U.S. Environmental Protection Agency Endocrine Disruptor Screening Program

Transcription

1 U.S. Environmental Protection Agency Endocrine Disruptor Screening Program Using High Throughput Assays and Computational Tools for Endocrine Screening Patience Browne, Ph.D. Senior Scientist, Office of Science Coordination and Policy Office of Chemical Safety and Pollution Prevention *does not necessarily reflect EPA policy September 18, 2015

2 Screening Tier 1 In vitro Tier 1 Screening Battery ER Binding Endocrine Pathway E + E A + A HPG Axis HPT Axis ERα Transcriptional Activation* AR Binding Steroidogenesis H295R* Aromatase Recombinant In vivo Uterotrophic* Hershberger* Pubertal Male Pubertal Female Amphibian Metamorphosis* Fish Short term Reproduction (male & female)* *OECD harmonized guidelines

3 Evolution of EDSP EDSP List Chemicals EDSP Chemical Universe 10,000 chemicals (FIFRA & SDWA) EDSP List 1 67 Chemicals Based on current pace it could take decades to screen all 10,000 chemicals in EDSP Universe Use predictive models and high throughput assasy to rapidly screen chemicals for potential bioactivity and exposure

4 EDSP Goals Use computational tools and models in the EDSP framework to: 1. Contribute to the weight of evidence evaluation of a chemical s potential bioactivity 2. Provide alternative data for specific endpoints in the EDSP Tier 1 battery Ultimately, these goals are common to the estrogen, androgen and thyroid pathways, however, estrogen bioactivity is the most mature model and is used to demonstrate the proposed approach. 4

5 Estrogen Receptor Bioactivity Model ER bioactivity model 18 HTS assays Detect receptor interaction at various points along signaling pathway Use a variety of technologies Capable of distinguishing true activity from cytotoxicity Values range from 0 to 1 ER agonists/antagonists

6 EDSP Tier 1 ER Agonist Assays vs. ToxCast ER Agonist Assays EDSP Tier 1 assays ToxCast High Throughput Screening ER assays Assay Name Biological Process Organism Tissue Assay Name Biological Process Organism Tissue NVS_NR_bER receptor binding bovine uterus RUC ER binding receptor binding rat uterus NVS_NR_hER receptor binding human NA NVS_NR_mERa receptor binding mouse NA OT_ER_ERaERa_0480 protein complementation human kidney OT_ER_ERaERa_1440 protein complementation human kidney OT_ER_ERaERb_0480 protein complementation human kidney OT_ER_ERaERb_1440 protein complementation human kidney OT_ER_ERbERb_0480 protein complementation human kidney OT_ER_ERbERb_1440 protein complementation human kidney OT_ERa_EREGFP_0120 gene expression human cervix OT_ERa_EREGFP_0480 gene expression human cervix ATG_ERa_TRANS_up mrna induction human liver ERTA gene expression human Ovary ATG_ERE_CIS_up mrna induction human liver Tox21_ERa_BLA_Agonist_ratio gene expression human kidney Uterotrophic Cell proliferation/ Rodent uterus Tox21_ERa_LUC_BG1_Agonist gene expression human ovary organ response ACEA_T47D_80hr_Positive cell proliferation human breast ER Agonism only ER Model (agonism and antagonism)

7 Judson et al Tox Sci Integrated Model of Chemical Perturbations of a Biological Pathway Using 18 In Vitro High Throughput Screening Assays for the Estrogen Receptor"

8 Kleinstreuer et al. 2015; EHP A Curated Database of Rodent Uterotrophic Bioactivity"

9 Browne et al. 2015; ES&T

10 Federal Register Notice, June 2015 EPA Solicits Comments on Use of High Throughput Assays and Computational Tools in Endocrine Disruptor Screening Program HQ OPPT g EPA is planning to incorporate scientific advancements and new tools incorporating validated ld dhigh throughput h h h assays and a computational model as an alternative for some of the current assays in the EDSP Tier 1 battery. Currently, EPA has partial screening results for over 1,800 chemicals that have been evaluated using the high throughput assays and computational model for the estrogen receptor pathway.

