1. Tox21: Transforming Environmental Health Protection Raymond Tice, Ph.D. Chief, Biomolecular Screening Branch (tice@niehs.nih.gov) LINCS Consortium Kick-Off Meeting Rockville, MD October 23-24, 2011

2. 200720062005 2004 20082009201020112012 NCGC NTP Vision & Roadmap EPA NCCT NAS Report: Tox in the 21 st Century ToxCast II (~1000 cmpds x 550 assays) ToxCast I (~300 cmpds x ~550 assays) Tox21 MOU FDA joins Tox21 qHTS I (~2800 cmpds) qHTS II (10K cmpds) Tox21 - a “Community Resource” Project EPA’s Strategic Plan 2 The Tox21 Screening Timeline

3. Area of ExpertiseNIEHSNCGCEPA FDA Lab Animal Toxicology Human Toxicology/Exposure Assessment Ultra High Throughput Screening Low to Mid Throughput Assays Stem Cell Assay Development Epigenetic Assays Engineered Tissue Models ‘Omic Based Systems Lower Organism Systems Genetic Variability in Response Databases & Informatic Tools Validation Experience 3 Tox21 Partners

4. Tox21 Goals Identify patterns of compound-induced biological response in order to: − characterize toxicity/disease pathways − facilitate cross-species extrapolation − model low-dose extrapolation Prioritize compounds for more extensive toxicological evaluation Develop predictive models for biological response in humans 4

5. Agency Points of Contact Christopher Austin, M.D. (NCGC) Thomas Colatsky, Ph.D. (FDA) Robert Kavlock, Ph.D. (EPA) Raymond Tice, Ph.D. (NTP)  Identify toxicity pathways & corresponding assays  Review nominated assays  Prioritize assays for qHTS  Characterize assay output and evaluate assay performance  Develop prioritization schemes and prediction models  Make all data publicly accessible via CEBS, PubChem, ACToR  Evaluate the relevance of prioritization schemes and prediction models  Prioritize substances for more complex testing  Extrapolate in vitro conc to in vivo dose  Establish a 10K DMSO soluble compound library for qHTS  Establish QC procedures  Establish libraries of mixtures and aqueous soluble compounds for qHTS Informatics Working Group Co-Chairs Ruili Huang, Ph.D. (NCGC) Richard Judson, Ph.D. (EPA) Jennifer Fostel, Ph.D. (NIEHS) Weida Tong, Ph.D. (FDA) Chemical Selection Working Group Co-Chairs William Leister, Ph.D. (NCGC) Donna Mendrick, Ph.D. (FDA) Ann Richard, Ph.D. (EPA) Cynthia Smith, Ph.D. (NTP) Targeted Testing Working Group Co-Chairs Kevin Crofton, Ph.D. (EPA) Michael DeVito, Ph.D. (NTP) David Gerhold, Ph.D. (NCGC) James Weaver, Ph.D. (FDA) Assays & Pathways Working Group Co-Chairs Kevin Gaido, Ph.D. (FDA) Keith Houck, Ph.D. (EPA) Kristine Witt, M.S. (NTP) Menghang Xia, Ph.D. (NCGC) 5

6. Tox21 Phase I – Proof of Principle NCGC screened 1408 compounds (1353 unique) from NTP and 1462 compounds (1384 unique) from EPA in >100 qHTS at 14 conc ( 5 nM to 92  M typical). EPA via ToxCast™ screened 320 compounds (309 unique, primarily pesticide actives and some endocrine active compounds) in ~550 assays. Data released to the scientific community via: − EPA ACToR (Aggregated Computational Toxicology Resource; http://epa.gov/actor) http://epa.gov/actor − NLM PubChem (http://pubchem.ncbi.nlm.nih.gov/)http://pubchem.ncbi.nlm.nih.gov/ − NTP CEBS (Chemical Effects in Biological Systems; http://www.niehs.nih.gov/research/resources/databases/cebs/index.cfm) 6

7. ToxCast 1.0 (April, 2007) Enzyme inhibition/receptor binding HTS (Novascreen) NR/transcription factors (Attagene, NCGC) Cellular impedance (ACEA) Complex cell interactions (BioSeek) Hepatocelluar HCS (Cellumen) Hepatic, renal and airway cytotoxicity (IVAL) In vitro hepatogenomics (IVAL, Expression Analysis) Zebrafish developmental toxicity (Phylonix) ToxCast 1.1 (January, 2008) Neurite outgrowth HCS (NHEERL) Cell proliferation (NHEERL) Zebrafish developmental toxicity (NHEERL) ToxCast 1.2 (June, 2008) NR Activation and translocation (CellzDirect) HTS Genotoxicity (Gentronix) Organ toxicity; dosimetry (Hamner Institutes) Toxicity and signaling pathways (Invitrogen) C. elegans WormTox (NIEHS) Gene markers from microscale cultured hepatocytes (MIT) 3D Cellular microarray with metabolism (Solidus) Zebrafish vascular/cardiotoxicity (Zygogen) HTS stress response (NHEERL+NCGC) 7 ToxCast TM Phase I Testing

