EPA ToxCast HTS Assays and Predictive M d l f E t A d Th id dModels for Estrogen, Androgen, Thyroid and

Steroidogenesis PathwaysD id DiDavid Dix Acting DirectorNational Center for Computational Toxicology

April 24, 2013RTP, NC

Office of Research and Development The views expressed in this presentation are those of the author and do not necessarily reflect the views or policies of the U.S. EPA

CompTox Goals

• Identify targets or pathways linked to toxicity• Develop high throughput assays for these targets or pathways• Develop high throughput assays for these targets or pathways• Develop predictive systems models

– in vitro → in vivo–in vitro → in silico

• Use predictive models:–Prioritize chemicals for targeted testing –Suggest / distinguish possible AOP / MOA for chemicals

• High throughput exposure predictions • High Throughput Risk Assessments

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Toxicity Forecaster (ToxCast)

• U.S.EPA research program profiling over 2,000 chemicals across >700 in vitro assays. http://www.epa.gov/ncct/toxcast/

– Phase-I: 309 data-rich chemicals (primarily pesticides) having over 30 years of traditional animal studies valued at $2B; in vitro signaturesyears of traditional animal studies valued at $2B; in vitro signatures defined by how well they can predict toxicity in the animal studies.

Phase II 776 chemicals from a broad range of so rces (e g ind strial– Phase-II: 776 chemicals from a broad range of sources (e.g., industrial and consumer products, food additives, failed drugs) to extend and apply first generation predictive models of toxicity.

– Phase-III: 1001 chemicals in a subset of assays, follow-up targeted testing

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ToxCast & Tox21 Chemical Inventories

ToxCastAvailable for download at: http://www.epa.gov/ncct/dsstox/

600eI

Iys eI

Large coverage of:• chemical structure• phys chem

Large coverage of:• chemical structure• phys chem

Phas

e

# A

ssay

Phas

e • phys. chem. property• use-category space

• phys. chem. property• use-category space

TOX21EDSP21

• toxicity AOPs• toxicity AOPs60

TOX21

83001060

Pesticides, cosmetics and personal care products, fragrances, antimicrobials,

# Cmpds311 1860

Office of Research and DevelopmentNational Center for Computational Toxicology

Pesticides, cosmetics and personal care products, fragrances, antimicrobials, food additives, failed drugs, chemicals of concern & green alternatives, industrial HPV & MPV, reference compounds (endocrine, repro/devtox, etc.) 4

ToxCastDB: 700+ HTS Assays

Assay ProviderACEA

Apredica Assay DesignBiological Responsecell proliferation and death

ll diff i i

Target FamilyResponse Element

TransporterCytokinesAttagene

BioSeekCellzDirectNCGC/Tox21NHEERL MESC

NHEERL NeuroTox

viability reportermorphology reporterconformation reporter

enzyme reportermembrane potential reporter

bi di t

cell differentiationmitochondrial depolarization

protein stabilizationoxidative phosphorylationreporter gene activationgene expression (qNPA)

CytokinesKinases

Nuclear ReceptorCYP450 / ADMECholinesterasePhosphatasesNHEERL NeuroTox

NHEERL ZebrafishNovaScreen

Odyssey Thera

binding reporterinducible reporter

gene expression (qNPA)receptor activityreceptor binding

pProteases

XME metabolismGPCRs

Ion Channels

SpeciesHumanRat

MouseZebrafish

Detection TechnologyqNPA and ELISA

Fluorescence & LuminescenceAlamar Blue Reduction 

Readout TypeSingle

MultiplexedMultiparametric

Tissue SourceLung              BreastLiver           VascularSkin              KidneyC i T tiZebrafish

SheepBoarRabbitCattle

Guinea pig

Cell FormatCell free Cell lines

Primary cellsComplex cultures

a a ue educt oArrasyscan / MicroscopyReporter gene activation

Spectrophotometry RadioactivityHPLC and HPEC

Cervix             TestisUterus            BrainIntestinal        SpleenBladder             OvaryPancreas        ProstateInflammatory Bone

Office of Research and DevelopmentNational Center for Computational Toxicology

p gpFree‐living embryos TR‐FRET

Inflammatory     Bone

5(http://actor.epa.gov/actor/faces/ToxCastDB)

ToxCast and Tox21Chemicals, Data and Release Timelines,

Set Chemicals Assays Endpoints Completion Available

ToxCast Phase I 293 600 1100 2011 NowToxCast Phase I 293 ~600 ~1100 2011 Now

ToxCast Phase II 767 ~600 ~1100 03/2013 09/2013

ToxCast Phase IIIa 1001 ~100 ~100 Just starting 2014

E1K (endocrine) 880 ~50 ~120 03/2013 09/2013

Tox21 8,193 ~25 ~50 Ongoing Ongoing

ys

~600

Ass

ay

0Office of Research and DevelopmentNational Center for Computational Toxicology 6

