EPA’s ToxCast Program for Predicting Toxicity and ...

May 14, 2008Office of Research and DevelopmentNational Center for Computational Toxicology

EPA’s ToxCast Program for Predicting Toxicity and Prioritizing Chemical Testing

http://www.epa.gov/ncct/toxcast

IJC SAB presentation

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.

1Office of Research and DevelopmentNational Center for Computational Toxicology

“…to integrate modern computing and information technology with molecular biology to improve Agency prioritization of data requirements and risk assessment of chemicals”

www.epa.gov/ncct


Too Many Chemicals Too High a Cost

Cancer

DevTox

NeuroTox

ReproTox

ImmunoTox

PulmonaryToxMillions $

The Problem

http://www.epa.gov/comptox/toxcast

1

10

100

1000

10000

100000

Data Collection

IRISTRIPesticide ActivesCCL 1&2Pesticide InertsHPVMPV CurrentMPV HistoricalTSCA Inventory

11,000

90,000

…and not enough data.


CancerReproToxDevTox

NeuroToxPulmonaryTox

ImmunoTox

Derive classifiers or signatures from hundreds of HTS, HCS and genomics assays to predict hazard…

Thousands $

… and use these toxicity predictions for prioritizing further testing of environmental chemicals.

The Solution: ToxCastTM

http://www.epa.gov/comptox/toxcast

Assays

End

poin

ts


ToxCast Website: www.epa.gov/ncct/toxcast


ToxCast Background

• Project of US EPA’s National Center for Computational Toxicology

• Formulated to address chemical screening and prioritization needs

• Screening approach based on experience of the pharmaceutical industry

• Comprehensive use of a broad range of current HTS technologies

• Phased approach to evaluate utility

• Committed to stakeholder involvement and publication of data


Phased Development of ToxCast

FY09$15-20k>400ValidationData Rich Chemicals>300IIa

FY09$15-20k>400ExtrapolationKnown Human Toxicants>100IIb

???

>400

>400

Number of Assays

Data poor

Expanded Structure and Use

Diversity

Data Rich(pesticides)

Chemical Criteria

FY11-12

FY10

FY07-08

TargetDate

$10-15k

$15-20k

$20k

Cost per Chemical

Prediction and Prioritization

Extension

Signature Development

PurposeNumber of Chemicals

Phase

ThousandsIII

>300IIc

320I

Affordable science-based system for categorizing chemicalsIncreasing confidence as database grows Identifies potential mechanisms of actionRefines and reduces animal use for hazard ID and risk assessment


Target Chemicals ~10,000

Chemicals with Toxicity Data(Training)

HTSCharacterization

Discover & Validate“Hazard Model”

ToxCast Phase I & II

Chemicals wo/Phenotype Data(Test)

Metabolite Prediction

HTSCharacterization

Apply“Hazard Model”

Mechanism-basedHazard Prediction

Prioritized Chemicals

ToxCast Phase II & III

ToxCast Screening and Prioritization


Relevance/ Cost/Complexity

Throughput/ Simplicity

High-Throughput Screening Assays

10s-100s/yr

10s-100s/day

1000s/day

10,000s-100,000s/day

LTS HTSMTS uHTS

batch testing of chemicals for pharmacological/toxicological endpoints using automated liquid handling, detectors, and data acquisition

Gene-expression


Biological Organization of ToxCast

Receptors& Enzymes

Biological Pathways

Cellular Processes

Tissue / Organ Endpoints

Chemicals

Direct Molecular Interactions


ToxCast Data Matrix:Predicting In Vivo from In Vitro

T1

Toxicity Endpoints

… TN

CN

…

C3

C2

C1

SN…S1CN…C1AN…A1PN…P1

Gene Expression Signatures

CellularAssays

In-vitro / Biochemical Assays

Physico-Chemical Properties

ToxCast320Chemicals

Chemical HTS HCS Genomics In Vivo

[320 chemicals] x [265 assays] x [multiple endpoints] x [multiple concentrations] = millions of datapoints

