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The Hamner Institutes for Health Sciences | ILSI FNSP Meeting | July 10, 2013 0
July 10, 2013
ILSI North America Food Nutrition & Safety Program
Russell Thomas, Ph.D.
Director, Institute for Chemical Safety Sciences
The Hamner Institutes for Health Sciences
Tox21 Update: Relevance of Tox21 to Nutrition
The Hamner Institutes for Health Sciences | ILSI FNSP Meeting | July 10, 2013 1
Broad-Based Movement in Toxicology
Towards In Vitro Testing
The Hamner Institutes for Health Sciences | ILSI FNSP Meeting | July 10, 2013 2
• A transformative redefinition of toxicity
testing is required to meet key design criteria
and take advantage of modern biology. • Broader coverage of chemicals, mixtures,
end-points, and life-stages
• Reduce cost and time
• Use fewer animals and reduce suffering
• More robust basis for risk assessment by
applying mechanistic data and relevant doses
• … a not-so-distant future where all routine
toxicity testing will be conducted in human
cells or cell lines in vitro by evaluating
perturbations of cellular responses in a suite
of toxicity pathway assays.
One Vision for Transforming Toxicity Testing
The Hamner Institutes for Health Sciences | ILSI FNSP Meeting | July 10, 2013 3
1
10
100
1000
10000
Nu
mb
er
of
Ch
em
icals
IRIS TRI
Pesticides Inerts
CCL 1 & 2 HPV
MPV
0
10
20
30
40
50
60
70
Pe
rce
nt
of
Ch
em
icals
Acute Cancer
Gentox Dev Tox
Repro Tox Judson, et al EHP (2010)
1920 1930 1940 1950 1960 1970 1980 1990 2000 2010 | | | | | | | | |
Acute toxicity studies
(LD50) developed to
standardized batches
of pharmaceuticals
Genotoxicity
assays
developed
Draize test
introduced for
eye irritants
Rodent cancer
bioassay
introduced
Continuous
breeding studies
for reproductive
toxicity
Reasons Underlying Effort to Modernize Tox
Testing
... and cannot efficiently assess safety of all the existing
chemicals or keep pace with those being developed
Current testing paradigm does not incorporate advances in
technology or biological understanding
The Hamner Institutes for Health Sciences | ILSI FNSP Meeting | July 10, 2013 4
ToxCast/Tox21 are U.S. Programs Using In Vitro
HTS to Address these Challenges
Tox21 ToxCast
• Began in 2005 with National Toxicology
Program and National Chemical Genomics
Center
• The U.S. Environmental Protection Agency
joined in 2006
• Five year MOU signed Feb 2008
• The U.S. Food and Drug Administration
joined in 2010
• Initiated inside a newly formed U.S.
Environmental Protection Agency Center –
National Center for Computational
Toxicology (NCCT)
• Began as a phased effort in 2007
• Planned Phase I 2008 - 2009
• Planned Phase II 2009 – 2010 (assay data
released by September, 2013)
The Hamner Institutes for Health Sciences | ILSI FNSP Meeting | July 10, 2013 5
What is In Vitro High-Throughput Screening and
How is it Being Applied to Toxicology?
