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10/18/2016
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Endocrine Disruption and the Environment Ellen Mihaich, Ph.D., DABT
PCPC Environmental Safety Workshop
October 25, 2016
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Endocrine Policy Forum
• Consortium of List 1 test order recipients and other interested/involved parties
• Self-funded
• Represents >95% of List 1 test order recipients
• Additional stakeholders include CLA, ACC, ACI, CSPA, API, and consulting companies
• Objective to address regulatory and policy issues, technical guidance and science advocacy
• To undertake collaborations to tackle common areas of interest
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US EPA’S LEGISLATIVE MANDATES (1996)
Food Quality Protection Act oMust screen pesticides (including inerts) for possible
estrogenic effects that may affect human health oMust use appropriate validated test systems or other
scientifically relevant information oCan include other endocrine effects
Safe Drinking Water Act (SDWA) oCan screen drinking water contaminants to which
substantial numbers of persons are exposed “Substantial numbers” and “may be found in sources
of drinking water “ not defined
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EDSP HISTORY: 1996 TO TODAY
1996-1998 Endocrine Disruptor Screening and Testing Advisory
Committee (EDSTAC)
o EPA multi-stakeholder advisory committee evaluates methods for priority setting,
screening, testing, and communications as key program elements
1999 to Today: Endocrine Disruptor Screening Program (EDSP)
o EPA adopts EDSTAC recommendations, creates EDSP, and in coordination with
OECD, works to standardize/validate screening and test protocols and guidelines
2009 1st Tier 1 List finalized: contained 67 pesticides and pesticide inerts
2013 Second Tier 1 List finalized; 109 chemicals (41 pesticides)
o Office of Management and Budget has not approved the Information Collection
Request (ICR) from EPA
o No test orders can be released until OMB approval of the ICR
Work is ongoing to prioritize the list of “10,000” chemicals for screening so
current List 2 chemicals may not be chosen ultimately
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EDSTAC Conceptual Framework
Tier 1 Screening ◦ Eleven In Vitro and In Vivo Assays
◦ Identify chemicals that can potentially interact with the endocrine
system (Estrogen, Androgen, and Thyroid pathways)
◦ Maximize sensitivity to minimize false negatives
◦ A battery of screening assays, with deliberate redundancy
Tier 2 Testing: ◦ Multigen studies in a range of species
◦ Confirm endocrine activity detected in Tier 1 and characterize
adverse effects and
◦ Establish NOAELs and LOAELs for risk assessment for regulation
of the substance
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OECD Conceptual Framework
• Compendium of data / test methods useful for the identification and assessment of potential endocrine disrupters
• Five levels with increasing information • Entering at all levels and exiting at all levels is possible
• Conceptual framework is not a test strategy • Enter and exit at any level
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OECD Conceptual Framework
Mammalian and Non-Mammalian Toxicology
Level 1
Existing Data and
Non-Test Information
• Physical & chemical properties, e.g. MW, reactivity, volatility,
biodegradability
• All available (eco)toxicological data from standardized or non-
standardized tests
• Read across, chemical categories, QSARs and other in silico predictions,
and ADME model predictions
Level 2
In vitro assays
providing data about
selected endocrine
mechanism(s)/
pathway(s)
(Mammalian and non-
mammalian methods)
• Estrogen or androgen receptor binding affinity
• Estrogen receptor transactivation (OECD TG 455 – [OECD TG 457])
• Androgen or thyroid transactivation (If/when TGs are available)
• Steroidogenesis in vitro (OECD TG 456)
• MCF-7 cell proliferation assays (ER ant/agonist)
• Other assays as appropriate
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OECD Conceptual Framework Mammalian Toxicology Non-Mammalian Toxicology
Level 3
In vivo assays providing
data about selected
endocrine
mechanism(s) /
pathway(s)
Uterotrophic assay (OECD TG 440)
Hershberger assay (OECD TG 441)
Xenopus embryo thyroid signalling assay (When/if TG is
available)
Amphibian metamorphosis assay (OECD TG 231)
Fish Reproductive Screening Assay (OECD TG229)
Fish Screening Assay (OECD TG 230)
Androgenized female stickleback screen (GD 140)
Level 4
In vivo assays providing
data on adverse
effects on endocrine
relevant endpoints
Repeated dose 28-day study (OECD TG 407)
Repeated dose 90-day study (OECD TG 408)
1-generation assay (OECD TG 415)
Male pubertal assay
Female pubertal assay
Intact adult male endocrine screening assay
Prenatal developmental toxicity study (OECD
