January 20, 2009Office of Research and DevelopmentNational Center for Computational Toxicology

2008 HESI Emerging Issue:Identification of Pharmaceuticals for Validation of ToxCastRobert KavlockDirector, National Center for Computational Toxicology, US EPA

1Office of Research and DevelopmentNational Center for Computational Toxicology

Too Many Chemicals Too High a Cost

Cancer

DevTox

NeuroTox

ReproTox

ImmunoTox

PulmonaryToxMillions $

Change Needed Because …..

1

10

100

1000

10000

100000

Data Collection

IRISTRIPesticide ActivesCCL 1&2Pesticide InertsHPVMPV CurrentMPV HistoricalTSCA Inventory

11,000

90,000

…and not enough data.

Judson, et al EHP submitted

2Office of Research and DevelopmentNational Center for Computational Toxicology

Future of Toxicity Testing

Bioinformatics/Machine Learning

in silico analysis

Cancer

ReproTox

DevTox

NeuroTox

PulmonaryTox

ImmunoTox

HTS -omics

in vitro testing

$Thousands

EPAs Contribution: The ToxCast Research Programwww.epa.gov/ncct/toxcast

3Office of Research and DevelopmentNational Center for Computational Toxicology

Cellular Assays

Biochemical Assays

Toxicology Endpoints

Physical chemical properties

Profile Matching

Correlating HTS to Toxicity

Genomic Signatures

In silico Predictions

4Office of Research and DevelopmentNational Center for Computational Toxicology

Key Challenges

•Find the Toxicity Pathways•Liver vs developmental

•Obtain HTS Assays for Them• Including metabolic capability

•Screen Chemical Libraries• Coverage of chemical properties

•Link Results to in vivo Effects• Gold standard and dosimetry

5Office of Research and DevelopmentNational Center for Computational Toxicology

Implications for Success

•Hazard Identification•Closing Data Gaps•Reductions in Cost•Hypothesis Generation•Reduced Animal Usage

•Risk Assessment•Providing MOA(s)•Targeted Testing •Identifying Susceptible Populations

•Ancillary Applications•Mixtures•Chirals•Nanomaterials•Green Chemistry•Lot variations

6Office of Research and DevelopmentNational Center for Computational Toxicology

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/

7Office of Research and DevelopmentNational Center for Computational Toxicology

Phased Development of ToxCast

FY09-10~$15-20K>200PMN Nanomaterials>12IId

FY09~$20-25k>400ExtrapolationKnown Human Toxicants>100IIb

FY09$10K166PilotNanomaterials15Ib

FY09~$20-25k>400ValidationData Rich Chemicals>300IIa

>300

>400

552

Number of Assays

Data poor

Expanded Structure and Use

Diversity

Data Rich(pesticides)

Chemical Criteria

FY11-12

FY10

FY08

TargetDate

~$15-20k

~$20-25k

$20k

Cost per Chemical

Prediction and Prioritization

Extension

Signature Development

PurposeNumber of ChemicalsPhase

ThousandsIII

>300IIc

320Ia

January 2009

8Office of Research and DevelopmentNational Center for Computational Toxicology

Phased Development of ToxCast

FY09-10~$15-20K>200PMN Nanomaterials>12IId

FY09~$20-25k>400ExtrapolationKnown Human Toxicants>100IIb

FY09$10K166PilotNanomaterials15Ib

FY09~$20-25k>400ValidationData Rich Chemicals>300IIa

>300

>400

552

Number of Assays

Data poor

Expanded Structure and Use

Diversity

Data Rich(pesticides)

Chemical Criteria

FY11-12

FY10

FY08

TargetDate

~$15-20k

~$20-25k

$20k

Cost per Chemical

Prediction and Prioritization

Extension

Signature Development

PurposeNumber of ChemicalsPhase

ThousandsIII

>300IIc

320Ia

January 2009

9Office of Research and DevelopmentNational Center for Computational Toxicology

$1B in Toxicology Now Stored in ToxRefDB

Martin, et al EHP, 2008

10Office of Research and DevelopmentNational Center for Computational Toxicology

ToxCast Phase I Datasets• 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 (June, 2008)– XME Gene Regulation (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 Zebrafish vascular/cardiotoxicity (Zygogen)– microarray with metabolism (Solidus)– HTS stress response (NHEERL+NCGC)

20 Assay sources554 Endpoints

11Office of Research and DevelopmentNational Center for Computational Toxicology

ToxCast Assays

• Cell lines– HepG2 human hepatoblastoma– A549 human lung carcinoma– HEK 293 human embryonic kidney

