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U.S. EPA Endocrine Disruptor Screening Program
David Dix, Ph.D.
Director, Office of Science Coordination and Policy
Office of Chemical Safety and Pollution Prevention
U.S. EPA Endocrine Disruptor Screening Program
David Dix, Ph.D.
Director, Office of Science Coordination and Policy
Office of Chemical Safety and Pollution Prevention
April 11th 2016
Expert Meeting to Reach Scientific Consensus on
Endocrine Disruptors
Berlin, Germany
April 11th 2016
Expert Meeting to Reach Scientific Consensus on
Endocrine Disruptors
Berlin, Germany
EDSP Prioritization, Screening & Testing
Prioritization
Bioactivity/Exposure
Screening
Bioactivity
Testing
Dose-Response/Adversity
Relies on:
• QSARs
• ToxCast/ExpoCast
• Monitoring data
• OSRI
Relies on:
• QSARs
• ToxCast
• EDSP Tier 1 data
• OSRI
Relies on:
• EDSP Tier 2 data
• OSRI
Prioritization and Screening for bioactivity
Testing for dose-response and adverse effects
More chemicals Fewer chemicals
Slide 2 of 18
Pathways:
Estrogen
Androgen
Thyroid
EPA EDSP Tier 1
EPA EDSP Tier 2
OECD Level 1 OECD Level 2 OECD Level 3
OECD Level 4
OECD Level 5
ToxicantMolecular
Interaction
Cellular Response
OrganOrgan
SystemOrganism Population
Toxicity Pathway (part of AOP)
Adverse Outcome Pathway
MIE Adverse OutcomeKey Events
EDSP Screening and Testing
Expert Meeting to Reach Scientific Consensus on Endocrine Disruptors April 11th 2016 Slide 3
En
do
crin
e P
ath
wa
y
Tier 1 Tier 2
ER
Bin
din
g
AR
Bin
din
g
ER
Tra
nsc
rip
tio
na
l
Act
iva
tio
n
Aro
ma
tase
Ste
roid
og
en
esi
s
Ute
rotr
op
hic
He
rsh
be
rge
r
Pu
be
rta
l M
ale
Pu
be
rta
l Fe
ma
le
Am
ph
ibia
n
Me
tam
orp
ho
sis
Fis
h S
ho
rt T
erm
Re
pro
du
ctio
n
Ra
t 2
-ge
n/
EO
GR
TS
ME
OG
RT
LAG
DA
JQT
T
E+ ■ ■■
■ ■ ■ ■ ■ ■ ■
E- ■ ■ ■ ■ ■ ■ ■ ■ ■
A+ ■■
■ ■ ■ ■ ■ ■ ■
A- ■■
■ ■ ■ ■ ■ ■ ■
HPT
Axis■ ■ ■ ■ ■ ■
EDSP Screening and Testing by Pathway
PopulationCellular
ResponseOrgan
Organ
System
Molecular
InteractionOrganism
Expert Meeting to Reach Scientific Consensus on Endocrine Disruptors April 11th 2016 Slide 4
EDSP Pivot Announcement
June 19, 2015
FRL-9928-69
“Use of High Throughput Assays
and Computational Tools;
Endocrine Disruptor
Screening Program;
Notice of Availability and
Opportunity for Comment”
https://www.federalregister.gov/articles/2015/06/19/2015-
15182/use-of-high-throughput-assays-and-computational-
tools-endocrine-disruptor-screening-program-notice
Expert Meeting to Reach Scientific Consensus on Endocrine Disruptors April 11th 2016 Slide 5
Developing Alternative EDSP Assays
EDSP Tier 1 Battery of Assays Model Alternative Development
Estrogen Receptor (ER) Binding ER Model FY 2015
Estrogen Receptor Transactivation (ERTA) ER Model FY 2015
Uterotrophic ER Model FY 2015
Androgen Receptor (AR) Binding AR Model FY 2016
Hershberger AR Model FY 2016
Aromatase STR Model FY 2016
Steroidogenesis (STR) STR Model 2016
Female Rat Pubertal ER, STR & THY Models FY 2017
Male Rat Pubertal AR, STR & THY Models FY 2017
Fish Short Term Reproduction ER, AR & STR Models FY 2017
Amphibian Metamorphosis THY Model FY 2017
ER = estrogen receptor; AR = androgen receptor; STR = steroidogenesis; THY = thyroid
Expert Meeting to Reach Scientific Consensus on Endocrine Disruptors April 11th 2016 Slide 6
The EDSP is Currently Working on
the Individual Puzzle Pieces
The EDSP is Currently Working on
the Individual Puzzle Pieces
Estrogen
