Office of Research and Development National Center for Computational Toxicology
Tox21 and ToxCast Screening of
Chemicals to Build Predictive and
Systems Models of Toxicity
David Dix
National Center for Computational Toxicology
ChemScreen
Amsterdam
Jan 18, 2012
Office of Research and Development National Center for Computational Toxicology
Too Many Chemicals Too Little Data (%)
Why Tox21?
1
0
10
20
30
40
50
60
Acute Cancer Gentox
Dev Tox Repro Tox
1
10
100
1000
10000
IRIS TRI Pesticides
Inerts CCL 1 & 2 HPV
MPV
Office of Research and Development National Center for Computational Toxicology
Toxicity Testing in the Twenty-First Century:
A Vision and a Strategy - National Academy of Sciences (2007) http://iccvam.niehs.nih.gov/docs/about_docs/NAS-Tox21.pdf
move away from animal testing to high-throughput assays
to understand how chemicals perturb cellular functions
establish relationships between in vitro perturbation (toxicity
pathways) and in vivo outcomes (adverse outcome
pathways)
broad coverage of chemicals and biological activities,
reduce cost and time for testing, and fewer animals (RRRs)
2
Why Tox21?
Office of Research and Development National Center for Computational Toxicology
3
High Throughput Screening 101
(HTS)
96-, 384-, 1536 Well Plates
Target Biology (e.g.,
Estrogen Receptor)
Robots
Pathway
Chemical Exposure
Cell Population
HTS: High Throughput Screening
3
Office of Research and Development National Center for Computational Toxicology
4
Toxicity Pathways
Receptors / Enzymes / etc.
Direct Molecular Interaction
Pathway Regulation /
Genomics
Cellular Processes
Tissue / Organ / Organism Tox Endpoint
Chemical
The Tox21 Community
• Identify mechanisms of
compound-induced biological
activity in order to:
− characterize toxicity/disease
pathways
− facilitate cross-species extrapolation
− provide input to models for low-dose
extrapolation
• Prioritize compounds for more
extensive toxicological evaluation
• Develop predictive models for
biological response in humans
Tox21 Goals
2007 2006 2005 2004 2008 2009 2010 2011 2012
NCGC
EPA NCCT ToxCast II
qHTS Phase I
ToxCast Phase I
qHTS II
Tox21 - a “Community
Resource” Project
Tox21 Timeline
Range of screening assays performed
Phenotype (Image-based
HCS, GFP, etc)
Pathway (Reporters, e.g.,
luciferase, b-lactamase)
Protein (Enzyme readouts, interactions, etc)
Extent of reductionism
8 8
qHTS Screening Format
r
c
96-well plate
384-well plate
4 x 96-well plates
1536-well plate
16 x 96-well plates
8 rows x 12 columns
88 test samples
16 rows x 32 columns
352 test samples
32 rows x 48 columns
1,408 test samples*
* wells remaining after subtraction of control wells; NCGC uses left 4 columns of 1536-well plate for controls
Tox21 Robot Ribbon-Cutting (March 10, 2011)
10
– Assay selection based on
− Phase I experience
− Information from in vivo toxicological investigations
− Advice of basic researchers and nominated assays
− Maps of disease-associated cellular pathways
– Stage I
− Nuclear receptor activation or inhibition (AR, AhR, ER, FXR, GR, LXR, PPAR,,
PXR, RXR, TR, VDR, ROR)
− Induction of stress response pathways (e.g., DNA damage, heat shock,
hypoxia, inflammation, oxidative)
– Stage II
− Other disease-associated pathways (e.g., obesity/diabetes, autism) and move
to HTS gene array assays applicable to all cell types
Phase II qHTS Strategic Screening Strategy
The Tox21 Phase II 10K Compound Library
NCGC
– Pharmaceutical
Collection
EPA
• ToxCast I and II
compounds
• Antimicrobial
Registration Program
• Endocrine Disruptor
Screening Program
• OECD Molecular
Screening Working
Group List
• FDA Drug Induced Liver
Injury Project
• Failed Drugs from
Pharma
NTP
• NTP-studied
compounds
• NTP nominations and
related compounds
• ICCVAM/NICEATM
validation and
reference compounds
• External collaborators
(e.g., Silent Spring
Institute, U.S. Army
Public Health
Command)
• Defined mixtures
12
Tox21 10K Library 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)
Tox21
111failed drugs
ToxCast_PhaseII
ToxCast_PhaseI
13
Collins et al. Science 319:906
14
Tox21: Critical Toxicity Pathway Information
Over 650 HTS assays testing environmental chemicals for
in vitro bioactivity.