Che

mic

als

Assays


Key Components of ToxCastProof of Concept

• Chemicals

• Traditional Toxicity Phenotypes

• HTS Assays covering Toxicity Pathways

• Data Analysis and Interpretation


ToxCast_320: Phase I Chemicals

MOA Classes with > 3 chemicals

Misc MOA classes with3 or fewer representatives

Acetylcholine esterase inhibitorsconazole fungicidesSodium channel modulatorspyrethroid ester insecticidesorganothiophosphate acaricidesdinitroaniline herbicidespyridine herbicidesthiocarbamate herbicidesimidazolinone herbicidesorganophosphate insecticidesphenyl organothiophosphate insecticidesaliphatic organothiophosphate insecticidesamide herbicidesaromatic fungicideschloroacetanilide herbicideschlorotriazine herbicidesgrowth inhibitorsorganophosphate acaricidesoxime carbamate insecticidesphenylurea herbicidespyrethroid ester acaricidesstrobilurin fungicidesunclassified acaricidesunclassified herbicides

Misc

Classification based on OPPIN

309 Unique StructuresReplicates for QC

291 Pesticide Actives9 Industrial Chemicals8 Metabolites

56/73 Proposed Tier 1 EDSP

14 HPV11 HPV Challenge


Toxicology Reference Database: ToxRefDB

• ToxRefDB is being developed as part of EPA’s ToxCast research program (www.epa.gov/ncct/toxcast).

• ToxRefDB captures existing information from Office of Pesticide Program (OPP) Data Evaluation Records (DER) into an electronic database that supports ToxCast and other analyses.

• DER are OPP's treatment-related effects summaries, based on the toxicology studies submitted by pesticide registrants.


In Vivo Chronic/Cancer Bioassay Effects and Endpoints in ToxRefDB

Effects & EndpointsTo

xCas

t Pha

se I

Che

mic

als


Common Phenotypes in Chronic Rodent Studies


The Home of TFomics TM

attageneattageneattageneattagene

Nine contracts provide chemical procurement; hundreds of biochemical, cellular, tissue and genomic assays; model

organisms; and the capacity to screen up to 10,000 chemicals

ToxCast Contracts for Generating HTS and Genomics Data

Compound Focus, Inc.a subsidiary of


Evolution of Phase I

• 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 (March, 2008)– Organ culture: liver, kidney, lung (Hamner Institutes)– HTS Genotoxicity (Gentronix)– Toxicity and signaling pathways (Invitrogen)– NR Activation and translocation (CellzDirect)– 3D Cellular microarray with metabolism (Solidus)

+5 Assay Sources& 32 Endpoints

+3 Assay Sources& 16 Endpoints

8 Assay Sources& 412 Endpoints

16 Assay Sources, 460 Endpoints


Transporter

GPCR

Enzyme, other

Ion channel

NR

Kinase

CYP450

Phosphatase

Protease

201 Assays

320 Chemicals

Activity (% of Control)

Bioactivity Profiling (NovaScreen)


Dopamine, dopamine transporter

Estrogen Receptor

Glucocorticoid receptor

Opioidreceptors

Progesterone receptor

Androgen receptor

HPTE

Methoxychlor

Bioactivity Profiling (NovaScreen)


PPARα

ERE, ERα

Fold Induction (log 2)

Hierachical Cluster Attagene Results

{

VDRE, PXR, PXRE

DR5, RARβ,RARα, RARγ,

BRE, AP1, NRF2/ARE

320

Che

mic

als

RTUs


PyraclostrobinInhibits mitochondrial

electron transport chain


Chemical Responses in HepG2 High Content Screening Assays

TV0001261 Hr

1.0×10-08 1.0×10-07 1.0×10-06 1.0×10-05 1.0×10-04 1.0×10-030

100

200

300p53c-jun activationH2AX activationCell Number

Conc (M)

Feat

ure

Sign

al

TV00012624 Hr

1.0×10-07 1.0×10-06 1.0×10-05 1.0×10-04 1.0×10-030

100

200

300p53c-jun activationH2AX activationCell Number

Conc (M)

% C

ontr

ol

TV0003031 Hr

1.0×10-08 1.0×10-07 1.0×10-06 1.0×10-05 1.0×10-04 1.0×10-030

100

200

300

400Cell NumberMicrotubule stabilityc-jun activationH2AX activation

Conc (M)