• Primary cells– Human endothelial cells– Human monocytes– Human keratinocytes– Human fibroblasts– Human proximal tubule kidney cells– Human small airway epithelial cells

• Biotransformation competent cells– Primary rat hepatocytes– Primary human hepatocytes

• Assay formats– Cytotoxicity– Reporter gene – Gene expression– Biomarker production– High-content imaging for cellular

phenotype

• Protein families– GPCR– NR– Kinase– Phosphatase– Protease– Other enzyme– Ion channel– Transporter

• Assay formats– Radioligand binding– Enzyme activity– Co-activator recruitment

Cellular AssaysBiochemical Assays

12Office of Research and DevelopmentNational Center for Computational Toxicology

Cellular Assays

• Types of Assays– Known toxicity pathways and targets

• biomarker measurements • reporter gene assays

– General cytotoxicity– Toxicity cellular phenotypes

• Cell lines and primary cells• Generally screened at up to 100 μM or used maximally

tolerated concentration defined by general cytotoxicity determination

• Concentration-response format used and EC50 generated

13Office of Research and DevelopmentNational Center for Computational Toxicology

Biochemical Assay Results228 A

ssays

320 Chemicals

Log IC50 (M)

14Office of Research and DevelopmentNational Center for Computational Toxicology

Methods described in:Judson et al 2008A comparison of machine learning algorithms for chemical toxicity classification using a simulated multi-scale data model.BMC Bioinformatics 9:241

N1 A1 E1 A2 N2 N3 N4 N5 C1 B1 B2 B3 G1 A3 E2HTS Assays

Positivecluster

Negativecluster

In Vivo

In VitroToxCastPredictive Modeling

of ChronicRat Liver

Apoptosis/Necrosis

(15)

(23)

A

B

15Office of Research and DevelopmentNational Center for Computational Toxicology

Beyond the Proof of Concept

Chemicals

ToxCast_320HighThroughput

Assay DataToxRef in vivobioassay data

17

Universe 13,247With structures 8,277Plausible P-chem (logP) 7,116

Current Additional

NTP 1353 ~1400

EPA 1330 ~2800

NCGC ~3000 drugs -

Target library, Summer 2009 ~10,000

* Sources include NTP, EPA HPV, CCL, OPPIN, OW, Inerts, ToxCast, DSSTox, EU Carcinogenomics, Pharmaceuticals, others

Tox21 Existing and Candidate Chemicals*

18Office of Research and DevelopmentNational Center for Computational Toxicology

Source to Outcome ContinuumSource to Outcome Continuum

Source/Stressor FormationSource/Stressor Formation

Environmental Conc.

External Dose Target Dose

Biological Event

Effect/OutcomeEffect/Outcome

ToxRefDB

ToxCast

ToxMiner

rTK

ACToR

ExpoCast

19Office of Research and DevelopmentNational Center for Computational Toxicology

Current Status of ToxCast and Tox21

• ToxRefDB - Relational phenotypic databases– Chronic rat and mouse studies (Martin, et al, EHP 2008)– Rat multigenerational studies (Martin, et al, submitted)– Rat and Rabbit developmental studies (Knudsen, et al, internal review)

• ToxCast– Submit manuscripts on v1.0 by Feb 1 2009– Data Summit

• RTP, May 14-15– Phase II launch

• Mid summer 2009• Major Pharma is considering supply +100 candidate drugs

• ACToR (Aggregated Computational Toxicology Resource) - www.epa.gov/actor– Released Jan 2009– Portal for public toxicity information, ToxRef and ToxCast data

• Tox21 – qHTS on +6000 chemicals starting in mid 2009

• Includes large collection of pharmaceuticals• One to two assays per week

20Office of Research and DevelopmentNational Center for Computational Toxicology

Emerging Issues Proposal

• SPECIFIC ACTIONS

– Coordinate Public-Private sector involvement in ToxCast predictions– Scoping meeting to articulate needs, timelines and boundaries of

involvement by participants

• DESIRED OUTCOME

– Successful deliberations and negotiations would result in:• Identify and provide chemicals (~100mg) for screening • Sharing of relevant pre-clincal and clinical data• [Cost sharing of screening costs]• Co-publications on predictive models

21Office of Research and DevelopmentNational Center for Computational Toxicology

Benefits of Proposal

• Creates a government – private sector effort with potential to significantly improve predictive basis of toxicity evaluations

• Enables utilization of a unique private sector knowledge

• Builds on the experience of EPA in computational toxicology

• Brings direct human relevance to HTS screening on environmental chemicals, which already involves the use of many human protein targets and cell types

• Significantly enables progress at reaching the vision of toxicity testing in the 21st envisioned by the National Research Council