ToxCast and the Use of Human Relevant In Vitro Exposures: Incorporating high-throughput exposure and toxicity testing data for 21 st century risk assessments Russell Thomas Director National Center for Computational Toxicology BTS April 4, 2017 The views expressed in this presentation are those of the author and do not necessarily reflect the views or policies of the U.S. EPA
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ToxCast and the Use of Human Relevant In Vitro Exposures
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ToxCast and the Use of Human Relevant In Vitro Exposures:Incorporating high-throughput exposure and toxicity testing data for 21st century risk assessments
Russell ThomasDirectorNational Center for Computational Toxicology
BTSApril 4, 2017
The views expressed in this presentation are those of the author and do not necessarily reflect the views or policies of the U.S. EPA
National Center forComputational Toxicology
Using the ‘Golden Circle’ for the Transition to 21st Century Risk Assessment
Why?
What?
How?
National Center forComputational Toxicology
Understanding ‘Why’ We Need to Innovate In This Space…
Why?
0
10
20
30
40
50
60
70
Per
cent
of C
hem
ical
s
Acute Cancer
Gentox Dev Tox
Repro Tox EDSP Tier 1
<1%
Modified from Judson et al., EHP 2010
$1,000
$10,000
$100,000
$1,000,000
$10,000,000
Cos
t
Number of Chemicals /Combinations
Ethics Concerns Economics
Lack of Data
National Center forComputational Toxicology
‘Golden Circle’ of 21st Century Risk Assessment
Why?
What?
How?
National Center forComputational Toxicology
4
Risk Assessments Generally Contain a Standard Set of Components
New technologies and approaches will also have to cover these basic components
Phys Chem
Exposure
HazardDose Response, PK, and PODs
Risk SummaryUncertainty
Variability
National Center forComputational Toxicology
5
It All Starts With Chemistry…
• Chemical structure database of >700,000 unique substances with QC flags to link chemical structure with names and identifiers
• Consensus QSAR models for a range of physical chemical properties, environmental fate, and hazard characteristics
• Comprehensive physical-chemical property database (experimental and predicted)
https://comptox.epa.gov/dashboard
National Center forComputational Toxicology
6
Adding the High-Throughput Hazard Screening Component
ToxCast
Concentration
Res
pons
e
~600 Cell & biochemical
assays
~1,000 Chemicals
Tox21
~30 Cell & biochemical
assays
~8,000 Chemicals
Set Chemicals Assays Completion
ToxCast Phase I 293 ~600 2011
ToxCast Phase II 767 ~600 2013
ToxCast Phase III 1001 ~100 Ongoing
E1K (endocrine) 880 ~50 2013
National Center forComputational Toxicology
7
Broad Success Derived from High-Throughput Screening Approaches
Provide Mechanistic Support for Hazard ID
Group Chemicals by Similar Bioactivity and Predictive Modeling
Prioritization of Chemicals for Further Testing
Assays/Pathways
Che
mic
als
IARC Monographs 110, 112, 113
FIFRA SAP, Dec 2014
National Center forComputational Toxicology
8
Selected Criticisms of ToxCast
• You don’t include metabolism in your in vitro assays
• You don’t measure my favorite endpoint
• You don’t cover all of biological space
• In vitro assays are not normal biology
• Assay (x) in your battery did not get the right answer for my chemical
• My assay disagrees with your assay (x), so your approach is flawed
• You can’t test my favorite chemicals because of limitations in your methods (e.g., solvents, high LogP)
• Your assay descriptions to do not allow me to reproduce your results
• I get different answers when I analyze your dataUpdated from Bob K’s original list
National Center forComputational Toxicology
9
Beginning to Address Concerns for Increased Biological Coverage
Requirements:
• Whole genome• 384 well• Automatable
• Low cost
Thousands of chemicals Multiple Cell Types
X
National Center forComputational Toxicology
10
Comparing Sequencing Platforms and Developing Analysis Approach
TruSeqr2 0.74
TempO-Seqr2 0.75
Low Coverager2 0.83
Currently capable of assigning to >40 MOAs/MIEs based on transcriptional responses
MOA/MIE Analysis Pipeline
• Large scale screen of 1,000 chemicals (ToxCast I/II) in single cell type this summer
• Additional screens across multiple cell types/lines• Additional reference chemicals and genetic
perturbations (RNAi/CRISPR/cDNA)
National Center forComputational Toxicology
11
Beginning to Address Metabolic Competence
“Extracellular”Approach
“Intracellular”Approach
Chemicals metabolism in the media or buffer of cell-based and cell-free assays
Capable of metabolizing chemicals inside the cell in cell-based assays
More closely models effects of hepatic metabolism and generation of circulating
metabolites
More closely models effects of target tissue metabolism
Integrated approach to model in vivometabolic bioactivation and detoxification
-9 -8 -7 -6 -5 -40
1
2
3
4
5
L o g M e th o x y c h lo r (M )
Fo
ld I
nd
uc
tio
n
A c tive In a c tiv e E m p ty
C Y P 3 A 4 m R N A (n g )
RL
U
0 5 0 1 0 0 1 5 00
5 0 0 0 0
1 0 0 0 0 0
1 5 0 0 0 01 2 H o u rs
1 8 H o u rs
2 4 H o u rs
3 6 H o u rs
4 8 H o u rs
Collaboration with Unilever
National Center forComputational Toxicology
12
Framework for Integrating Hazard Components…
Tier 2Select In Vitro
Assays
Tier 1High-Throughput Transcriptomic
Assay
No Defined Biological Target or Pathway
Defined Biological Target or Pathway
Tier 3
Organotypic Assays and Microphysiological
Systems
Estimate Point-of-Departure Based on Likely Tissue- or Organ-level Effect without
AOP
Estimate Point-of-Departure Based on Pathway
Transcriptional Perturbation
Orthogonal confirmation
Identify Likely Tissue-, Organ-, or Organism
Effect and Susceptible Populations
In VitroAssays for other KEs
and Systems Modeling
Existing AOP No AOP
Estimate Point-of-Departure Based on AOP
Multiple Cell Types+/- Metabolic Competence
MOA/MIE Identification
-4
-3
-2
-1
0
1
2
3
-4 -3 -2 -1 0 1 2 3
Min
imum
In V
ivo
ToxR
efLo
w E
ffect
Lev
el
for R
at O
nly
(mg/
kg/d
)
Minimum In Vitro Rat Oral Equivalent Dose (mg/kg/d)
National Center forComputational Toxicology
Adding the High-Throughput Toxicokinetic Component
Rotroff et al., Tox Sci., 2010Wetmore et al., Tox Sci., 2012
Reverse Dosimetry
Oral Exposure
Plasma Concentration
In Vitro Potency Value
Oral Dose Required to Achieve Steady
State Plasma Concentrations
Equivalent to In VitroBioactivity
Human Liver Metabolism
Human Plasma Protein Binding
Population-Based IVIVE Model
Upper 95th Percentile CssAmong 100 Healthy
Individuals of Both Sexes from 20 to 50 Yrs Old
EPA ToxCast Phase I and II Chemicals • Currently evaluated ~700 ToxCast Phase I and II
chemicals• Models available through ‘“httk” R package