% C

ontr

ol

TV00030324 Hr

1.0×10-08 1.0×10-07 1.0×10-06 1.0×10-05 1.0×10-04 1.0×10-030

100

200

300

400Cell NumberMicrotubule stabilityc-jun activationH2AX activation

Conc (M)

Feat

ure

Sign

al

Compund A Compound B

Compound BCompund A


ACToR: Aggregated Computational Toxicology Resource

Assay GenomicsDevToxDB ToxMiner

Tabular Data,Links to Web Resources Specialized Toxicology Databases

MicroArrayData

Data Mining

BioRefDB

Biological Reference Data

Chemical ID, StructureChemical

Internet Searches

ACToR API

ToxRefDB …

ACToR Web Browser


Comparing Activities by Chemical Class

NAM

E

CYP

2C19

CY

P2C

9

CY

P3A

1

Dop

amin

e Tr

ansp

orte

r (H

uman

)

CY

P2D

2

And

roge

n R

ecep

tor

Dop

amin

e Tr

ansp

orte

r (R

at)

CYP

2B6

CY

P2D

1

CY

P3A4

Prog

este

rone

Rec

epto

r

Ben

zodi

azep

ine

Rec

epto

r

Cyproconazole 1 1 1 1 1 0 1 0 0 1 0 0Difenoconazole 1 1 1 1 1 0 0 1 1 0 0 0Diniconazole 1 1 1 0 1 0 0 0 1 1 1 0Fenbuconazole 1 1 0 0 0 0 0 0 0 1 0 0Flusilazole 1 1 1 0 1 1 0 1 1 NA 1 1Hexaconazole 1 1 1 1 1 0 1 1 1 NA 1 0Imazalil 1 1 1 1 1 1 1 1 1 1 1 1Myclobutanil 1 1 1 1 0 0 0 0 0 NA 0 0Paclobutrazol 1 0 1 1 0 1 1 0 1 1 0 0Prochloraz 1 1 1 1 1 1 1 1 1 NA 1 1Propiconazole 1 1 1 0 0 0 0 1 0 NA 0 1Tetraconazole 1 1 1 0 1 1 0 1 0 1 1 0Triadimefon 1 1 0 1 1 1 1 0 0 1 0 1Triadimenol 1 0 0 1 0 1 1 0 0 0 0 0Triflumizole 1 1 1 1 1 1 0 1 1 1 1 1Triticonazole 1 1 1 1 0 1 1 0 0 NA 0 0Totals 16 14 13 11 10 9 8 8 8 8 7 6

ConazoleFungicides vs. NovaScreen Assays


ToxCast Predictive Signatures

• Use Machine Learning methods– SLR: Stepwise Logistic Regression– LDA: Linear Discriminant Analysis– SVM: Support Vector Machines– Many others

• For each binary endpoint, build models of form– Predictor = F(assay values)– If

• Predictor for a chemical meets criteria– Then

• Predict endpoint to be positive for the chemical

Assay 1

Assay 2

LDA

TNFN

FPTP

Truth

Test+

-

+ -


ToxCast Expansion BeyondProof of Concept

Chemicals

ToxCast_320HTS

Assay DataToxRef in vivobioassay data

Phase II


MOU, IAG, MTA and CRADA are providing additional biochemical, cellular and tissue assays; model organisms; and other resources.

ToxCast External Partners


Moving ToxCast Forward

• Complete data acquisition and data mining for Phase I

• Publish and release all Data

– Core papers on datasets (including 2 on ToxRefDB)

– Predictive signatures

• OECD Molecular Screening Initiative (June, Bilthoven)

• Security and Prosperity Partnership (SPP) in North America

• ToxCast Data Summit, Fall/Winter 2008

• MOU partnership with NTP/NIEHS and NCGC/NHGRI

– Addition of 2816 chemicals to current 2816 at NCGC

• Superset of ToxCast Phase I-III chemicals

– Coordinated deployment of assays with ToxCast

• Mapping toxicity pathway responses, and modeling their dose response…


Science, Feb 15, 2008

Transforming Toxicology

EPA’s ToxCast Program for Predicting Toxicity and ...

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