• Use robotics to screen compounds in 96, 384, or
1536-well plates
• Screening assays are either cell-free (i.e.,
biochemical), cell-based, or use lower-order
organisms (e.g., zebrafish)
• Generally use re-purposed drug discovery
assays
• Performed in concentration-response format
called qHTS (quantitative HTS)
• Used as a broad survey of potential proximal
biochemical and cellular targets for chemicals
HTS
Throughput
Molecular mechanism
Immediate organismal relevance
10’s/day
1000’s/day
10,000’s/day
100,000’s/day
“MTS”“LTS”
The Hamner Institutes for Health Sciences | ILSI FNSP Meeting | July 10, 2013 6
Chemicals in the ToxCast and Tox21 Libraries • ToxCast Phase I library – 309 unique
chemicals
• ToxCast Phase II library – 776 unique
chemicals
• The ToxCast Phase I chemicals are
primarily pesticides with a small
number of industrial chemicals
• ToxCast Phase II are pesticides,
industrial chemicals, and failed drugs
• Tox21 Phase I library – 2737
chemicals
• Tox21 Phase II library – 8193 unique
chemicals
• Compounds particularly relevant to
food and nutrition
• 160 FDA GRAS substances
• 905 FDA EAFUS substances
Kavlock et al., Chem Res Toxicol., 2012
NCCT Presentation, May 14, 2009
The Hamner Institutes for Health Sciences | ILSI FNSP Meeting | July 10, 2013 7
Assays in the ToxCast and Tox21 Portfolio
• ToxCast Phase I – 667 in vitro
assays across 10 platforms
• ToxCast Phase II – ~700 in vitro
assays across 13 platforms
• The ToxCast assays cover 327
gene targets with at least one gene
in 293 out of 592 KEGG pathways
• The ToxCast assays are primarily
re-purposed pharmaceutical assays
run by commercial vendors
• Tox21 – ~50-100 in vitro assays
• The Tox21 in vitro assays are
primarily cell-based reporter and
cytotox assays run at the National
Chemical Genomics Center
Kavlock et al., Chem Res Toxicol., 2012
Tox21 Presentation, June 2010
Nuclear Receptor; 30; 25%
Genetox; 14; 12%
Cytotoxicity; 32; 27%
Signaling Pathway; 9; 8%
Apoptosis; 32; 27%Ion Channel; 1; 1%
Nuclear Receptor
Genetox
Cytotoxicity
Signaling Pathway
Apoptosis
Ion Channel
The Hamner Institutes for Health Sciences | ILSI FNSP Meeting | July 10, 2013 8
ToxCast and Tox21 Data Analysis and Release
ToxCast
• Release of data through multiple peer-
reviewed publications
• Many analysis related publications
Tox21
• Release of data primarily through on-line
repositories PubChem and CEBS
• Fewer analysis related publications
The Hamner Institutes for Health Sciences | ILSI FNSP Meeting | July 10, 2013 9
Analysis of the ToxCast In Vitro Screening Data
for Predicting Hazard
Model #1
... Model #84
60 In Vivo Endpoints
Chemical
Structural
Descriptors
~600 In Vitro High
Throughput
Screens
Broad-based Evaluation
of In Vitro-to-In Vivo
Predictive Performance
Thomas et al., Tox Sci., 2012
8 Classification Algorithms,
~12 Feature Selection, 84
Classification Model
Combinations
309 EPA ToxCast
Chemicals
The Hamner Institutes for Health Sciences | ILSI FNSP Meeting | July 10, 2013 10
Current High-Throughput In Vitro Assays Have
Limited Capability of Predicting In Vivo Hazard In Vitro Assays
Chemical Structurep = 0.0709
Range of
performance
across the 84
statistical
models
The ideal is a balanced
accuracy of 1
60 In Vivo Endpoints
0.5 is like flipping a
coin
Thomas et al., Tox Sci., 2012
The Hamner Institutes for Health Sciences | ILSI FNSP Meeting | July 10, 2013 11
But, In Vitro HTS Assays May Be Useful to Identify
Potential Molecular Initiating Events
In VivoAnimal Response
In Vitro
Assay
Response
Positive Negative
Positive A B
Negative C D
Odds Ratio = (A/B)/(C/D) Log2 Odds Ratios for All Significant In Vitro Assay: In Vivo Endpoint
Combinations
In Vivo Endpoint In Vitro Assay Odds
Ratio
p-value
Chronic Study, Rat
Acetylcholineaserase
Inhibition
Biochemical, Rat
Acetylcholinesterase Binding
87.0 < 0.0001
Biochemical, Human
Acetylcholinesterase Binding
60.6 < 0.0001
Biochemical, Human
Butyrylcholinesterase
Binding
12.8 0.0003
Biochemical, Bovine
Progesterone Receptor
Binding
9.6 0.0007
Chronic Study, Mouse
Liver Tumors
Cellular, Human Peroxisome
Proliferator Activated
Receptor Alpha Reporter
27.8 0.0021
Biochemical, Guinea Pig
Opioid Receptor, Kappa 1
Binding
22.4 0.0074
Biochemical, Human
Serotonin Transporter
Binding
22.4 0.0074
aThe in vitro assays for each in vivo endpoint were filtered to remove those
with odds ratios < 5 and p-values > 0.01.
Thomas et al., Tox Sci., 2012
The Hamner Institutes for Health Sciences | ILSI FNSP Meeting | July 10, 2013 12
Current Limitations of the ToxCast andTox21
Approach
• The current in vitro assays may not capture the biochemical and cellular
responses in the in vivo tissues with adequate fidelity (e.g., metabolic
competence, transporter expression , cell-cell interactions)
• The current suite of assays may not reliably capture context-specific
outcomes.
