tox21 & toxcast chemical landscapes: laying the foundation for 21 st century toxicology

Office of Research and DevelopmentNational Center for Computational Toxicology

August 25, 2011

Tox21 & ToxCast Chemical Landscapes: Laying the Foundation for 21st Century Toxicology

5th Meeting on U.S. Government Chemical Databases & Open Chemistry, Frederick, MD

Ann RichardNational Center for Computational Toxicology

Office of Research and DevelopmentNational Center for Computational Toxicology 2

NCCT’s ToxCast Program

StructuresHTS

In Vitro

In VivoQSAR


Generic Chemicals

Test Substances

NCCT Public Data http://www.epa.gov/ncct/

ToxRefDBToxCast

ACToR DSSTox

Tox21

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



ToxRefDBToxCast

Chemical Structure AnnotationChemical Management

Chemical QC

DSSToxACToR

Tox21




ToxRefDB

Tox21

ToxCast

DSSTox

Data IntegrationChemical – Assay Linkage

In vivo data LinkageAnalysis tools

ACToR



2007 2009

2008 2010

ToxCast ProjectTo

xCas

t con

cept

pap

er To

xSci

EPA NCCT launches ToxCast

ToxC

ast P

hase

I co

ntra

cts

issue

d

ACToR launchedNAS Report released

Phas

e I t

estin

g be

gins

Phase I data results

ToxC

ast D

ata A

nalys

is

Sum

mit

ToxRefDB web access w/in ACToR

Full Phase I data publication/release

Phase I (309) Phase II: (700)


Tox21 Project

2005 2007 2009

10K

5.6K

1.4K

Tox21 Chemical Library2.8K

2006 2008 20102004

2011

EPA Tox21 Plate A

NTP HTS Plate A

ToxCast Phase I NCGC Drug

Plates A,B

Tox21 EPA Plates A’,B,C

NTP Tox21Plates A’,B,C

NCGC DrugPlates A’,B’,C

ToxCast Phase II


Tox21 Chemical x Assay Landscape

# Chemicals>500

~1000 ~10,000

# Assays100

ToxCast Phase I & II

960 Chem x >500 assays

Tox21

~10,000 Chem x 50-100 assays

1408 Chem x >100 assays

NTP HTSPlate A

Drugs

NCCT/EPA NIEHS/NTP NIH/NCGCEnvironmentalIndustrialPesticidesFood UseDrugsToxicology

1536 well microplate format(1408 cmpds/plate) x 9 plates

FDACFSAN/CDER


e1k 820 unique(1000 samples)

To be tested in endocrine-related subset of ToxCast assaysToxCast_

960

Phase I & II(a,b)

111 (+24 PhIIc) failed drugs, CFSAN, pesticides, EPA high interest compounds tested in >500 HTS assays

100

IIc

Tox21: EPA Chemical Inventories

TOX21_EPA_3726


Tox21: EPA ToxCast Chemicals ToxCast Phase I (293 unique)

EPA pesticidal actives w/ rich in vivo data PFOAs, BPA, metabolite/parent pairs

ToxCast Phase II a,b (776) +c (100) EPA pesticides, high interest EPA and stakeholder inventories, data rich

chemicals (EDSP, OPPT, Antimicrobials, Inerts, …) 135 Pharma failed drugs w/ pre-clinical & clinical tox data FDA CFSAN data rich, NCTR LTKB Priority 1 drugs Toxicity reference chemicals, data-rich chemicals, NTP immunotox

Broaden selections from ToxCast Phase II nomination lists to include in Tox21 EPA set


211 volatile/ insufficient sample

COA/MSDS review

Unable to procureToo expensive

4371 compounds Able to purchase

3726 unique substances

EPA Tox21 Inventory

e.g., EPA Tox21 Chemical Procurement

~7,000 compounds Candidates for procurement Complex mixturesIll-defined substancesNo structureInsoluble (est. LogP)Volatiles (est. VP)InorganicsExplosiveReactivePolymers …

435 Insoluble in DMSO (approx 10%)

Solubilize in DMSO

HPV, MPV SubstancesAntimicrobials

Pesticide actives Pesticide inertsWater contaminants

Industrial chemicals

NCCT Programs EU, FDAIn vivo data availability (EPA, NTP, FDA)

Endocrine disruptors

~19,000 compounds EPA Tox21/ToxCast Phase II Chemical Nominations (over 100 lists)

Green Chemistry

11


Tox21 Chemicals: EPA Selection Strategy

Nominations & tracking of inventory overlaps by CAS Prioritize for procurement Cost & availability Filter by MW, physchem, volatility, MSDS cautions DMSO solubility


13

ToxCast PhI&PhII 960:# Compounds per Inventory (960 x 16)

PesticideInerts

Water

Consumer

Antimicrobials

Green Chemistry

HPV

MPV

TRI

IRIS

EDSP

GRAS

AIR

PesticideIn-erts; Series1;

243

Water; Se-ries1; 217

Consumer; Series1; 188

Antimi-crobials; Series1;

91

Green Chem-istry; Series1;

