Tox21 & ToxCast Chemical Landscapes: Laying the Foundation for 21 st Century Toxicology

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

2. NCCT’s ToxCast Program Structures HTS In Vitro QSAR In Vivo

3. Generic Chemicals Test Substances NCCT Public Data http://www.epa.gov/ncct/ DSSTox ACToR ToxRefDB ToxCast Tox21

4. Chemical Structure Annotation Chemical Management Chemical QC NCCT Public Data http://www.epa.gov/ncct/ DSSTox ACToR ToxRefDB ToxCast Tox21

5. NCCT Public Data http://www.epa.gov/ncct/ Data Integration Chemical – Assay Linkage In vivo data Linkage Analysis tools DSSTox ACToR ToxRefDB ToxCast Tox21

6. ToxCast Project Phase I (309) ToxCast concept paper ToxSci ToxCast Data Analysis Summit ToxCast Phase I contracts issued Phase I testing begins Phase II: (700) 2007 2009 2008 Phase I data results 2010 EPA NCCT launches ToxCast Full Phase I data publication/release NAS Report released ACToR launched ToxRefDB web access w/in ACToR

7. 10K Tox21 Chemical Library 5.6K 2.8K 1.4K 2004 2010 2006 2008 2007 2009 2005 2011 Tox21 Project NTP HTS Plate A ToxCast Phase II ToxCast Phase I Tox21 EPA Plates A’,B,C NCGC Drug Plates A,B EPA Tox21 Plate A NTP Tox21 Plates A’,B,C NCGC Drug Plates A’,B’,C

8. Tox21 Chemical x Assay Landscape Tox21 NTP HTS Plate A ToxCast Phase I & II ~10,000 Chem x 50-100 assays 1408 Chem x >100 assays 960 Chem x >500 assays 100 # Assays >500 ~1000 ~10,000 # Chemicals 1536 well microplate format (1408 cmpds/plate) x 9 plates Drugs Environmental Industrial Pesticides Food Use Drugs Toxicology NCCT/EPA NIEHS/NTP NIH/NCGC FDA CFSAN/CDER

9. Tox21: EPA Chemical Inventories TOX21_EPA_3726 ToxCast_960 e1k 820 unique (1000 samples) 100 To be tested in endocrine-related subset of ToxCast assays Phase I & II(a,b) 111 (+24 PhIIc) failed drugs, CFSAN, pesticides, EPA high interest compounds tested in >500 HTS assays IIc

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

12. 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

13. 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

14. 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

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

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

17. 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. ToxCast/Tox21 Chemical Registry Analog Searching External Resources Structure-based Data Mining AIM DSSTox PubChem ChemSpider EPA ACToR DSSTox t u r e Chemical structure - CID Substance details - SID Project inventory record - RID S t r u c CID Table SID Table Tox21 ToxCast ToxRefDB RID Table SAR Modeling Assay Results Structures Test Sample 7

19. ToxCast/Tox21 Chemical Registry DSSTox Chemical structure - CID Substance details - SID Project inventory record - RID Bottle_ID DSSTox_RID COA_ID Soln_ID QC_ID DSSTox RID Bottle ID ( COA ID) Solution ID ( QC ID) Tox21 Sample Tracking Database Assay Results Structures Test Sample 8

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

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 Toxicology literature Public databases Cheminformatics resources Chemical Annotation Layer CAS SMILES InChI / Structure Substance details Toxicology Chemical Name

23. Chemical Information & Sample QC Sample Annotation & QC Procure from Chemical Supplier • No CAS or wrong • Name wrong • Structure wrong • Not same as generic chemical Salt, isomer, … • Name does not match info on COA hydrate, stereo • Wrong MW • COA expired Name, CAS, purity (COA) MW, dose • Purity <90% • Active impurities • Sample degrades • Rxn with solvent • Hydrolysis IC 50 Chemical Annotation of Public resources Generic Chemical of Toxicological Interest • 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

24. Features Classes Descriptors Properties Tox21 Cheminformatics Chemical Substance: SID CAS/Name Mixture IDs 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 2D, 3D Conformers Tautomers Structure: CID Parent: PID Salts Complexes (Metabolites?) Groups: MapID Analogs, classifiers, stereo family, etc. Assay Results Tox21/ToxCast Bottle IDs Analytical QC Solution IDs Plate IDs, Plate Addresses

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

26. Tox21 Structural Library Feature enrichment Feature diversity

27. 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. Tox21: Molecular Weight Distributions Tox21 NTP & EPA Mean = 231 NTP & EPA collections enriched with lower MW compounds compared to NCGC drug collection # Molecules Tox21 NCGC Mean = 304 Molecular Weight

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

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

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

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

33. Expanding Applicability QSAR In vitro/HTS Defined organics Virtual chemicals Volatiles DMSO insolubles Reactives Defined structure + Biological profile Mixtures Formulations Proprietary substances Organometallics Metals Salts metabolism ADME In Vivo

34. 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 EPAIndiraThillainadarajah – EPA:SEE • PatraVolarath – 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.