Phase-I: 309 data-rich chemicals (primarily pesticides) having
over 30 years of traditional animal studies valued at $2B; in
vitro signatures defined by how well they predict outcomes in
the mammalian studies.
Phase-II: currently screening a total of 1,100 chemicals from a
broad range of sources (eg, industrial and consumer
products, food additives, failed drugs) to validate, expand,
and apply predictive models of toxicity.
e1k or EDSP21: currently screening a total of 2,100 chemicals
in a subset of ToxCast assays relevant to potential for
endocrine disruption.
15
ToxCast
Current ToxCast / Tox21
HTS Chemical Space
16
…
ToxCast
EDSP21
Tox21
1,000 2,000 10,000
500 ToxCast
assays ToxCast EATS
assays
Tox21 Assays
Pesticides (active and inert), industrial chemicals, consumer products,
marketed and failed pharmaceuticals, food additives, water contaminants,
natural human metabolites
17
The Home of TFomics TM
attageneattageneattageneattagene
Over 500 ToxCast HTS Assays from
Contractors and Collaborators
Compound Focus, Inc. a subsidiary of
EPA/ORD/NHEERL
Predictive Model dose response and
reverse dosimetry exposure metrics
and QSAR
Assay dimension (ToxCastDB)
18
CYP450s GPCRs Kin-Phs NRs proteases TRs / ICs
Biochemical HTS (292)
cellular
impedance
cellular
properties NPAs
trans-
activation
Cell-based assays (147)
human 1o cell
co-cultures
zebrafish
embryos murine ES cells
Complex culture / SMOs (179)
ToxRefDB
In vivo endpoints
~338,000 chemical-assay pairs tested in Phase-I
Organizes what we know about chemicals: in vitro, in vivo, pathways and exposure
Integrates critical information from disparate sources
Flexible tool for ranking chemicals based on weight-of-evidence for different prioritization tasks
SOURCE: Reif et al. 2010, Env Hlth Persp 118: 1714-1720 19
Toxicological Prioritization Index: ToxPi
Example: ToxPi for endocrine disruption
ToxPi: Prioritizing Chemicals for
EDSP21 and Potential Endocrine Activity
Prioritization Index = ToxPi = f(HTS assays + Chemical properties + Pathways)
Bisphenol A Tebuthiuron
Ingenuity pathways
ER
AR
TR Other
XME/ADME
KEGG pathways
LogP_TPSA
Predicted CaCO-2
Disease classes
Other NR
Reif et al (2010) Endocrine Profiling and Prioritization of
Environmental Chemicals Using ToxCast Data. Environ
Health Perspect. Epub PMID: 20826373
20
ToxPi Ranking of Chemicals
ToxScore
Reif et al, 2010
21
HPTE
Methoxychlor
Pyrimethanil
Tebuthiuron
30
9 C
he
mic
als
so
rte
d b
y T
oxP
i
highest lowest
Bisphenol A
Linuron
Bioactivity Signature Workflow
ToxCast HTS data
(AC50s)
ToxRefDB
UNIVARIATE ASSOCIATIONS • t-test (continuous assay data)
• classifier (2x2 contingency table)
• Fisher’s exact test (dichotomous data)
MULTIVARIATE MODELS • machine learning
• 5-fold cross-validation (80/20 split)
• sensitivity analysis (ROC curves)
MULTICELLULAR MODELS • knowledge-base (VT-KB)
• computer simulations (VT-SE)
• biological inferences / mechanisms
(+)ve (-)ve
(+)ve TP FP
(-)ve FN TN
23
Multigeneration Reproductive Toxicity Study (MGR)
Low Mid High
Dose
24
Predictive Model of Reproductive Toxicity
98 total chemicals w/ reproductive LOAEL (rLOAEL ≤ 500 mg/kg/day)
86 from 256 acceptable studies in ToxRefDB
12 from 39 unacceptable studies in ToxRefDB
Martin et al 2011 BOR
25
Model Features
Classification Rate = TP / Total # of Positives
Misclassification Rate = FP / Total # of Negatives Martin et al 2011 BOR
26 Classification Rate = TP / Total # of Positives
Misclassification Rate = FP / Total # of Negatives
Anti-/Androgenicity
Steroid Metabolism
Estrogenicity
Steroid/
Cholesterol
Metabolism
Steroidogenesis
Xenobiotic Metabolism
FSH-R or LH-R
Inhibitors???