TG 414)
Chronic toxicity and carcinogenicity studies
(OECD TG 451-3)
Reproductive screening test (OECD TG 421 if
enhanced)
Combined 28-day/reproductive screening
assay (OECD TG 422 if enhanced)
Developmental neurotoxicity (TG 426)
Fish sexual development test (OECD TG 234)
Fish Reproduction Partial Lifecycle Test (when/If TG is
Available)
Larval Amphibian Growth & Development Assay (OECD
241)
Avian Reproduction Assay (OECD TG 206)
Mollusc Partial Lifecycle Assays (TGs 242 and 243)
Chironomid Toxicity Test (TG 218-219)
Daphnia Reproduction Test (with male induction) (OECD
TG 211)
Earthworm Reproduction Test (OECD TG 222)
Enchytraeid Reproduction Test (OECD TG 220)
Sediment Water Lumbriculus Toxicity Test Using Spiked
Sediment (OECD TG 225)
Predatory mite reproduction test in soil (OECD TG 226)
Collembolan Reproduction Test in Soil (TG OECD 232)
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OECD Conceptual Framework
Mammalian Toxicology Non-Mammalian Toxicology
Level 5
In vivo assays
providing more
comprehensive data
on adverse effects on
endocrine relevant
endpoints over more
extensive parts of the
life cycle of the
organism
Extended one-generation reproductive
Toxicity Study (OECD TG 443)
2-Generation assay (OECD TG 416 most
recent update)
FLCTT (Fish LifeCycle Toxicity Test) (when
TG is available)
Medaka Extended One-Generation Test
(MEORGT) (TG 240)
Avian 2 generation reproductive toxicity
assay (OCSPP 890.2100)
Mysid Life Cycle Toxicity Test (when TG is
available)
Copepod Reproduction and Development
Test (when TG is available)
Sediment Water Chironomid Life Cycle
Toxicity Test (TG 233)
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Tier 1 * Tier 2
In vitro
ER binding
ER transcriptional activation
AR binding
Steroidogenesis, H295R
Aromatase, recombinant
In vivo (toxicology)
Uterotrophic assay 2-generation rat reproduction study
Hershberger assay (or extended 1-generation study)
Pubertal male assay
Pubertal female assay
In vivo (ecotoxicology)
Fish short-term screening assay Medaka extended 1-generation study
Amphibian metamorphosis assay Larval amphibian growth and development test
Avian 2-generation reproductive test
* Tier 1 screens cost $750,000 to $1 million per chemical
Current EDSP Test Guidelines
List 1 Results
52 chemicals (primarily pesticides) were screened in most, if not all, of the tier 1 screens
oEPA evaluated all of the data and wrote weight of evidence reports All reports and study summaries are publically available on the EPA
website
There was no evidence for potential interaction with any of the endocrine pathways for 20 chemicals
For 14 chemicals that showed potential interaction with one or more pathways, EPA already has enough information to conclude that they do not pose risks.
Of the remaining 18 chemicals, all 18 showed potential interaction with the thyroid pathway, 17 with the androgen pathway, and 14 with the estrogen pathway.
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EPA Overall Conclusions
For several of the chemicals displaying activity in the Tier 1 screening assays, EPA determined they have enough information to conclude that they do not pose risks. EPA recommended the following higher tier studies: o A comparative thyroid assay for 4 chemicals that showed interaction
with the thyroid pathway in mammals (not one of the validated Tier 2 tests)
o A medaka extended one-generation reproductive test (MEOGRT) for 13 chemicals that showed potential interaction with the estrogen or androgen pathways in wildlife
o A larval amphibian growth and development assay (LAGDA) for 5 chemicals that showed potential interaction with the thyroid pathway in wildlife
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A Tier 2 Quandary
• Tier 2 is for determining adverse effects and to provide data for risk assessment
• In regulatory toxicology we test at levels that achieve some effect
• How will we determine if that effect is specifically endocrine related? • Not as much of a problem in the US where regulations are based
on risk but,
• In Europe, deciding if the effect is “endocrine” or may have implications for authorization based solely on hazard
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Ecotoxicological Hazard and Risk Assessment
SETAC Pellston™ Workshop
• 48 global experts brought together to address the controversy
over hazard and risk Some scientists believe that EDS can be reliably assessed using the standard risk
assessment paradigm,
Others do not believe this is sufficiently precautionary and propose risk
management on the basis of hazard alone
• 6 case studies evaluated
• Cross-cutting issues identified
• Conclusion: Provided that environmental exposure, relevant
taxa and life-stages, delayed effects, and concentration-
response relationships are adequately characterized then
conducting environmental risk assessment of EDC is
scientifically sound.
