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ToxCast and Tox21: High Throughput Screening for Hazard & Risk of Environmental Chemicalsfor Hazard & Risk of Environmental Chemicals
David Dix
Office of Research and DevelopmentNational Center for Computational Toxicology
This work was reviewed by EPA and approved for presentation but does not necessarily reflect official Agency policy.
NLSOT, 07oct2010
Future of Toxicity Testing and Ultimately, Environmental Risk Assessments
Science, February 2008
EPA strategy, March 2009
CTRP 2nd gen plan, Sept 2009
Science, August 21, 2009, g ,
Office of Research and DevelopmentComputational Toxicology Research Program 1
Grand Challenge for Computational Toxicology:P di ti H T i itPredicting Human Toxicity
Complex Cellular and
Tissues
Biochemical HTS
Cellular and HCS HTS
ToxRefDB
ToxicityCellChanges
CellularNetworks
Cellular SystemsTissueDose
HTS
Exposure MolecularTargets Changes NetworksDose
MolecularPathways
p Targets
Cell-Based
HTSModel
Organism MTS
Office of Research and DevelopmentComputational Toxicology Research Program
Virtual Tissues2
Too Many Chemicals Too Little Data (%)609912
40
50
60
1000
10000
9912
20
30
40
100
1000
0
10
20
1
10
0
Acute Cancer GentoxDev Tox Repro Tox
IRIS TRI PesticidesInerts CCL 1 & 2 HPV
Office of Research and DevelopmentComputational Toxicology Research Program
MPV
3
High Throughput Screening 101 (HTS)(HTS)
96-, 384-, 1536 Well Plates
Chemical Exposure
96 , 38 , 536 e atesRobotic Platform
Cell Population
Pathway
Cell Population
Target Biology (e.g., Estrogen Receptor)
Office of Research and DevelopmentComputational Toxicology Research Program4
HTS: High Throughput Screening4
HTS in Drug Development
Id tifMake
difi tiHTS tests
100 000C tIdentify target,
pathway, or cellular h t
modifications to most active chemicals to
make suitable f i i
Test in animals for
safety, effectiveness
Test in humans for
safety, effectiveness
>100,000 chemicals with
no known activity
Create testing system (aka,
“ ”)phenotype for in vivo testing
effectiveness effectivenessfor effect on
target“assay”)
Office of Research and DevelopmentComputational Toxicology Research Program 5
HTS i T i lHTS in Toxicology
Obt i
Test prioritized chemicals in
animals Chemicals Id tifObtain or create testing
systems (“ ”)
HTS tests chemicals for effect
on assays
C e ca swith known
or suspected
toxicity/
Identify toxicity
pathways, cellular
h t
Computational analysis
&Synthesis
(“assays”)on assays yactivityphenotypesof HTS resultsCategorize as
inactive subject to further testing
Office of Research and DevelopmentComputational Toxicology Research Program 6
ToxCast and Tox21 High Throughput Screening of Chemical Bioactivity
• Addresses chemical screening and prioritization• Addresses chemical screening and prioritization needs for chemicals regulated by EPA
• Comprehensive use of HTS technologies to generate biological fingerprints and predictive signatures
• Committed to stakeholder involvement and transparencyp y• Communities of Practice- Chemical Prioritization;
Exposure• Release of all data upon peer review publication
Office of Research and DevelopmentComputational Toxicology Research Program
• Release of all data upon peer review publication7
G l f T C t d T 21Goals of ToxCast and Tox21• Identify toxicity pathwaysy y p y
• Obtain HTS assays for pathways
• Screen a large chemical library
• Initially link HTS results to adverse effects- Toxicity signatures
• Ultimately identify points of departure from HTS data - Toxicity pathways
Office of Research and DevelopmentComputational Toxicology Research Program 8
Tox21 Screening Throughputg g p
8 rows x 12 columns 88 test samples
16 rows x 32 columns 352 test samples
96-well plate 32 rows x 48 columns 1,408 test samples
384-well plate4 x 96-well plates
1536-well plate16 x 96-well platesIf @ 100 microtiter plates per day:
Time to screen 1 MM samples
samples§/day(wells/day)
Plate format Time to screen 1 MM samples
samples§/day(wells/day)
Plate format
Office of Research and DevelopmentComputational Toxicology Research Program
§ wells remaining after subtraction of control wells; NCGC uses left 4 columns of a 1536-well plate for control wells7 days140,800 (153,600)1,536-well
4 weeks35,200 (38,400)384-well
4 months8,800 (9,600)96-well
7 days140,800 (153,600)1,536-well
4 weeks35,200 (38,400)384-well
4 months8,800 (9,600)96-well
9
Comparison of VolumesComparison of Volumes
TotalV l
ppfor for Screening Compounds Screening Compounds in 7 Concentrationsin 7 Concentrations
Volume
96-well plate: 100 l x 7 pts = 700 l
384-well plate: 40 l x 7 pts = 280 l
1536-well plate: 5 l x 7 pts = 35 l
Office of Research and DevelopmentComputational Toxicology Research Program 10
• 1536 well HTS• 10,000 chemicals• 25 assays per year
Office of Research and DevelopmentComputational Toxicology Research Program 11
ToxCast Phase I and II, Tox21
ToxCastPhase I
ToxCastPhase II Tox21
Number of Chemicals
Actives 272 120 700
Inerts 24 100 1000
Antimicrobials 33 100 500Antimicrobials 33 100 500
HPV 35 170 1300
MPV 7 60 1500
Green 4 60 500
PCCL 73 150 500
Pharmaceuticals 0 100 2500Consumer Products /Food additives 0 0 1500
Total 309 ~700 ~10000
