlorobenzenes Methyl phenols Nitrobenzenes Aromatic amines Phthalates Perfluorinates…

26. Office of Research and Development National Center for Computational Toxicology Tox21 Structural Library Feature enrichment Feature diversity

27. Office of Research and Development National Center for Computational Toxicology Tox21 Reaction Features: Commonalities NTP Tox21 NCGC Tox21 EPA Tox21 Drugs Food additives Antimicrobials Water contaminants HPVs Toxicants … Metabogen reaction features generated using “MOSES” software by Molecular Networks

28. Office of Research and Development National Center for Computational Toxicology Tox21 NTP & EPA Tox21 NCGC # Molecules Molecular Weight NTP & EPA collections enriched with lower MW compounds compared to NCGC drug collection Mean = 231 Mean = 304 Tox21: Molecular Weight Distributions

29. Office of Research and Development National Center for Computational Toxicology ToxCast_PhaseI ToxCast/Tox21 property distributions Chemical properties computed using “Adrianna” software by Molecular Networks. LOG P = Octanol/Water partition coefficient TPSA = log (Total Polar Surface Area) Complexity = log (complexity based on paths, branching, atoms) LOG P = Octanol/Water partition coefficient TPSA = log (Total Polar Surface Area) Complexity = log (complexity based on paths, branching, atoms)

30. Office of Research and Development National Center for Computational Toxicology ToxCast/Tox21 property distributions Chemical properties computed using “Adrianna” software by Molecular Networks. LOG P = Octanol/Water partition coefficient TPSA = log (Total Polar Surface Area) Complexity = log (complexity based on paths, branching, atoms) LOG P = Octanol/Water partition coefficient TPSA = log (Total Polar Surface Area) Complexity = log (complexity based on paths, branching, atoms) ToxCast_PhaseI ToxCast_PhaseII

31. Office of Research and Development National Center for Computational Toxicology ToxCast/Tox21 property distributions Chemical properties computed using “Adrianna” software by Molecular Networks. LOG P = Octanol/Water partition coefficient TPSA = log (Total Polar Surface Area) Complexity = log (complexity based on paths, branching, atoms) LOG P = Octanol/Water partition coefficient TPSA = log (Total Polar Surface Area) Complexity = log (complexity based on paths, branching, atoms) 111 failed drugs ToxCast_PhaseII ToxCast_PhaseI

32. Office of Research and Development National Center for Computational Toxicology ToxCast/Tox21 property distributions Chemical properties computed using “Adrianna” software by Molecular Networks. LOG P = Octanol/Water partition coefficient TPSA = log (Total Polar Surface Area) Complexity = log (complexity based on paths, branching, atoms) LOG P = Octanol/Water partition coefficient TPSA = log (Total Polar Surface Area) Complexity = log (complexity based on paths, branching, atoms) Tox21 111 failed drugs ToxCast_PhaseII ToxCast_PhaseI

33. Office of Research and Development National Center for Computational Toxicology Expanding Applicability QSAR In vitro/HTS Defined organics Virtual chemicals Volatiles DMSO insolubles Reactives Mixtures Formulations Proprietary substances Organometallics Metals Salts metabolism ADME Defined structure + Biological profile

34. Office of Research and Development National Center for Computational Toxicology Acknowledgements: EPA NCCT ToxCast Team: Robert Kavlock - Director David Dix Keith Houck Matt Martin (ToxRefDB) Richard Judson (ACToR) EPA NCCT DSSTox: Maritja Wolf– Lockheed Martin, Contractor to the EPA Indira Thillainadarajah – EPA:SEE Patra Volarath – EPA Post Doc External Collaborators: Chihae Yang, FDA/CFSAN Chris Austin & colleagues, NCGC/NIH Ray Tice & colleagues, NTP/NIEHS This work was reviewed by EPA and approved for publication but does not necessarily reflect official Agency policy.