Androgen
Steroidogenesis
Thyroid
Toxicokinetics Exposure
Expert Meeting to Reach Scientific Consensus on Endocrine Disruptors April 11th 2016 Slide 7
ER Model: Performance Based
Approach to Establish Scientific Confidence
Judson et al. 2015, Tox Sci: “Integrated Model of Chemical
Perturbations of a Biological Pathway Using 18 In Vitro High
Throughput Screening Assays for the Estrogen Receptor"
Kleinstreuer et al. 2015, EHP: “A Curated Database of
Rodent Uterotrophic Bioactivity"
Browne et al. 2015, ES&T: “Screening Chemicals for Estrogen
Receptor Bioactivity Using a Computational Model"
Expert Meeting to Reach Scientific Consensus on Endocrine Disruptors April 11th 2016 Slide 8
Current Status on the Estrogen
Piece of the Puzzle
Current Status on the Estrogen
Piece of the Puzzle
ER Receptor
Binding
(Agonist)
Dimerization
Cofactor
Recruitment
DNA
Binding
RNA
Transcription
Protein
Production
ER-induced
Proliferation
R3
R1
R5
R7
R8
R6
N1
N2
N3
N4
N5
N6
A1
A2
A3
A4
A5
A6
A7
A8
A9
A10
A12
A13
A14
A15
A16
ε3
A11
Receptor (Direct
Molecular Interaction)
Intermediate Process
Assay
ER agonist pathway
Interference pathway
Noise Process
ER antagonist pathway
R2
N7
ER Receptor
Binding
(Antagonist)
A17
A18
Dimerization
N8
N9DNA
Binding
Cofactor
Recruitment
N10Antagonist
Transcription
Suppression
R4
R9
18 In Vitro Assays Measure ER-
Related Activity
Judson et al., Tox Sci. 2015
Browne et al., ES&T. 2015
Kleinstreuer et al., EHP 2016
True Positive 26 (25)
True Negative 11 (11)
False Positive 1 (0)
False Negative 2 (2)
Accuracy 0.93 (0.95)
Sensitivity 0.93 (0.93)
Specificity 0.92 (1.0)
True Positive 29 (29)
True Negative 8 (8)
False Positive 5 (1)
False Negative 1 (1)
Accuracy 0.86 (0.95)
Sensitivity 0.97 (0.97)
Specificity 0.67 (0.89)
In Vitro Reference Chemicals*
In Vivo Reference Chemicals*
*Values in parentheses exclude
inconclusive chemicals
Expert Meeting to Reach Scientific Consensus on Endocrine Disruptors April 11th 2016 Slide 9
Current Status on the Androgen
Piece of the Puzzle
Current Status on the Androgen
Piece of the Puzzle11 In Vitro Assays Measure AR-
Related Activity
Kleinstreuer et al. 2016 SOT poster #2651
True Positives 8
True Negatives 20
False Positives 1
False Negatives 0
Accuracy 0.97
Sensitivity 1.00
Specificity 0.95
Agonism
True Positives 18
True Negatives 8
False Positives 0
False Negatives 2*
Accuracy 0.93*
Sensitivity 0.90*
Specificity 1.00
Antagonism
*Two false negatives were correctly identified
when Tox21 AR antagonist screen was
evaluated at both agonist concentrations
Expert Meeting to Reach Scientific Consensus on Endocrine Disruptors April 11th 2016 Slide 10
Current Status on the Steroidogenesis
Piece of the Puzzle
Current Status on the Steroidogenesis
Piece of the Puzzle
Karmaus et al. 2016 SOT poster #2637
• 13 hormones were quantified using HPLC-MS/MS
• 2060 chemicals screened at single concentration
• 403 chemicals selected for concentration response (altered > 4 hormones)
• 120 additional chemicals selected for concentration response based on
other needs
ToxCast H295R Steroidogenesis Assay
Computational Model Under
Development (includes integration
with aromatase, TSPO/PBR assays)
Expert Meeting to Reach Scientific Consensus on Endocrine Disruptors April 11th 2016 Slide 11
Current Status on the Thyroid
Piece of the Puzzle
Current Status on the Thyroid
Piece of the PuzzleTHR Assays
TRHR Assay
Undergoing Online Validation (Tox21)
