What Can ToxCast Tell Us About Endocrine Disruption?

1. What Can ToxCast Tell Us About Endocrine Disruption? Keith Houck, PhD ISRPT 2009 Endocrine Workshop 9-10 Sept 2009 This work was reviewed by EPA and approved for presentation but does not necessarily reflect official Agency policy. Mention of trade names or commercial products does not constitute endorsement or recommendation by EPA for use.

2. What Can ToxCast Tell Us About Endocrine Disruption? • Probably not much but our focus is chemical prioritization for targeted testing

3. 90,000 11,000 Millions $ Our Challenge: Too Many Chemicals Too High a Cost Cancer DevTox NeuroTox ReproTox ImmunoTox PulmonaryTox …and not enough data. 2 Judson, et al EHP 117(5):685-95 (2009)

4. ToxCastTM Background • Research program of EPA’s National Center for Computational Toxicology • Addresses chemical screening and prioritization needs for pesticidal inerts, anti-microbials, CCLs, HPVs and MPVs • Comprehensive use of HTS technologies to generate biological fingerprints and predictive signatures • Coordinated with NTP and NHGRI/NCGC via Tox21 • Committed to stakeholder involvement and public release of data • Communities of Practice- Chemical Prioritization; Exposure • NCCT website- http://www.epa.gov/ncct/toxcast • ACToR- Aggregated Computational Toxicology Resource • http://www.epa.gov/actor/

5. Acetylcholine esterase inhibitors conazole fungicides Sodium channel modulators pyrethroid ester insecticides organothiophosphate acaricides dinitroaniline herbicides pyridine herbicides thiocarbamate herbicides imidazolinone herbicides organophosphate insecticides phenyl organothiophosphate insecticides aliphatic organothiophosphate insecticides amide herbicides aromatic fungicides chloroacetanilide herbicides chlorotriazine herbicides growth inhibitors organophosphate acaricides oxime carbamate insecticides phenylurea herbicides pyrethroid ester acaricides strobilurin fungicides unclassified acaricides unclassified herbicides ToxCast_320 Phase I Chemicals 309 unique structures Replicates for QC 8 metabolites 291 total pesticide actives 273 registered pesticide actives 22 pesticide inerts 33 antimicrobials 54 Tier 1 EDSP 23 IUR 13 HPV 73 OW PCCL 11 CCL1 10 CCL2 25 CCL3 MOA Classes with > 3 chemicals Misc

6. EDSP Chemicals in ToxCast 320 Select Other Chemicals in ToxCast 320

7. Types of Endocrine Assays Tested In ToxCast • Radioligand Receptor Binding (NRs) • Single conc at 25 mM followed by 7-point concentration response for actives • Multiplexed Reporter Gene Assay (Attagene) • Trans: GAL4-Ligand Binding Domain NR Assays • Cis: NR Response Element Assays • 5-point concentration-response up to 100 mM (unless limited by cytotoxicity) • Nuclear Receptor Coactivator Recruitment (NRs) • Single conc at 25 mM followed by 7-point concentration response for actives • CYP Enzyme Activity Assays • Single conc at 10 mM followed by 7-point concentration response for actives • NR Target Gene Assays (in primary human hepatocytes) • 5-point concentration-response up to 40 mM

8. Endocrine Related Assays

9. Other Endocrine Related Assays

10. Other Endocrine Related Assays

11. Example NR Binding Assay Actives hERa hAR hTRa

12. Overview of Assay Results • Generally found expected actives for ERa and AR binding assays • Little activity for Thyroid Receptor • Some species and/or assay sensitivity differences seen, e.g. low sensitivity of rat AR binding assay • Not always agreement between binding and cellular reporter assays • Many CYP inhibitors including those from expected chemical classes, i.e. conazoles and organophosphates • Potency mostly in the micromolar to tens of micromolar range; few nanomolar actives

13. Tox21 • Tripartite Collaboration: EPA/NTP/NCGC • Quantitative HTS Assays at NCGC • Accepting Nominations for Assays from All Sources • Chemical Libraries Being Screened: • EPA Library of 1462 Substances • Contains Toxcast 309 • Contains many NR reference compounds, e.g. pharmacological tools and drugs • Many different environmental chemicals, e.g. bisphenols, phthalates, PFAAs • 1408 substances in NTP library also screened • Expanding to 10,000 this year including all human pharmaceuticals

14. Tox21 Assays Nuclear Receptor Cellular Reporter Gene Assays • GAL4-Ligand Binding Domain System--principally ligand detection • Receptors screened in agonist and antagonist modes • 11-15 concentrations (highest 92 mM) • antagonist mode data interpretation confounded by cytotoxicity • Receptors screened: • Human • ERa, AR, TRa, VDR, MR, RXRa, PPARa, PPARb, PPARg, PXR, (AhR) • Rat • PXR

15. Tox21 Results EPA Library Actives: Agonists

16. Tox21 EDSP Chemicals Agonist (All)

17. Tox21 EPA Library Antagonist Actives

18. Partial Agonist Examples: ERa

19. Importance of Biotransformation ERa radioligand binding assay ERa cellular (HEK293) transactivation assay

20. In vivo/In vitro Correlation Physical chemical properties In silico Predictions Cellular Assays Data Normalization Biochemical Assays 19 Toxicology Endpoints Genomic Signatures

21. ToxRefDB website: http://www.epa.gov/ncct/toxrefdb/

22. Reproductive and Endocrine Organ Toxicity Endpoints from ToxRefDB

23. Using the Data for Prioritization In vitro assays (ToxCast) Chemical properties (descriptors) In vivo endpoints (ToxRefDB) Pathways (endocrine)

24. Bisphenol A Fenitrothion Symclosene Using the Data for Prioritization Perfluorooctane sulfonic acid Linuron

25. Summary of Preliminary Findings • Suite of assays useful in prioritization • endocrine-active chemicals score higher across assays • Individual assays may not be sufficient on their own • Moderate concordance across different assay types for same target • Differences in biotransformation between assays • Assay-specific false positives/false negatives • Error • Differences in biology we don’t understand • Sensitivity range of different assay types presents a challenge to interpret biological significance

26. NCCT/ORD/USEPA Matt Martin David Dix Richard Judson David Reif Ann Richards Robert Kavlock NCGC/NIH Menghang Xia Ruili Huang Chris Austin ACKNOWLEDGEMENTS: