The Tox21 Program
Christopher P. Austin, M.D.
Director, NIH Chemical Genomics Center
The Future of Chemical Toxicity Testing in the U.S.:
Creating a Roadmap to Implement the NRC’s Vision
and Strategy
June 21, 2010
The Tox21 Community
has become….
Tox21
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The Tox21 Community
Tox21
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Interagency Coordination on Biomolecular Screening
• Scientific collaboration began 2005 (NTP-NCGC) – 2006 (NTP-NCGC-EPA)
• Memorandum of Understanding on “High-Throughput Screening, Toxicity Pathway
Profiling and Biological Interpretation of Findings” (http://ntp.niehs.nih.gov/go/28213)
– Signed February 14, 2008 by:
• NIH/NIEHS/NTP by Dr. S. Wilson
• NIH/NHGRI by Dr. F. Collins
• EPA/ORD by Dr. G. Gray
– Points of Contact
• EPA - Bob Kavlock (Director, National Center for Computational Toxicology)
• NCGC - Chris Austin (Director)
• NTP - Ray Tice (Chief, NTP Biomolecular Screening Branch)
– Public status made available through monthly meetings of the EPA CPCP (Chemical Prioritization
Community of Practice)
• Leverage
– Pools resources for common goal
– Overcomes the resource limitations of a single agency
– Builds on existing expertise
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– Avoids the need to create a new administrative and support structure
The Tox21 Community
Activities
NTP
NCGC
Historical Toxicology Data
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Experimental Toxicology
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Ultra High-Throughput Testing
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Mid- to High Throughput Systems
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EPA
Lower Organism Model System
 C. elegans
 Zebrafish
In Vitro 3-D Model Systems
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Effect of Human/Rodent Genetic
Background on Toxic Effects
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Computational Toxicology
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Validation Experience
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(NICEATM-ICCVAM)
Tox21 activity matrix
Historical
EPA and
NTP data
Rodent
in vivo
Human
in vivo
Tox21
Rodent
in vitro
Human
in vitro
Approved
drug data
Tox21
Tox21: Organization
• Leadership: meets every 2 wks
– B. Kavlock (EPA), R. Tice (NTP), C. Austin (NCGC)
• Working Groups: chairs meet together every 4 wks
– Compounds, Assays, Informatics, Targeted Testing
– Co-leads from each agency
• Community: meets every 3 months
– Larger group of interested parties from 3 agencies
• Oversight: component Scientific Advisory Boards
– Reports at least once/yr
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The Current Tox21 Compound Library
• All have been evaluated in one or more toxicological tests
• NTP 1408 (1353 unique compounds)
– 1206 with NTP test data, 147 ICCVAM reference substances
– MW = 32-1168, calculated log p = -3 to 13.2
• EPA: 1462 (1384 unique compounds)
– MW = 58-516, calculated log p = -2.8 to 8.2
• ~400 compound overlap
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NTP compounds
Proof of Principle Toxicology qHTS
Unsupervised clustering in
combination with Dunn’s
cluster validity index is a
robust method for
identifying mechanisms of
action without requiring a
priori knowledge about
mechanisms of toxicity.
Full Tox21 Chemical Library: Fall 2010
Universe
13,247
With structures
8,277
Plausible P-chem (logP)
7,116
Current
Additional
NTP
1353
~1400
EPA
1330
~2800
NCGC
~3000 drugs
Library, Fall 2010
~10,000
Sources include NTP, EPA HPV, CCL, OPPIN, OW, Inerts, ToxCast,
DSSTox, EU Carcinogenomics, Pharmaceuticals, others
Tox21 IDs
ToxCast Phase I
Tox21
ToxCast Phase II
>50
NCCT/EPA
NIEHS/NTP
>500
309
# Chemicals
~1000
Environmental
Industrial/Tox
Drugs
Food Use
Environmental
Industrial/Tox
NIEHS/NTP
~10,000
Drugs
NIH/NCGC
NCCT/EPA
Tox21_3_######
Tox21_2_######
Office of Research and Development
National Center for Computational Toxicology
Tox21_1_######
NIH/NCGC
NIH Chemical Genomics Center
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75 scientists: biology,
chemistry, informatics,
robotics
>100 collaborations
with investigators
worldwide
Chemical genomics:
biological profiles of
chemical activity
Chemical probes of
novel targets,
rare/neglected diseases
NCGC Screening System 1:
BSL1/Kalypsys