75

HPV; Se-ries1; 232

MPV; Se-ries1; 83

TRI; Series1; 216

IRIS; Se-ries1; 240

EDSP; Se-ries1; 120

GRAS; Se-ries1; 26

AIR; Series1; 90

SUM

580

94

202

SUM; Se-ries1; 0

580; Se-ries1; 0

94; Se-ries1; 0

202; Se-ries1; 0

Series1; 0

SUM; Se-

ries2; 580

580; Se-

ries2; 94

94; Se-

ries2; 202

202; Se-

ries2; 111

Se-ries2; 329

960 Total chemicals 2740 total overlaps across 16 diverse inventories Excellent coverage of multiple high-interest inventories Broad diversity of chemical-use and types Large overlap with data-rich inventories


14

ToxCast_960 Pharmaceuticals: Multipurposing (255)

111 Failed pharmaceuticals – no overlaps 144 additional cmpds classified as drugs; on other lists with other uses 137 of these appear on 2 or more lists Caffeine appears on 18 lists

HPVCSI_1081CPDBAS_677EPAFHM_276

NTP_ToxCarcLong_SubstanceNTP_ReproTox

NTP_Phthalates_IURNTP_ImmunoTox

NTP_DevToxOPPIN_DER

Nongenotoxic_hepatocarcinogensFDA_CFSAN_repro_dev

FDA_CFSAN_chronic_subchronicFDA_CFSAN_carc

FDA_GRASfragrance

PFCsOECD

NTP_ImmunotoxLTKB_Priority1,2_PhaseII

ECHA_organic_CMR_2010FAR_EDSP_173

EPA_Fooduse_InertsEPA_PCCL

EPA_OW_Priority2EPA_DNT

EPA_AntimicrobialsEPA_OPPT-RAD_All

OPPT_Categoriesv-Liver_22+14ref

v-EmbryoSuperfund_PPRTV

IRIS+NominExposure_Summit

EPA_DfEGreen Plasticizers_Nisha

phthalate_metabolitePhthalates

0 20 40 60 80 100 120 140 160


Chemical Sample Registration Workflow

Place chemical orders Procure 2x100mg

compound in tared, barcoded vials

Store neat

Solubilize in DMSO

+

Partition to Matrix

racks

Solution Plates

Insoluble

Review COA

QC structure

Register in DSSTox Inventory

Register in Sample Tracking

Database

X

Bottle IDs

Remove from DSSTox

Update

Solution IDs

Update

AssayProviders

ACToR/PubChem

NCGC

Plate Address

Plate ID

Assay Results

Analytical QC

Analytical QC


Publish QC summary results in association with assay data 16

Tox21 Analytical QC

LC-MS

GC-MS

PASS = Confirm parent ion peak and >90% purity

Fail, inconclusive or analytical method inappropriate

Retest at later time point under assay conditions for stability

A copy of each parent Tox21 assay plate (352 cmpds/plate) will be subjected to analytical QC for assessing purity, identity, stability


Analytical QC Summary PDF results will be available for each sample

NCGC Analytical Chemist (W. Leister) will review all preliminary LC results and QC “FAIL” compounds, and supervise follow-up testing

Prime objective of QC is to inform analysis & interpretation of assay results:

high confidence low confidence fail


Structure-based Data MiningPubChem ChemSpider EPA ACToR

Analog SearchingAIM DSSTox

External Resources

S t r u c

t u r

e

Chemical structure - CIDSubstance details - SIDProject inventory record - RID

Cl

Cl

OO

OH

OH

Cl

NH2NH

N

N

N

PNHO

S OO

NN N

Cl

N

OH

OO

Cl

F

FFN+

O

O-

NO

O

NHS

DSSTox

Test Sample

AssayResults

Structures

ToxCast/Tox21 Chemical Registry

SAR Modeling

CID Table

RID Table

SID Table

Tox21ToxCast

ToxRefDB

7

Office of Research and DevelopmentNational Center for Computational ToxicologyTox21 Sample Tracking Database

DSSTox RID Bottle ID ( COA ID) Solution ID ( QC ID)


Cl

Cl

OO

OH

OH

Cl

NH2NH

N

N

N

PNHO

S OO

NN N

Cl

N

OH

OO

Cl

F

FFN+

O

O-

NO

O

NHS

DSSTox

Test Sample

AssayResults

Structures


Bottle_ID

Soln_IDQC_ID

DSSTox_RID

COA_ID

8


ACToR/ToxMiner

Solution ID Plate ID Plate Address ID

DSSTox RID Assay nameAssay detailsAssay outcome

AssayResults

DSSTox RID Bottle ID ( COA ID) Solution ID ( QC ID)

Tox21 Sample Tracking Database


Cl

Cl

OO

OH

OH

Cl

NH2NH

N

N

N

PNHO

S OO

NN N

Cl

N

OH

OO

Cl

F

FFN+

O

O-

NO

O

NHS

DSSTox

Test Sample

Structures


9

PubChem_CID

S t r u c t u

r e

PubChem_SID

Tox21_ID

Chemical Information & Sample QC

“Whoever is careless with the truth in small matters cannot be trusted with important matters.”