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A Cross-Cutting Issue: Challenges in Assigning an Endocrine MOA
• Use of Weight of Evidence
• Establishing Biological Plausibility • Computational/in vitro approaches • In vivo concordance
• Essentiality of Key Events • Adverse Outcome Pathway (AOP)
• Empirical Evidence • Do the pieces fit??
• Confounding Factors • Systemic/overt toxicity • Stress • Infection • Organ specific toxicity • Nutritional status
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WHO/IPCS Definition
• WHO IPCS 2002:
An endocrine disruptor is an exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact organism, or its progeny, or (sub)populations.
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In silico
In vitro
In vivo screening
In vivo definitive
Weight of Evidence = Endocrine disruptor +
Mechanism
Incr
easi
ng
po
wer
Adverse Effect
Increasin
g po
wer
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The Dilemma of Many Endpoints
Chance of a Clean Chemical Screening “Clean”
(1- p)n
• Tier 1 Endocrine Screening Battery:
• 89 endpoints; if all independent
(0.95)89 = 0.01
(0.99)89 = 0.41
• 89 endpoints; if every 4th independent:
(0.95)22 = 0.32
(0.99)22 = 0.80
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If Only WoE Was This Easy!
Courtesy of Tim Ward 19
WOE Conceptual Framework
1. Define Explicit Hypotheses • 8 Total for Tier 1
• Potential E+ / E- / A+ / A- / T+ / T- /Steroidogenesis
induction/Steroidogenesis inhibition
2. Systematic Literature Review
3. Evaluate Data Quality
4. Weight Endpoints Quantitatively or Rank-Ordered
based on Explicit Criteria and Data
5. Weight Results within the context of known positives
and negatives
6. Develop Narrative Interpretation listing all assumptions
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Framework: Borgert et al. 2011 Reg Tox Pharm 61:185-191
Relevance weightings: Borgert et al. 2014, Birth Defects Research, Part B, 101:90-113
Ranking Endpoints
• No hypothesis can be decided on the results of a single assay – Battery Approach
• Rank the relevance (Wrel) of each endpoint for deciding each hypothesis • Rank #1: specific & sensitive for the hypothesis; interpretable
without other endpoints; in vivo endpoints rarely confounded by artifacts or non-specific activity.
• Rank #2: specific & sensitive for the hypothesis; interpretable alone, but less informative than Rank #1, often due to potential confounding; in vitro assays, many in vivo endpoints
• Rank #3: Relevant but only when corroborative of Rank #1 and #2 endpoints; many apical in vivo endpoints.
• No Response (NR)
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Relevance Rankings By Hypothesis: Estrogen Agonist
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Estrogen Agonist Hypothesis
Rank 1 Endpoints Rank 2 Endpoints Rank 3 Endpoints
FSTRA ERTA ER binding
Vitellogenin: increased in males ER agonism ER competitive binding
Uterotrophic Assay FSTRA FSTRA
Increased uterine weight (wet/blotted) Secondary sexual characteristics: decreased
tubercle score: males
Fecundity
Gonad histopathology: males Female behavior
Behavior: males GSI: males, females
Pubertal Female Assay Gonad histopathology: Follicular atresia
Age&weight @vaginal opening: reduced Fertilization success
Ovary weight: reduced Estradiol
Age at first estrous: reduced Testosterone
Pubertal Male Assay Pubertal Female Assay
Testes weight Growth
Testes histopathology: atrophy Estrous cyclicity
Pubertal Male Assay
Growth
Ventral prostate weight
Epididymides histopathology
Steroidogenesis Assay
Estradiol levels
Relevance Rankings By Hypothesis: Estrogen Antagonist
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Estrogen Antagonist Hypothesis
Rank 1 Endpoints Rank 2 Endpoints Rank 3 Endpoints
No rank 1 endpoints identified
ER Binding Aromatase
ER competitive binding Aromatase inhibition
Pubertal Female Assay Steroidogenesis
Age&weight @vaginal opening:
increased
Estradiol levels
Age at first estrous Pubertal Female Assay
FSTRA Estrous cyclicity
Vitellogenin: reduced in females Ovary histopathology: atrophy
Gonad histopathology: females Ovary weight: reduced, with atrophy