11 Performance Based Approach to Establish Scientific Confidence Reference eee cechemical ca set that includes a range geof structures and potencies that are accurately detected in vitro reference chemicals In vivo reference chemicals New methods compared with ihcurrent methods Bioactivity model versus Tier 1 results Evaluated by independent, external peer review FIFRA Scientific Advisory Panel meetings

12 ER bioactivity model: in vitro reference chemicals 40 in vitro ER reference chemicals with independently confirmed activity (OECD 2012) # True Pos 26 (25) # True Neg 11 (11) # False Pos 1 (0) # False Neg 2 (2) Accuracy 0.93 (0.95) Sensitivity 0.93 (0.93) Specificity i 092(10) 0.92 (1.0) Browne et al. 2015, ES&T Judson et al. 2015, Tox Sci

13 ER bioactivity model: in vivo reference chemicals 43 in vivo ER reference chemicals with independently confirmed activity (Kleinstreuer et al. 2015) Active >1 POS result Inactive >1 NEG result and no POS result equivocal > 1 POS result and >1 NEG result # True Pos 29 (29) # True Neg 8 (8) # False Pos 5 (1) # False Neg 1 (1) Accuracy 0.86 (0.95) Sensitivity 0.97 (0.97) Specificity 0.67 (0.89) Browne et al. 2015, ES&T Kleinstreuer et al., 2015, EHP

14 ER bioactivity model versus Tier 1 ER model performs as well or better than existing methods Model evaluated with 45 reference chemicals T1 ER binding: 23 (35% were not were not consistent with expected outcome) T1 ERTA: 12 T1 UT: 7 ER model in 100% agreement with Tier 1 ER, ERTA, and Uterotrophic results for List 1 chemicals (very low or no ER activity) ERmodel may be more sensitive than Tier 1 assays due to redundancy

15 ER Agonist Activity

16 Path Forward Determine how well existing models predict intact animal results Comparison to other Tier 1 endpoints Additional Tier 1 assay substitution? Use additional computational tti ltools to develop Adverse Outcome Pathway models for estrogen, androgen, and thyroid hormones Integrate more assays Integrate more key events Expand reference chemicals with defined potencies for performance based test guidelines incorporating computational tools Curation of high quality in vivo data from peer reviewed literature

17 EDSP Tier 1 AR Assays vs. ToxCast AR Assays EDSP Tier 1 assays Assay Name Biological Process Organism Tissue RPC AR binding receptor binding rat prostate Hershberger organ response Rodent AST ToxCast High Throughput Screening ER assays Assay Name Biological Process Organism Tissue NVS_NR_hAR receptor binding human NA NVS_NR_rAR receptor binding rat prostate OT_AR_ARSRC1_0480 protein stabilization human kidney OT_AR_ARSRC1_0960 protein stabilization human kidney ATG_AR_TRANS_up transcription factor human liver Tox21_AR_BLA_Agonist_ratio gene expression human kidney Tox21_AR_LUC_MDAKB2_Agonist gene expression human breast Tox21_AR_BLA_Antagonist_ratio gene expression human kidney Tox21_AR_LUC_MDAKB2_Antagonist gene expression human breast AR Agonism/Antagonism AR Model (agonistm/antagonism) i

18 Evolution of Screening and Testing in the EDSP EDSP Tier 1 Battery of Assays High Throughput Assays and Computational (current) Model Tier 1 Battery Alternatives Estrogen Receptor (ER) Binding ER Model (alternative) Estrogen Receptor Transactivation (ERTA) ER Model (alternative) Uterotrophic ER Model (alternative) Androgen Receptor (AR) Binding AR Model (Future) Hershberger AR Model (Future) Aromatase STR Model (Future) Steroidogenesis i (STR) STR Model l(future) Female Rat Pubertal ER, STR, THY Models (Future) Male Rat Pubertal AR, STR, THY Models (Future) Fish Short Term Reproduction ER, AR, STR Models (Future) Amphibian Metamorphosis THY Model (Future) EDSP Tier 2 Tests High Throughput Assays and Computational Model Tier 2 Battery Alternatives Rat 2 gen/eogrt ER, AR, STR, THY(Future) MEOGRT ER, AR, STR (Future) LAGDA THY (Future) Quail ER, AR, STR, THY (Future) ER = estrogen receptor; AR = androgen receptor; STR = steroidogenesis; THY = thyroid