TSHR Assay
Undergoing Online Validation (Tox21)
Paul et al., In Review
Expert Meeting to Reach Scientific Consensus on Endocrine Disruptors April 11th 2016 Slide 12
Current Status on the Thyroid
Piece of the Puzzle
Current Status on the Thyroid
Piece of the Puzzle
Paul et al., In Review
Single-concentration Screen
Concentration Response Screen
Stratify by Selectivity
Simmons et al. 2016 SOT poster #1886
• Rat thyroid microsomes
• Fluorescent peroxidase
substrate (Amplex Ultra
Red)
• Validated against existing
kinetic guaiacol assay
• Luciferase, cytotoxicity
counterscreens
TPO Assay Details
Expert Meeting to Reach Scientific Consensus on Endocrine Disruptors April 11th 2016 Slide 13
Current Status on the Thyroid
Piece of the Puzzle
Current Status on the Thyroid
Piece of the Puzzle
Paul et al., In Review
NIS Inhibition (hNIS-HEK293T-EPA)
log [Compound] (M)
-10 -8 -6 -4 -20
50
100
150NaClO4
NaSCN
NaNO3
NaF
KClO4
KPF6
NaBF4
NaBr
High-Throughput Human NIS Screen
(with Secondary Screen in FRTL-5 rat follicular
cells)
Buckalew et al. 2016 SOT poster #1887
Murr et al. 2016 SOT poster #1888
Hallinger et al. 2016 SOT poster #1889
High-Throughput Deiodinase Screen
(In Development)
M. Hornung
Expert Meeting to Reach Scientific Consensus on Endocrine Disruptors April 11th 2016 Slide 14
Current Status on the Thyroid
Piece of the Puzzle
Current Status on the Thyroid
Piece of the Puzzle
SLCO1B1: thyroid hormone transporter in the liver
SULT2A1: not main SULT that metabolizes TH, but
demonstrated to have some activity
UGT1A1/1A6: mediate T(4) glucuronidation
THRSP: thyroid hormone-inducible hepatic protein
HIF1a: downstream to TRB1 activation via T3 or T4
signaling
HepaRG Gene Expression Assay
Bayesian model for NR regulation
Paul et al., In Review
J. Franzosa
J. Wambaugh
• HepaRG cells
• Treated for 48 h
• Gene expression measured
using Fluidigm 96.96
• Cytotoxicity measured in
parallel
LTEA Assay Details
Expert Meeting to Reach Scientific Consensus on Endocrine Disruptors April 11th 2016 Slide 15
Addressing the Metabolic
Competence Challenge
Addressing the Metabolic
Competence ChallengeAlginate Immobilization of Metabolic Enzymes (AIME)
Prototype Lids
CYP1A2 CYP2B6 CYP2C9 CYP3A40
10
20
30
Fo
ld A
cti
vit
y/F
ree
S9
(1µ
g)
Hours
Perc
en
t A
cti
vit
y
0 1 2 3 4 50
50
100
150 CYP1A2
CYP2B6
CYP2C9
CYP3A4
-3 -2 -1 0 1 2 30
50
100
150
CYP1A2
[Compound] log µM
Perc
en
t A
cti
vit
y
-3 -2 -1 0 1 2 30
50
100
150
CYP2B6
[Compound] log µM
Perc
en
t A
cti
vit
y
-3 -2 -1 0 1 2 30
50
100
150
CYP3A4
[Compound] log µM
Perc
en
t A
cti
vit
y
-3 -2 -1 0 1 2 30
50
100
150
CYP2C9
[Compound] log µM
Perc
en
t A
cti
vit
y
Furafylline
Thio-TEPA
Tienilic Acid
Ketoconazole
Amount of XME Activity in Microspheres
Small Molecule Inhibition of XME Activity
DeGroot et al. 2016 SOT poster #3757
Expert Meeting to Reach Scientific Consensus on Endocrine Disruptors April 11th 2016 Slide 16
The Toxicokinetics
Piece of the Puzzle
The Toxicokinetics
Piece of the Puzzle
Wambaugh et al., Toxicol Sci, 2015
• Additional in vivo rat TK data collected for 26 chemicals by NHEERL
collaborators and RTI contractors
• Literature in vivo rat TK data curated by TNO collaboratorsIn
Vit
ro P
red
icte
d (
ng
/ml)
In V
itro
Pre
dic
ted
(L
)
Hughes et al. 2016 SOT poster #3436
Expert Meeting to Reach Scientific Consensus on Endocrine Disruptors April 11th 2016 Slide 17
Current Status on the Exposure