– Albert Einstein

Gene-expression

Chemical SupplierLot/Batch

Certificate of AnalysisSolubility

Purity & Stability

Chemical Sample Details

Chemical Name Toxicology literature Public databases

Cheminformatics resourcesCAS SMILES

InChI / StructureSubstance details

ChemicalAnnotation

LayerCl

Cl

O

O

OH

Name is misspelled or incorrect

CAS is invalid or retired CAS and name do not

agree Name and structure do

not agree Name is insufficient for

structure assignment Insufficient description

of substance

Chemical Annotation of Public resources

Generic Chemical of Toxicological

Interest

Chemical Information & Sample QC

Sample Annotation & QC

Not same as generic chemical

Salt, isomer, … Name does

not match info on COA

hydrate, stereo Wrong MW COA expired

Purity <90% Active impurities Sample degrades Rxn with solvent Hydrolysis

Procure from

ChemicalSupplier

Name, CAS, purity (COA)

MW, dose

IC 50

No CAS or wrong Name wrong Structure wrong


Tox21 Cheminformatics

Plate IDs, Plate Addresses

Solution IDs

Bottle IDs

Assay Results

Analytical QC

Tox21/ToxCast

FeaturesClassesDescriptorsProperties

Mixture IDs Chemical Substance: SID

Structure: CID

Parent: PID

2D, 3DConformersTautomers

SaltsComplexes

(Metabolites?)

CAS/Name

Groups: MapID

Analogs, classifiers, stereo family, etc.

Creating SAR-ready files: How to process salts, complexes, charged species How to aggregate results for resulting structure “duplicates” How to deal with enriched features & “families” of structures within dataset


DSSTox TOX21E1_3619

NCGC PharmaceuticalsNTP compoundsFDA compoundsEPA compounds

13 macromolecule155 mixture or formulation1 unspecified or multiple forms45 inorganics100 organometallics173 complexes219 salts12 no structures

91 “parent” groupings (2-3)

MW ranges from 30 (formalin) to 1700 (tannic acid)

AlkylbenzenesChlorobenzenesMethyl phenolsNitrobenzenesAromatic aminesPhthalatesPerfluorinates…


Tox21 Structural Library

Feature enrichment

Feature diversity


Tox21 Reaction Features: Commonalities

NTP Tox21

NCGC Tox21

EPA Tox21

DrugsFood additivesAntimicrobialsWater contaminantsHPVsToxicants…

Metabogen reaction features generated using “MOSES” software by Molecular Networks


Histogram

Column 1

0 200 400 600 800 1000 1200

Cou

nt

0

100

200

300

400

500

600

700

Count Histogram

Column 1

0 200 400 600 800 1000 1200

Cou

nt

0

10

20

30

40

50

Count

Histogram

Col um n 1

0 200 400 600 800 10 00 120 0

Cou

nt

0

10

20

30

40

50

Coun t

Tox21 NTP & EPA

Tox21 NCGC

# M

olec

ules

Molecular Weight

NTP & EPA collections enriched with lower MW compounds compared to NCGC drug collection

Mean = 231

Mean = 304

Tox21: Molecular Weight Distributions


-5

0

5

1015

LOGP

0

1

2

3

TPSA

1

2

3

4

CO

MP

LEX

ITY

ToxCast_PhaseI

ToxCast/Tox21 property distributions

Chemical properties computed using “Adrianna” software by Molecular Networks.

LOG P = Octanol/Water partition coefficient

TPSA = log (Total Polar Surface Area)

Complexity = log (complexity based on paths, branching, atoms)


-5

0

5

1015

LOGP

0

1

2

3

TPSA

1

2

3

4

CO

MP

LEX

ITY






ToxCast_PhaseIToxCast_PhaseII


-5

0

5

1015

LOGP

0

1

2

3

TPSA

1

2

3

4

CO

MP

LEX

ITY






111 failed drugs

ToxCast_PhaseIIToxCast_PhaseI


-5

0

5

1015

LOGP

0

1

2

3

TPSA

1

2

3

4

CO

MP

LEX

ITY






Tox21111 failed drugs

ToxCast_PhaseIIToxCast_PhaseI


Expanding Applicability

QSAR In vitro/HTS

In Vivo

Defined organicsVirtual chemicalsVolatilesDMSO insolublesReactives

MixturesFormulations

Proprietary substancesOrganometallics

MetalsSalts

metabolismADME

Defined structure + Biological profile


Acknowledgements:

EPA NCCT ToxCast Team:Robert Kavlock - DirectorDavid Dix Keith Houck Matt Martin (ToxRefDB)Richard Judson (ACToR)

EPA NCCT DSSTox:Maritja Wolf– Lockheed Martin, Contractor to the EPAIndira Thillainadarajah – EPA:SEEPatra Volarath – EPA Post Doc

External Collaborators: Chihae Yang, FDA/CFSANChris Austin & colleagues, NCGC/NIHRay Tice & colleagues, NTP/NIEHS

This work was reviewed by EPA and approved for publication but does not necessarily reflect official Agency policy.

tox21 & toxcast chemical landscapes: laying the foundation for 21 st century toxicology

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