Capacity:
3.0MM Assay Wells
5.0MM Compound
Wells
Throughput:
1400 plates/day
Readers:
ViewLux (2)
Acumen (2)
Envision (2)
New
Capabilities:
Automatic Loading and
Unloading stations
using commercially
available plate stackers
Dispense Inspection
Systems using
integrated CCD
cameras
“LTS”
“MTS”
HTS
10’s/day
1000’s/day
10,000’s/day
100,000’s/day
Throughput
Molecular mechanism
Immediate organismal relevance
HTS as done for drug discovery is not
suitable for toxicity testing
•
•
•
•
High false positive rate: up to 90%
High false negative rate: up to 70%
Prior probability of activity low
Cannot reliably “bank” or computationally
synthesize results
• Purpose is to generate a few leads for
subsequent chemical optimization
Quantitative High-Throughput Screening (qHTS)
• Conventional HTS done at single
concentration
– typically 10 uM
• qHTS tests all compounds at 15
concentrations
– Range = 5nM – 92uM
– Assay volumes 2-6 uL in 1536well plate format
– Concentration-response curve
generated for each compound
from primary screen
• Produces robust activity
profiles of all compounds
– Dramatically reduced FP and FN
• Throughput >200,000
concentration-response profiles
(2M wells) per week
– Entire Tox21 collection is tested
at 15 concs in single day
Tox21 assays screened at NCGC to date
• General toxicity
•
–
–
–
–
–
–
–
– Cytotoxicity assays
• Cell viability assay (measures
ATP)
– Apoptosis assays
• Caspase assays (measure activity
of Caspase 3/7, 8, 9)
– Membrane integrity assay
• LDH release
• Protease release
“Tox Pathways”
•
Targets
–
– Mitochondrial Toxicity assay
• Mitochondrial membrane potential
– Gene tox assays
–
• Micronucleus
• DNA repair
•
CREB
ER stress
HRE/Hypoxia
NFkB
P53
ARE
HSE
Nuclear receptor assays: AR, AhR,
ER, FXR, GR, LXR, PPARδ,
PPARγ, PXR, RXR, TRβ, VDR,
ROR
hERG channel
Inter-individual variation
–
87 HapMap lines
Ion Channel; 1; 1%
Apoptosis; 32; 27%
Nuclear Receptor; 30; 25%
Nuclear Recep
Genetox
Cytotoxicity
Signaling Pathw
Apoptosis
Signaling Pathway; 9; 8%
Ion Channel
Genetox; 14; 12%
Cytotoxicity; 32; 27%
Rapidly access (inhouse) biological profiles of chemical series
Browse via interactive heatmap that provides details of assay response
Cluster heatmap by assay response and chemical similarity
Approaches for Identifying Key Toxicity Pathways
- Toxicogenomic data
- Human disease - genetic associations
- The Pathway Universe
- Contract Research Organizations
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Limitations
• Not all in vitro assays are suitable for HTS and not all
•
substances can be tested in vitro (volatiles, solvent
requirement, practical concentration limitation)
Responses are for the most part limited to cellautonomous effects of parent compound
• Exposure (route, extent), metabolism
• Genetic heterogeneity relating to differences in
•
sensitivity
(A gene is not a pathway, a pathway is not a cell, a
cell is not an organ, an organ is not an animal……)
Ways to address these limitations
• View all results with skepticism“All results are
artifacts until proven otherwise”
• Confirm results in other assays of same
phenomenon and/or lower-throughput more
physiological systems
• Test same pathway/readout in multiple different
assays/approaches
• Move from cell lines to primary cells
• Incorporate metabolism (e.g., co-culture, S9)
• Determine responses in cells from differing
genetic backgrounds
Needs
• Determine which cell types (human vs rodent cell lines,
primary cells, stem cell-derived cells, etc.) are most useful
for HTS
• Develop comprehensive battery of pathway and phenotypic
assays for testing
• Incorporate metabolism and genetic heterogeneity
• Secondary assays to evaluate compounds ID’ed in HTS
(e.g., in vitro 3D organ models, C. elegans, zebrafish?)
• Obtain existing in vitro, experimental animal, and human
data on compounds from industry
• Developing cross-assay meta-analysis informatics
algorithms/browsers that enable identification of correlations
among the many in vitro assays and in vivo readouts
• Validate resulting testing strategies for reliability and
relevance, develop strategies for incorporation into
regulatory decision-making
Prediction is very difficult, especially if it's about the future.
- Niels Bohr
Contact Information
Chris Austin, NCGC:
austinc@mail.nih.gov
Ray Tice, NTP:
tice@niehs.nih.gov
Bob Kavlock, EPA:
kavlock.robert@epamail.epa.gov