Xenobiotic
Metabolism
Reserpine-like MOA
(Hypothalamus/Pituitary)
ReproTox Predictions for Conventional
and Alternative Plasticizers
27
Biol Reprod. 2011 Aug;85(2):327-39. Epub 2011 May 12.
Predictive model of rat reproductive toxicity from ToxCast high throughput screening.
Martin MT, Knudsen TB, Reif DM, Houck KA, Judson RS, Kavlock RJ, Dix DJ.
28
Systems Modeling: Blood Vessel Development
Endothelial proliferation,
migration & sprouting
(VEGF, Chemokines)
• Extracellular matrix
degradation
(uPAR, PAI-1, MMPs)
Neovascular stabilization
(Ang/Tie2)
29
Environ Health Perspect. 2011 Jul 25. [Epub ahead of print]
Environmental Impact on Vascular Development Predicted
by High Throughput Screening.
Kleinstreuer NC, Judson RS, Reif DM, Sipes NS, Singh AV,
Chandler KJ, Dewoskin R, Dix DJ, Kavlock RJ, Knudsen TB.
Cell-agent-based models
30
• stochastic cellular behaviors • specified cellular activities • PDE solvers for biochemical gradients • toolbox of morphogenetic processes • executes collective cell behavior • enables emergent properties
CompuCell3D.org
31
Molecular Signals and Cellular Behaviors of Angiogenesis
32
Angiogenetic field at time=0
ECs
ECt
MC
IC
SOURCE: Kleinstreuer et al. (in preparation)
33
CCL2
CXCL10
Ang1/
Tie2
VEGF
121
sFlt1
VEGF
165
MMPs
Virtual angiogenesis
5HPP-33 5HPP-33
34
In vitro SOURCE: Noguchi et al. 2005, Bioorg Med Chem Lett. 15 :5509-13. (experimental, 100 uM)
Thalidomide 5HPP-33 control
In silico SOURCE: Kleinstreuer et al. 2011, in preparation
(ToxCastDB, 40 uM)
quantitative prediction emergent from ToxCast HTS data input (AC50s)
TEST CASE: Thalidomide
and 5HPP-33
35
PFOS
PFOS
PFOS
PFOS
PFOA
PFAA_Alt1
PFAA_Alt2
PFAA_Alt3
PFAA_Alt4
36
HUVEC Proliferation: PFAA_Alt2 HUVEC Proliferation: PFAA_Alt1
HUVEC Proliferation: PFOS HUVEC Proliferation: PFOA
Inhibition of
Endothelial Cell Proliferation
PFOA, PFOS, and PFAA Alternatives:
in silico vasculogenesis assay
37
PFOA PFOS
PFAA_Alt1 PFAA_Alt2
Quantitative Effects: in silico chemical exposure
38
Total Cell Number Total VEGF Concentration
High-Throughput Risk Assessment (HTRA)
• Uses HTS data for initial, rough risk assessment of data poor chemicals
• Risk assessment approach
– Estimate upper dose that is still protective (~ RfD, BMD)
– BPAD (Biological Pathway Altering Dose)
– Compare to estimated steady state exposure levels
• Contributions of high-throughput methods
– Focus on molecular pathways whose perturbation can lead to
adversity
– Screen 100s to 1000s of chemicals in HTS assays
– Estimate oral dose using High-Throughput PK modeling
• Incorporate population variability and uncertainty
See Judson et al, 2011 ChemResTox
39
HTRA Outline
40
Identify biological pathways linked to adverse effects
Measure Biological Pathway Altering Concentration (BPAC) in vitro (ToxCast)
Estimate in vivo Biological Pathway Altering Dose (BPAD) (PK modeling)
Incorporate uncertainty and population variability estimates
Calculate BPAD lower limit – Estimated health protective exposure limit
Example concentration-response curves
41
Sample curves for BPA in two ER assays
Note that full concentration-response profiles can be measured, at
arbitrary spacing and to arbitrarily low concentrations (at moderate
cost for a given chemical)
In collaboration with Hamner Institutes / Rusty Thomas
Reverse ToxicoKinetics (rTK)
Human
Hepatocytes
(10 donor pool)
Add Chemical
(1 and 10 mM)
Remove
Aliquots at 15,
30, 60, 120 min
Analytical
Chemistry
-5
-4
-3
-2
-1
0
1
2
3
0 50 100 150
Ln C
on
c (u
M)
Time (min)
Nifedipine
1 uM initial
10 uM initial
Hepatic
Clearance
Human
Plasma
(6 donor pool)
Add Chemical
(1 and 10 mM)
Analytical
Chemistry
Plasma Protein
Binding
Equilibrium
Dialysis
42
Combine experimental data with PK Model to estimate
dose-to-concentration scaling Rotroff et al, 2010
Wetmore et al, 2011
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Triclosan Pyrithiobac-sodium
log (
mg/k
g/d
ay)
Rotroff, et al. Tox.Sci 2010 Wetmore et al Tox Sci 2012
43
Range of in vitro AC50
values converted to human
in vivo daily dose
Actual Exposure (est. max.)
margin
Combining in vitro activity and dosimetry
BPAD Probability Distribution • “Biological Pathway Altering Dose”
• BPAD = BPAC / Css / DR
• BPAC and Css / DR ~ log-normal
– BPAC: lowest AC50 for pathway assays
– directly measured in vitro
• Estimate protective BPAD as the lower 99% tail (BPAD99) of population
distribution
• Add in uncertainty and take the lower 95% bound on BPAD99 to give a
more protective lower bound
– BPADL99 (“L” for lower)
44
BPAD Distribution
Adverse Effect
Toxicity Pathway
Key Events
MOA
HTS Assays
Intrinsic
Clearance
Plasma Protein
Binding
PopulationsPK Model
Biological Pathway Activating
Concentration (BPAC)
Probability Distribution
Dose-to-Concentration
Scaling Function (Css/DR)
Probability Distribution
Probability Distribution
for Dose
that Activates
Biological Pathway
BPADL
Pharmacodynamics Pharmacokinetics
High Throughput Risk Assessment (HTRA)
46 BPAD Distribution
Pathway / Target / Model
CAR/PXR Pathway
ER / AR / Endocrine Targets
ReproTox Signature
DevTox Signature
Vascular Disruption Signature
Thyroid Cancer Signature
Exposure / Dose Low High
Lower no effect dose
Critical Effect
No detect No detect
No detect
HTRA Report Card For Chemical: ABC
Estimated Exposure
Conazoles and Liver
Hypertrophy • Conazoles are known to cause liver hypertrophy and
other liver pathologies
• Believed to be due (at least in part) to interactions with
the CAR/PXR pathway
• ToxCast has measured many relevant assays
• Calculate BPADL for 14 conazoles
– Compare with liver hypertrophy NEL/100
47
Conazole CAR/PXR Results
48
LEL, NEL
BPAD Range
Exposure estimate
NEL/100
BPAD Distribution
Conazole HTRA Summary • Rough quantitative agreement
– Significant BPADL vs. NEL/100 rank correlation (p=0.025)
– 12 of 14 chemicals have BPADL within 10 of NEL/100
– For only 3 is BPADL significantly less protective than
NEL/100
– All BPADL > Exposure estimate
• Some apples to oranges: human BPADL, rat NEL
– Rat RTK underway for some of these chemicals
49
• explicitly translate information on pathways of toxicity into endpoints
meaningful to populations (development, reproduction, survival)
• pathway from initiating molecular event (MIE) through a sequential
series of biological activities to an adverse outcome of relevance to
human or ecological risk assessment
SOURCE: Ankley et al. 2010
50
SOURCE: Ankley et al. 2010
51
Ways to think about AOP utility …
• build ‘AOP Cards’ for distinct chemicals and outcomes;
proposed AOPs will evolve from a global conceptual
framework
• Communication: link AOP cards to a systems dashboard or
Effectopedia (direct linkages to VT-KB?)