Office of Research and DevelopmentComputational Toxicology Research Program 12
ToxCast HTS AssaysCellular Assays
~500 Total Endpoints
• Cell lines– HepG2 human hepatoblastoma– A549 human lung carcinoma– HEK 293 human embryonic kidney
• Protein familiesGPCR
Biochemical Assays
y y
• Primary cells– Human endothelial cells– Human monocytes
H k ti t
– GPCR– NR– Kinase– Phosphatase
– Human keratinocytes– Human fibroblasts– Human proximal tubule kidney cells– Human small airway epithelial cells– Rat hepatocytes
– Protease– Other enzyme– Ion channel– Transporter p y
– Mouse embryonic stem cells (Sid Hunter)
• Biotransformation competent cells– Primary rat hepatocytes
• Assay formats– Radioligand binding
Enzyme activity – Primary human hepatocytes
• Assay formats– Cytotoxicity
Reporter gene
– Enzyme activity– Co-activator recruitment
Primarily Human / RatE ti Z b fi h d l t (St h i P dill )
Office of Research and DevelopmentComputational Toxicology Research Program
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– Reporter gene – Gene expression– Biomarker production– High-content imaging for cellular phenotype
Exception: Zebrafish development (Stephanie Padilla)
The ToxCast In Vitro Data SetAssays
Many hits – median value=50Chemicals
Many hits – median value=50
Fewer in cell-free HTS (Novascreen, red)
Many hits are at or near top of tested concentration range
A few are in nanomolar rangeA few are in nanomolar range
Office of Research and DevelopmentComputational Toxicology Research Program 14
ToxCast Data Analysis
Assays
ls
AC50
Emax
Che
mic
al
AC50828 Assay-Chemical Pairshad AC50s of less than 1µM
: Assay-Chemical Hit
500 Assays X 320 Chemicals X {5-18} Concentrations X{1-3} Replicates X {1-3} Time Points ≈3.2 Million Data Points
3 2 1 04…
Concentration
Office of Research and DevelopmentComputational Toxicology Research Program 15Emax: Maximal efficacy/response/activity
AC50: Concentration whereby 50% of maximal response was achievedConc (uM): Micromolar concentrations of chemical
Digitizing Legacy in Vivo Data in ToxRefDB
Chronic/CancerMultigenationMultigenationDevelopmental
mic
als
Che
m
Martin et al 2009a,bKnudsen et al 2009
Office of Research and DevelopmentComputational Toxicology Research Program 16
SOT 2010
30 years and more than $2B worth of data 16
Predicting Toxicity and Disease from HTS Data
ToxRefDB ToxCast Human Disease
from HTS Data
Office of Research and DevelopmentComputational Toxicology Research Program 17
ToxCast: Multiple Assays and Technologies per Target
Office of Research and DevelopmentComputational Toxicology Research Program 18
ToxCast: Multiple Targets per Pathway
Biologically Multiplexed Activity Profiling (BioMAP)
Multiplex Transcription Reporter Assay
Cell-free HTS Assays
Cell-based HTS Assays
High Content Cell Imaging Assays
Office of Research and DevelopmentComputational Toxicology Research Program
19
Building Toxicity Signatures forR t Li Hi t th lRat Liver Histopathologyfrom Chronic Bioassays
37
21
No PathologyProliferative LesionsPre-neoplastic Lesions
68
Neoplastic Lesions
N = 248 Chemicals in ToxRefDB
Office of Research and DevelopmentComputational Toxicology Research Program 20
ToxCast Identied Genes Associated with Progression of Rat Liver Disease
132 60 21
Any Lesion Pre-Neoplastic Neoplastic
Office of Research and DevelopmentComputational Toxicology Research Program 21Liver Disease Progression
Judson et al, EHP (2010)
Toxicity Pathways
Receptors / Enzymes / etc.Chemical
p yDirect Molecular Interaction
Pathway Regulation / Genomics
Cellular Processes
Tissue / Organ / Organism Tox EndpointTissue / Organ / Organism Tox Endpoint
Office of Research and DevelopmentComputational Toxicology Research Program 22
Predictive Signatures from ToxCast for Chronic, Developmental and Reproductive Toxicityp p y
• Chronic/Cancer endpoints from rat, mouse and dog
• Developmental endpoints from rat and rabbit
• Reproductive endpoints from rat
Office of Research and DevelopmentComputational Toxicology Research Program 23
ToxPi: Prioritizing Chemicals for P t ti l E d i A ti itPotential Endocrine Activity
Prioritization Index = ToxPi = f(HTS assays + Chemical properties + Pathways)( y p p y )
Bisphenol A TebuthiuronER
TROther XME/ADME
AROther NR
Ingenuity pathways
KEGG
LogP_TPSA
Predicted CaCO-2
Disease classes
KEGG pathways
Office of Research and DevelopmentComputational Toxicology Research Program
Reif et al, EHP, 2010
ToxPi Ranking of ChemicalsHPTE
Linuron
ToxP
i sco
re
Pyrimethanil
als
sorte
d by
Tebuthiuron309
Che
mic
a
ERTROther
XME/ADME3
hi h
Ingenuity path
AR
KEGG path
LogP_TPSA
CaCO-2
Disease classes
Other NR
Office of Research and DevelopmentComputational Toxicology Research Program
ToxScore
ToxPi
highestlowest
Reif et al, 2010
Expanding the ToxPi Approach for Prioritization of Toxicity Testing Based on ToxCast Chemical ProfilingTesting Based on ToxCast Chemical Profiling
CANCERDEVELP th
Identify in vitro assays, targets, genes and In vitro assays
Pathwaysassociated with CANCER in vivo endpoints g , g
pathways associated with multiple sectors of in vivo toxicity
In vitro assaysassociated with CANCER in vivo endpoints
of in vivo toxicity.