8. Phenotypic readouts − Cytotoxicity − Apoptosis: caspase 3/7, 8, 9 − Membrane integrity: LDH, protease release − Mitochondrial toxicity (membrane potential) − Genetox: p53, ELG1, DNA damage gene deficient lines (DT40 lines and mouse) Cell Signaling − Stress response: ARE, ESRE, HSP, Hypoxia, AP-1 − Immune response: IL-8, TNF , TTP − Other: AP-1, CRE, ERK, HRE, JNK3, NFkB, LDR Epigenetics − Locus DeRepression (LDR) Drug metabolism − CYP1A2, CYP2C19, CYP2C9, CYP2D6, CYP3A4 Target specific assays − Nuclear receptors: AR, AhR, ER , FXR, GR, LXR, PPAR , PPARδ, PPARγ, PXR, RXR, TRβ, VDR, ROR , RORγ − hERG channel − Isolated molecular targets: 12hLO, 15hLO1, 15hLO2, ALDH1A1, HADH560, HPGD, HSD17b4, APE1, TDP1, DNA polymerase III, RECQ1 helicase, RGS4, BRCA, IMPase, O-Glc NAc Transferase, Caspase-1/7, CBFβ-RUNX1, PK, Tau, Cruzain, β-Lactamase, PRX, YjeE, NPS, Proteasome, SF1, SMN2, beta-globin splicing, Anthrax Lethal Factor, TSHR Genetic variation: 87 HapMap CEPH Panel Phase I NCGC qHTS Assays 8

9. Differential Compound Toxicity Among 13 Cell Types Xia et al., EHP 116:284, 2008 IC50 Values HHMHHHRMHHHHR 9

10. Tox21 Phase II EPA’s ToxCast™ Phase II: ~1000 compounds in ~550 assays. NCGC qHTS Phase II: >10K compounds 3x at 14 conc for: − nuclear receptor activation or inhibition (AR, AhR, ER, FXR, GR, LXR, PPAR, PXR, RXR, TR, VDR, ROR) − induction of stress response pathways (e.g., DNA damage, heat shock, hypoxia, inflammation, oxidative) Assay selection based on − Information from in vivo toxicological investigations − Phase I experience, advice of basic researchers, and nominated assays − Maps of disease-associated cellular pathways Future focus on disease-associated pathways (e.g., obesity/diabetes, autism) using stem cells/differentiated cells and high throughput gene array assays 10

11. Tox21 Phase II qHTS 10K Library NCGC Drugs Drug-like compounds Active pharmaceutical ingredients EPA ToxCast I and II compounds Antimicrobial Registration Program Endocrine Disruptor Screening Program OECD Molecular Screening Working Group List FDA Drug Induced Liver Injury Project Failed Drugs NTP NTP-studied compounds NTP nominations and related compounds NICEATM/ICCVAM validation reference compounds for regulatory tests External collaborators (e.g., Silent Spring Institute, U.S. Army Public Health Command) Formulated mixtures 11

12. The Tox21 Genomes Project (with I. Rusyn, UNC) Assessment of variation within and between populations Mapping of genomic regions associated with variation of responses to individual chemicals or classes In a cell-based system, with carefully controlled growth and environmental conditions, the assay may serve as an endo-phenotype, with a greater proportion of variation explained by genomic variation than for a typical complex trait http://en.wikipedia.org/wiki/1000_Geno mes_Project Status: Phase I – 87 CEPH panel x 240 cmpds x 12 conc x 2 assays (cytotox & caspase 3/7) Phase II – 1090 lines (9 racial groups) x 180 cmpds x 8 conc x 1 assay (cytotox) 12

13. The NTP DrugMatrix Rat Toxicogenomics Database Integrated Collection of Data –637 unique chemicals (mostly drugs) –5600 drug-treatment transcript profiles in rat organs –127,000 histopathology measurements –100,000 blood chemistry measurements –60,000 literature facts Over 500 validated signatures –Mode of action and pathology Comprehensive data mining –Formulate 100,000’s questions (phenotypes) –Test for ability to classify using transcript data only ~122,000 frozen tissues Automated genomics analysis Steatotic Non-steatotic 13 Drugmatrix website: https://ntp.niehs.nih.gov/drugmatrix ToxFx website: https://ntp.niehs.nih.gov/toxfx/https://ntp.niehs.nih.gov/toxfx/

14. The NCGC Universe of Human Pathways ~1100 human pathways mapped to the pathway globe Detailed view of a pathway Gene information Pathways 14

15. Development of an Integrated Prediction System Collaboration between NIEHS, Leadscope Inc., Lhasa Limited, and MultiCASE Inc. System to support the prioritization of chemicals through human-relevant toxicity predictions Designed for use by scientists with different backgrounds Brings together toxicity data and predictions from multiple geographically distributed locations − qHTS data from the Tox21 project − Historical in vitro and in vivo data − (Q)SAR models for human adverse event endpoints as well as in vitro and in vivo endpoints

16. NTP Workshop: Role of Environmental Chemicals in the Development of Diabetes and Obesity January 11-13, 2011 Michael Gallo, Workshop Chair Dept. of Environmental & Occupational Health, University of Medicine & Dentistry of New Jersey Kristina Thayer, Director NTP Office of Health Assessment and Translation http://cerhr.niehs.nih.gov/evals/diabe tesobesity/ 16 Identifying Disease Pathways

17. Well-characterized chemical libraries (identity, purity, concentration, stability) Well-characterized assays in terms of reliability and relevance Ability to incorporate xenobiotic metabolism Informatic tools to integrate and mine robust data from multiple sources Understanding the relationships between pathways and disease in animal models and humans Making the data freely accessible as quickly as possible Scientific outreach and training the next generation Success depends on: 17