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2007

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. The Tox21 Screening Timeline. Tox21 MOU. NTP Vision & Roadmap. FDA joins Tox21.

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2007

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

  2. The Tox21 Screening Timeline Tox21 MOU NTP Vision & Roadmap FDA joins Tox21 EPA’s Strategic Plan NAS Report: Tox in the 21st Century NCGC qHTS II (10K cmpds) qHTS I (~2800 cmpds) ToxCast I (~300 cmpds x ~550 assays) ToxCast II (~1000 cmpds x 550 assays) EPA NCCT 2005 2006 2007 2008 2009 2010 2011 2012 2004 Tox21 - a “Community Resource” Project 2

  3. Tox21 Partners 3

  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. 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) 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) 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) 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) 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 • Establish a 10K DMSO soluble compound library for qHTS • Establish QC procedures • Establish libraries of mixtures and aqueous soluble compounds 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 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) • NLM PubChem (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 1.2 (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) ToxCastTM Phase I Testing 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)

  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 IC50 Values H M H H H H R M H H H H R Xia et al., EHP 116:284, 2008 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 • 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 NCGC • Drugs • Drug-like compounds • Active pharmaceutical ingredients

  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_Genomes_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 • Drugmatrix website: https://ntp.niehs.nih.gov/drugmatrix • ToxFx website: https://ntp.niehs.nih.gov/toxfx/ 13

  14. Detailed view of a pathway The NCGC Universe of Human Pathways Pathways Gene information ~1100 human pathways mapped to the pathway globe 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. Identifying Disease Pathways 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/diabetesobesity/ 16

  17. Success depends on: • 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 17

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