Chemicals ~8,2000

ToxCast Data Analysis -Overview

• Amount and complexity of data requires robust informatics process

Data ~15 labs in multiple formats

Data Pipeline

R D t

Overview

• Data ~15 labs in multiple formats• Multiple normalization, background subtraction, outlier detection methods need to be used

• Total ToxCast data set is 27M data points >1 M

Raw Data

Corrected

• Total ToxCast data set is 27M data points, >1 M chemical‐assay concentration response curves

• Data Workflow8 L l l i

Normalized

Modeled

• 8‐Level analysis process• Assays treated similarly after normalization • Results are converted to common format• Data is stored in Toxminer/ToxCastDB

Summarized

Meta‐AnalyzedData is stored in Toxminer/ToxCastDB

• All data and software will be made publicly available during 2013

Stored

Presented

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1 2 3 4Data Pipeline, Curve Fits, Hit Calling

1 2 3 4Pipeline - takes raw data files to consistent formats for curve fitting, includes: • standard file formatting • automated chemical and assay mapping,• provides plate/batch effect correctionData is never removed, only filtered or• provides plate/batch effect correction,• assay specific data normalization

Curve Fits & Hit Calling includes filtering data: • Cytotoxicity and outlier detection masking

Data is never removed, only filtered or ‘tagged” in the datasets

Transparency is a core tenet y y g• Data confounder detection (e.g., fluorescence or non-specific activity)• Final manual calls • assay specific data normalization

All data and analysis programming code is publicly available

Curve-fittingHit-calling

SystematicAlterations

ManualAlterations

Outlier DetectionData Correction

5X

X

Office of Research and DevelopmentNational Center for Computational Toxicology

8

56 7 8

Approximate geographic distribution of current ToxCast contract assay providersy p

9dix.david@epa.gov

Current NCCT ToxCast external dcontract assay providers

• OdysseyThera 1 QMP 18 SOPsy y• Vala Sciences 1 QMP 16 SOPs• BioReliance 1 QMP 15 SOPs• CeeTox 1 QMP 28 SOPs• LifeTechnologies 1 QMP 13 SOPs

E i A l i 1 QMP 72 SOP• ExpressionAnalysis 1 QMP 72 SOPs• BIOSEEK 1 QMP 39 SOPs• Attagene 1 QMP 19 SOPsAttagene 1 QMP 19 SOPs• ACEA 1 QMP 20 SOPs• Perkin Elmer 1 QMP >200 SOPs

10dix.david@epa.gov

Hazard Predictions

ToxCast/Tox21 methods beginning to bear fruit on predicting hazard (Martin et al

Discrete chemicals(26,379 structures)

predicting hazard (Martin et al. 2011, Sipes et al. 2011, Kleinstreuer et al. 2012)

HTS Chemical Library(8,126 Structures)

Chemicals w/o HTS that have no structural similarity

Structural neighbors to

Structure Clustering

Methods exist for approximately converting in vitro results to daily doses needed to produce similar levels in a human (Rotroff

( , )

Exposure-only prioritization

y(N2)

AOP-targeted HTS Data

gHTS library (N1)

similar levels in a human (Rotroffet al. 2010, Wetmore et al. 2012)

We have ToxCast/Tox21 data for 

Active chemicals and structural neighbors

(N3)

Inactive chemicals and structural

neighbors (N4)

thousands of chemicals – use chemical features to relate tens of thousands of other chemicals to these data High, Medium, Low

i it biVery Low priority bin

Detailed Exposure Evaluation

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to these data priority bins

Detailed Pathway Modeling

• Focus on a single pathway–Estrogen Receptor is an exampleEstrogen Receptor is an example

• Use multiple assays to get complete picture of chemical action

• Different assays tell different stories about the pathway• No assay is perfect – balance strengths and weaknessesweaknesses

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Pro‐ligand

Using multiple lines of evidence to test for ER activity

Odyssey Thera and A

ERActive ligand

Novascreen

Attagene assays have metabolic capacity

Non‐ligand‐mediatedti ti f

Odyssey Thera

Oxidative stress

pathways

Non‐ER‐mediatedcell proliferation

pathways

activation of ER activity

Cofactor

p y

Odyssey

ER‐regulated gene expression

Odyssey Thera

Office of Research and DevelopmentNational Center for Computational Toxicology

Cell proliferation

Attagene AttageneNCGC ACEA 13

R3A1

A2

Receptor (Direct Molecular Interaction)

Intermediate Process

ER Pathway Model

ER Receptor Binding(Agonist)

R1

A3

A4

3

Intermediate Process

Assay

Noise ProcessR2ER Receptor 

Binding(Antagonist)