19 Metabolism Metabolite prediction software Generation of likely metabolites for five human Phase I biotransformation ti cytochrome P450 enzymes (cyps) Parent chemicals run through metabolism predictor software Phase I metabolism only CYP dependent Parent metabolite pairs identified in literature used to validate Parent + 1 o metabolites + 2 o metabolites run through ER QSAR List QSAR models trained on 1800 ToxCast ER results Reference chemicals of parent compounds with known estrogenic metabolites used to validate Bioactivity of parent metabolite pairs evaluated QSAR results from several models normalized and average Relative predicted estrogen agonist bioactivity examined Parent Chemical Primary Metabolite(s) Secondary Metabolite(s) X

20 QSAR ER model Prediction of Activity of Estrogenic Metabolites Parent compound Methoxychlor Mestranol Trans-stilbene Azobenzene Diphenyl Diphenylmethane 2,2-Diphenylpropane Permethrin Cypermethrin Benzo[a]pyrene Benz[a]anthracene Chrysene Fluorene Phenanthrene Pyrene Naphthalene Fluoranthene Benzophenone Benzophenone 3 Estrogenic Metabolite Mono-hydroxymethoxychlor HPTE 17α-ethynylestradiol Trans-4-hydroxystilbene Trans-4,4'-dihydroxystilbene 4-Hydroxyazobenzene 4-Hydroxydiphenyl 3-Hydroxydiphenyl 4,4'-Dihydroxydiphenyl 4-Hydroxydiphenylmethane 4,4'-Dihydroxydiphenylmethane 2-(4-Hydroxyphenyl)-2-phenylpropane 4,4'-Dihydroxydiphenylpropane 3-Phenoxybenzylalcohol 3-(4 -Hydroxyphenoxy)-benzyl alcohol 3-Phenoxybenzaldehyde 3-Phenoxybenzaldehyde 3-Hydroxybenzo[a]pyrene 9-Hydroxybenzo[a]pyrene 8-Hydroxybenzo[a]pyrene 7-Hydroxybenzo[a]pyrene 3-Hydroxybenz[a]anthracene 4-Hydroxybenz[a]anthracene 10-Hydroxybenz[a]anthracene 1-Hydroxychrysene 2-Hydroxychrysene 3-Hydroxychrysene 2-Hydroxyfluorene 2-Phenanthrol 9-Phenanthrol 1-Hydroxypyrene 1-Naphthol 2-Naphthol 3-Hydroxyfluoranthene 4-Hydroxybenzophenone 2,4-Dihydroxybenzophenone 2,3,4-Trihydroxybenzophenone Metabolites failed to be predicted are indicated in red

21 Summary Pivot to using high throughput and computational methods in EDSP Endocrine pathway models will continue to be revised and improved as more data are available (ER, AR, thyroid ) Provides bioactivity i i predictions i for thousands of chemicals Allows resources to be focused on chemicals more likely to have endocrine effects List 1 chemicals have limited estrogen and/or androgen receptor mediated bioactivity Prioritizeschemicalsbased onbioactivity (and exposure) Provides alternative to current Tier 1 screening Multi century project becomes multi year

22 Acknowledgements EPA OSCP Kristan Markey Caroline Pinto David Dix Jane Robbins EPA ORD Richard Judson Rusty Thomas John Wambaugh NIEHS/NICEATM Warren Casey Nicole Kleinstreuer (NICEATM/ILS)