• The current set of assays may not provide sufficient coverage of pathways,
protein targets, and cell types.
• Significant cross-species differences between the in vitro assays and in vivo
endpoints being predicted.
• The current analysis does not capture assay efficacy (only potency)
• The number of positive chemicals present for each endpoint may not be
sufficient to redundantly capture the broad array of mechanisms leading to
in vivo toxicity.
The Hamner Institutes for Health Sciences | ILSI FNSP Meeting | July 10, 2013 13
An Example of Why Context is Important from a
Dietary Perspective
The Hamner Institutes for Health Sciences | ILSI FNSP Meeting | July 10, 2013 14
An Example of Why Context is Important from a
Dietary Perspective
13 men
Pre-diet
Low Fat
High Fiber Diet
Exercise
Prostate Cancer Cell Growth
Apoptosis
TP53 Levels
The Hamner Institutes for Health Sciences | ILSI FNSP Meeting | July 10, 2013 15
An Example of Why Context is Important from a
Dietary Perspective
Tymchuk et al. J Urology 2001
Ngo et al. Cancer Causes Cntrl 2002
In addition, TP53 protein was induced in the diet
and exercise group.
Does this mean that diet and exercise would be
considered toxic/hazardous?
The Hamner Institutes for Health Sciences | ILSI FNSP Meeting | July 10, 2013 16
A Project is in Progress to Provide Dietary Context
to the Tox21 and ToxCast Datasets
• Screening ~30 fruit and vegetable juices in concentration
response format across ToxCast and Tox21 in vitro assays • 6-8 concentrations with maximum of 10%
• Screen in a subset of cell and biological assays (Bioseek, ACEA,
Attagene, Novascreen assays)
• Follow protocol of Charles et al., Food Chem Toxicol (2002)
The Hamner Institutes for Health Sciences | ILSI FNSP Meeting | July 10, 2013 17
Screening Fruit and Vegetable Extracts
Charles et al., Food Chem Toxicol, 2002
The Hamner Institutes for Health Sciences | ILSI FNSP Meeting | July 10, 2013 18
A Project is in Progress to Provide Dietary Context
to the Tox21 and ToxCast Datasets
• Screening ~30 fruit and vegetable juices in concentration response
format across ToxCast and Tox21 in vitro assays • 6-8 concentrations with maximum of 10%
• Screen in a subset of cell and biological assays (Bioseek, ACEA, Attagene,
Novascreen assays)
• Follow protocol of Charles et al., Food Chem Toxicol (2002)
• The NTP Tox21 group is interested in collaborating and want to also
screen the juices across a subset of the NCGC assays
• The juices will be obtained from “organic” fruits and vegetables
obtained from the local supermarket, but will also be screened for
pesticide residues and heavy metals • Support provided by ILSI Technical Committee on Food and Chemical
Safety.
The Hamner Institutes for Health Sciences | ILSI FNSP Meeting | July 10, 2013 19
Acknowledgements My Lab
ACC-LRI
ILSI
Funding
Barbara Wetmore
Reetu Singh
Bethany Parks
Linda Pluta
Chad Deisenroth
Filipe Muhale
Joe Trask
Briana Foley
Michael Black
Eric Healy
Longlong Yang
Joshua Harrill
External Collaborators (cont)
Jay Zhao (EPA)
Ila Cote (EPA)
David Dix (EPA)
Daniel Rotroff (EPA)
John Wambaugh (EPA)
Richard Judson (EPA)
Keith Houck (EPA)
Bob Kavlock (EPA)
Matt Martin (EPA)
David Reif (EPA)
Cornelia Smith (CellzDirect)
Kimberly Freeman (CellzDirect)
Stephen Ferguson (CellzDirect)
Harvey Clewell
Mel Andersen
Mark Sochaski
Brittany Allen
Katherine Cantwell
Ed LeCluyse
Institute Collaborators
External Collaborators
Russ Wolfinger (SAS)
Lili Li (SAS)
Wenjun Bao (SAS)
Tzu-Ming Chu (SAS)
Bruce Allen (BAC)
Jeff Gift (EPA)
Woody Setzer (EPA)
Scott Wesselkamper (EPA)
Nina Wang (EPA)
Jason Lambert (EPA)
Janet Hess-Wilson (EPA)
Dan Petersen (EPA)