Piece of the Puzzle
Current Status on the Exposure
Piece of the Puzzle3
rdG
en
• Averaging multiple exposure
models
• Challenge is to know which
exposure model to use for
which chemical
• Chemical use identifies
relevant pathways
SHEDS-HT
Literature
Models
CPcat Database
NHANES
Urine, Blood
and Serum
Data
~2
0,0
00
Ch
em
icals
Cu
rate
d D
SS
tox
+ F
orm
ula
tion
Chemical
Information
Exposure
PredictionsPredicted
Pathway
Non-Targeted
Analytical Screening
of Consumer
Product
Composition
Machine Learning
Approaches to
Predict Chemical
Use
Use Database
(FUSE)
Pilot Study
• 20 Product types x
5 products/type
• 2D GCxTOF/MS
Isaacs et al. 2016 SOT poster #1679
Phillips et al. 2016 SOT poster #1680
Wambaugh et al. 2016 SOT poster #1682Expert Meeting to Reach Scientific Consensus on Endocrine Disruptors April 11th 2016 Slide 18
Working to Assemble the EDSP
Screening Puzzle
Working to Assemble the EDSP
Screening Puzzle
Estrogen Androgen
SteroidogenesisThyroid
Toxicokinetics Exposure
Expert Meeting to Reach Scientific Consensus on Endocrine Disruptors April 11th 2016 Slide 19
OECD GD 34, Validation and International Acceptance of New or
Updated Test Methods
Validation is a process by which the reliability and relevance of a
test method are established for a specific purpose.
Validation
Expert Meeting to Reach Scientific Consensus on Endocrine Disruptors April 11th 2016 Slide 20
OECD GD 34, Validation and International Acceptance of New or
Updated Test Methods
Relevance and reliability should be characterized against data
generated with a list of reference chemicals (tested in the
original methods) accepted by regulatory agencies.
Reference chemicals: Chemicals selected for use in the
validation process, for which responses in the in vitro or in
vivo reference test system or the species of interest are
already known.
Validation
Expert Meeting to Reach Scientific Consensus on Endocrine Disruptors April 11th 2016 Slide 21
40 In Vitro Reference Chemicals
ER Model Validation- In VitroE
R A
UC
ER AUC Rank Order
True Positive 25
True
Negative
12
False
Positive
0
False
Negative
3
Accuracy 0.95
Sensitivity 0.89
Specificity 1.00
Inactive
Active
In Vitro (Lit)
Expert Meeting to Reach Scientific Consensus on Endocrine Disruptors April 11th 2016 Slide 22
77 In Vivo Reference Chemicals
ER Model Validation- In Vivo
ER AUC Rank Order
ER
AU
C
True Positive 29
True
Negative
46
False
Positive
1
False
Negative
1
Accuracy 0.97
Sensitivity 0.97
Specificity 0.97
Inactive
Active
Uterotrophic
Expert Meeting to Reach Scientific Consensus on Endocrine Disruptors April 11th 2016 Slide 23
Same Study Design (Immature Rat): BPA
Uterotrophic Reproducibility
LEL
or
MD
T (
mg
/kg
/da
y)
Injection Oral
Inactive
Active
Uterotrophic
Expert Meeting to Reach Scientific Consensus on Endocrine Disruptors April 11th 2016 Slide 24
Androgen Receptor
AR AUC Rank Order
AR
AU
C
InactiveActive
In Vitro
Expert Meeting to Reach Scientific Consensus on Endocrine Disruptors April 11th 2016 Slide 25
Chemicals with High Throughput Data
for Endocrine Screening
0 1000 2000 3000
NUMBER OF CHEMICALS
Current Anticipated
Estrogen Receptor
Androgen Receptor
Steroidogenesis
Thyroid
Metabolism:
• Pathway based
• Ongoing data generation,
analysis and validation
• Performance-Based Test
Guidelines being developed
Expert Meeting to Reach Scientific Consensus on Endocrine Disruptors April 11th 2016 Slide 26