• Prototype: Disruption of embryonic vascular development;
hypothesis to guide experimental work and computational
models
52
Embryonic Vascular Disruption (pVDCs in ToxCast)
pVDC model Kleinstreuer et al. 2011, EHP 119: 1596-03
53
Herbert and Stainier. Nature Reviews 2011.
Vasculogenesis: de novo formation of embryonic blood vessels
Angiogenesis: sprouting of new blood vessels from pre-existing vascular structures
Adults (pathological or physiological): tumor growth, wound healing, uterine cycle, etc
54
e-library for vasculogenesis/angiogenesis prototype at: vembryowiki.epa.gov
55
• searched the Gene Ontology (GO) and Mammalian Phenotype (MP)
browsers of the Mouse Genome Informatics database
(http://www.informatics.jax.org/ ) for terms affiliated with the
disruption of vascular development
– abnormal vasculogenesis [MP:0001622; 72 genotypes, 73 annotations]
– abnormal angiogenesis [MP:0000260; 610 genotypes, 894 annotations]
– genes linked to ToxCast assays (65 target genes with bona fide roles in
vasculogenesis or angiogenesis, 50 of which had evidence of abnormal
embryonic vascular development based on genetic mouse models)
56
57 SOURCE: Knudsen and Kleinstreuer, 2011, Birth Def Res C (in press)
58
59
Fluazinam AOP: Embryonic Vascular Disruption
Placenta
Nutrient exchange
Altered physiology
Impaired blood flow
Embryo-Fetus
Altered
hemodynamics
Impaired growth
Dysmorphogenesis
Altered differentiation
Macrophage cells
↓cell motility
↓growth factor
release ECM
interactions
Chemokine
pathway
Endothelial cells
↓cytoskeletal cycle
↓angiogenic sprouts
Vessel
remodeling
VDCs
TGF-beta
Mural cells
↓cell recruitment
↓vessel stabilization
Rat Prenatal
Outcomes Cleft Palate,
Jaw/Hyoid
Fetal weight
reduction
Skeletal axial and
cranial
malformations
Trunk, Renal
defects
Rabbit Prenatal
Outcomes
Skeletal axial and
cranial
malformations
Pregnancy loss
Fluazina
m
VCAM,
CXCL10,
CCL2,Il1,
TNFa
uPA, uPAR,
PAI-1, MMP9
60
Butralin AOP: Embryonic Vascular Disruption
Rabbit Prenatal
Outcomes
Fetal weight
reduction
General fetal
pathology
Skeletal cranial
malformations
Placenta
Nutrient exchange
Altered physiology
Impaired blood flow
Embryo-Fetus
Altered
hemodynamics
Impaired growth
Dysmorphogenesis
Altered differentiation
Macrophage cells
↓cell motility
↓growth factor
release ECM
interactions
Chemokine
pathway
Endothelial cells
↓cytoskeletal cycle
↓angiogenic sprouts
VDCs
Mural cells
↓cell recruitment
↓vessel stabilization
Butrali
n
PAI-1, MMP9,
uPAR, uPA
VCAM, CCL2,
CXCL10
61
EPA CompTox Research
Benefits
-Less expensive
-More chemical
-Fewer animals
-Solution Oriented
ToxCast testing Bioinformatics/
Machine Learning
Bisphenol A Tebuthiuron
Thousand
chemicals
Chemical Toxicity Profile
-Innovative
-Multi-disciplinary
-Collaborative
-Transparent