R4

DimerizationN1

A5

A6

A7

ER agonist pathway

Interference pathway

ER antagonist pathway

N7Dimerization

R4

CofactorRecruitment

R5

N2

A7

A8

A9

N8CofactorRecruitment

DNA Binding

RNA Transcription

R5

R6N3

N4

A10

A12

A13

A11

N9DNA Binding

R9Transcription

Protein Production

R7

N5

A13

A14

A15

A17

A18N10

AntagonistTranscriptionSuppression

ER‐inducedProliferation

R8N6

A15

A16 14dix.david@epa.gov

36 Reference Chemicals: ER

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36 Reference Chemicals: ER

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High Agonist  Activity Active‐like StructureBin I – Blue (81)

High Agonist  Activity NOT Active‐like Structure

Bin IV – Blue (13)

13 0 81

ER PathwayChemical Prioritization

POSITIVE (81)St

Novel Active Class

13 81Chemical Prioritization

NEGATIVE‐BAI (186)Broad Spectrum Pathway or Assay 

Interference

Intermediate Agonist  Activity

Active‐like Structure Bin II (Gray) (54)

Significant Alternate Receptor Activity 

Active‐like StructureBin II, III – Cyan, Red (60)

Strong

34

2031

29

POSITIVE‐W/S (113)Significant Alternate Receptor Activity 

Intermediate Agonist  Activity

NOT Active‐likeNovel Active

Novel Active

3983

29

Significant Assay ActivityActive‐like Structure

Weak  / SERM

Significant Assay ActivityNOT Active‐like Structure

NOT Active‐like StructureBin V,VI – Cyan, Red (86)

NOT Active like Structure 

Bin V (Gray) (39)

Active Class

Active Class 03

47Active like StructureBin II, III – Yellow (145)

No Activity Active‐like StructureBin III – Gray (382)

No Activity NOT Active‐like Structure

Bin VI – Gray (720)

NOT Active like StructureBin V,VI – Yellow (190)

190 98

382 720NEGATIVE‐NAI (288)Narrow Spectrum Assay Interference

NEGATIVE (1102)Total 1770

382 720

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Active

One assay

Multiple assays

Multiple Technologies

Single Technology

Activity Pattern & Chemical Class Information

Broad Spectrum Pathway or Assay 

Interference

Weak True Positive or SERM

Narrow Spectrum Assay Interference

Or Noise

Strong True Positive

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ER Agonist Scores for 1770 ToxCast Chemicals

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Bisphenol A: ER Results

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Vinclozolin: AR Results

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AdverseOutcomesTRH R

Hypothalamus

TR3

1

Hypothalamic Pituitary

TSH ThyroidHyperplasia

ThyroidTumors

Outcomes

4Thyroid Gland

Pituitary

TSH RBlood

2

Hypothalamic Pituitary Feedback 

Thyroperoxidase

NIS

Thyroid Receptors

FreeT3 & T4T4 & T3

Synthesis

4

35Altered

M t b li

Hepatic Nuclear

TissueTH Changes

Phase 2Catabolism

9

6

8

Bound TransportProteins Cellular TR

Signaling

Metabolism

Hepatic NuclearXenoreceptors

CellularTransporters

BiliaryElimination10 Altered

Development

DeiodinasesNeurologicalDevelopment

CellularT4 T3

Conversion AmphibianMetamorphosis

7

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Steroidogenesis: H295R Assayg y• Chemical exposures done in 96-well format (CeeTox)

10uM forskolin-stimulated selected based on pilot study0 1% DMSO0.1% DMSO100mM stock-solution (new to toxcast; enables 100uM top concentration)MTT cytotoxicity assay performed iteratively in duplicate starting at 100uM & reducing 10 fold until viability >80%starting at 100uM & reducing 10-fold until viability >80%Viable samples sent for steroid analysis

• Able to detect 13 of the steroid hormones in the steroidogenic pathway using multiplexed MS (OpAns)ste o doge c pat ay us g u t p e ed S (Op s)

• Current plans for ToxCast–Run at single concentration across all ~2100 ToxCast

chemicals as an initial screen (in duplicate)Run in concentration response format (1/3rd of all chemicals)–Run in concentration response format (1/3rd of all chemicals)

–Single-screen completed/near-completed for ToxCast Phase I and II Chemicals

–Concentration-response follow-up performed on Phase I chemicals

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chemicals

Key Messages

• Significant progress has been made in high throughput screening thousands of chemicals for potential endocrine h dhazard

• Predictive toxicology and systems models using a combination of biology chemistry and statistics arecombination of biology, chemistry and statistics are required for risk characterization

• Initial models point the way to real-world applications– example EDSP21

• Further research needed:–More chemicals assays pathways–More chemicals, assays, pathways–Systems-level models for hazard and exposure–Targeted testing approaches to follow up on prioritizations

Office of Research and DevelopmentNational Center for Computational Toxicology

– High Throughput Risk Assessment24

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