REPRO SYSTEMICOffice of Research and DevelopmentComputational Toxicology Research Program 26
REPRO SYSTEMIC
ToxPi Scores for Antimicrobial Pesticide Actives
One of the methods be developed to use ToxCast data for classifying and prioritizing antimicrobials and inerts.
Office of Research and DevelopmentComputational Toxicology Research Program
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Future ToxPi (Toxicological Prioritization Index)
ToxPi = f(Exposure + Chemical properties + In vitro assays + Pathways)
Incorporate additional components (slices) from other domains:( )
- Exposure- Chemical properties- QSAR
Office of Research and DevelopmentComputational Toxicology Research Program 28
Range of in vitro AC50 l t d t s
TriclosanPyrithiobac-sodium
values converted to human in vivo daily dose
kine
tics
g/da
y)
oxic
ok
log
(mg/
kg
rse
To
Margin
Rev
e
xazo
lem
ectin
ofez
inha
late
trobi
nat
hion
xabe
nod
ium
azon
em
phos
2,4-
Det
hrin
MG
Kaz
ine
omac
ilxy
carb
nuro
nat
hion
clos
anen
one
rodi
nil
fluto
lem
iprid
amid
eDi
uron
sulid
e oz
olin
ne D
Hop
hos
buzi
nm
efon
azop
yrip
hos
anid
inen
ol-A
achl
oroc
hlor
zoxo
nor
vos
-oxo
n
Estimated Exposure
Office of Research and DevelopmentComputational Toxicology Research Program
Etox
Emam
Bupr
oDi
buty
l pht
hPy
racl
ost
Para
Isox
Pryr
ithio
bac-
soBe
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Prop
etam
S-Bi
oalle
Atra
Bro
Feno
xFo
rchl
orfe
nM
ethy
l Par
aTr
icRo
teCy
prIs
oxaf
Acet
amZo
xa DBe
nsVi
nclo
Oxy
tetra
cycl
inDi
crot
oM
etri
Tria
dim
Thia
Fena
mCl
othi
aBi
sphe Al
aAc
eto
Diaz
Dich
lCh
lorp
yrip
hos-
Rotroff, et al. Tox.Sci. In Press
29
Systems Approaches to Modeling Toxicity:From Pathways to Virtual Tissues
chemicals pathways networks cell states tissue function
Moving beyond empirical models, to
Identify Key Targets and Pathways For Prioritization
Quantitative Dose-Response
Models Next Generation
Risk assessments
multi-scale models of complex biological systems: vLiver,
vEmbryo…
Office of Research and DevelopmentComputational Toxicology Research Program
30SOT 2010
vEmbryo…
HIGH THROUGHPUT SCREENING and CHARACTERIZATION OF HAZARD & RISKCHARACTERIZATION OF HAZARD & RISK
Tissues
Cellular SystemsToxicityCell
ChangesMolecular
TargetsCellularNetworks
TissueDose
Molecular
Exposure
Pathways
IN VITRO: concentration responseIN VITRO: concentration response
Animal IN VIVO: exposure dose response
Human IN SILICO: exposure dose response
Office of Research and DevelopmentComputational Toxicology Research Program 31
SOT 2010
Animal IN VIVO: exposure dose response
ToxCast Phase I Publications & Data Release
Office of Research and DevelopmentComputational Toxicology Research Program
32SOT 2010http://epa.gov/ncct/toxcast/
Acknowledgements
Tom KnudsenImran ShahJ h W b h
Robert Kavlock Keith HouckMatt Martin
Ray TiceChris AustinJohn WambaughMatt Martin
Richard JudsonAnn RichardDavid Reif
Chris Austin
Daniel RotroffWoody SetzerHolly MortensonA d B
http://www.epa.gov/ncct/
Andrew Beam
Office of Research and DevelopmentComputational Toxicology Research Program 33
SOT 2010