FSTRA
Fecundity
GSI
Behavior
Fertilization success
Estradiol
Testosterone
Relevance Rankings By Hypothesis: Androgen Agonist
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Androgen Agonist Hypothesis
Rank 1 Endpoints Rank 2 Endpoints Rank 3 Endpoints
Hershberger Pubertal Male Aromatase
Weights of Cowper's gland, seminal
vesicle, LAB, glans penis, ventral
prostate: concordance of all endpoints
Age&weight @preputial separation: if
accelerated
Aromatase inhibition
FSTRA Seminal vesicle + coagulating gland
weight (wet/blotted)
FSTRA
Secondary sexual characteristics:
decreased tubercle score: females
Ventral prostate weight Fecundity
Dorsolateral prostate weight Testosterone and estradiol levels
LABC muscle complex weight GSI
Epididymis weight Behavior
Testes weight Fertilization success
Testes histopathology Pubertal Female
Epididymides histopathology Growth
FSTRA Age&weight @vaginal opening
Vitellogenin: reduced in females Uterus weight
Gonad histopathology Ovaries weight
AR Binding Adrenals weight
Competitive binding - NO Uterus histopathology
Hershberger assay Ovary histopathology
Concordance of two or more endpoints
(see rank 1)
Pubertal Male
Growth
Testosterone levels
Steroidogenesis
Testosterone levels
Hershberger assay
Only one of five endpoints respond (see
Rank 1)
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Relevance Rank-FSTRA Endpoints
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Endpoint E+ E- A+ A- T+ T- Steroid.
induct.
Steroid.
inhib.
Vitellogenin R1; m R2; f R2; f R2; f NR NR NR R2; f
2o Sex
Characteristic
s
R2; m
tubercle
score
NR R1: f
tubercle
score
R2; m
tubercle
score
NR NR NR NR
Fecundity R3 R3 R3 R3 NR NR NR R3
Estradiol Opt.R3 Opt.R3 Opt.R3 Opt.R3; m NR NR NR Opt.R3
Testosterone Opt.R3 Opt.R3 Opt.R3 Opt.R3; f NR NR NR Opt.R3
Gonad
Histopatholog
y
R2 m R2; f R2 R2 NR NR NR R2; m
GSI R3; m
f
R3 R3 R3 NR NR NR R3
Behavior R2 m
R3 f
R3 R3 R3 NR NR NR R3
Fertilization
Success
R3 R3 R3 R3 NR NR NR R3
Adult Survival NR NR NR NR NR NR NR NR
Evaluating the Responses
• Positive or negative responses in Rank 1 • Preliminary indication of support for or against hypothesis
• Consistent positive or negative responses in Ranks 1 and 2 • Sufficient support for or against the hypothesis
• Consult Rank 3 for positive, not needed for negative
• Positive responses in Rank 2, but not Rank 1 • Strength of response and corroboration with Rank 3 critical, case-
by-case evaluation needed
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Hypothesis-Based WoE Framework • Uses explicit relevance rankings of endpoints derived a
priori
• hypothesis-based versus hypothesis-generating
• Transparent, objective and consistent interpretation of results for each hypothesis
• Systematic method to resolve inconsistencies in the data and focus on the most definitive information
• Can be applied to Tier 2 and other MoA as well
• Can be updated and altered with new information
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Helping Establish Biological Plausibility: High-Throughput Methodologies
Toxicity Testing in the 21st Century
o Criticism of EPA for how long the EDSP has taken and an interest in reducing animal testing has prompted new technologies and processes
o ToxCast™ program at the heart of EPA screening methodology for bioactivity
Battery of more than 700 in vitro, high-throughput screens
Approximately 15% are directly relevant to EDSP
Over 1,800 chemicals already screened and data in the public domain (actor.epa.gov/dashboard)
oExpoCast™ to be used with the
bioactivity screens in order to consider
exposure
Currently only for human health
assessment
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0.0
01
0.0
10.1 1
10
A R
C e ll a d h e s io n m o le c u le s
C e ll c y c le
C y p
C y to k in e
D e v e lo p m e n t
D N A b in d in g
E R
G P C R
G R
M R
N u c le a r re c e p to r
P R
P ro te a s e
P X R
S te ro id h o rm o n e
T ra n s p o rte r
E E 2 in th e T o x C a s t B a tte ry
A C 5 0 v a lu e s (M )
low
er l
imit
of
cy
toto
xic
ity
Cy
toto
xic
ity
po
int
Understanding Essentiality of Key Events: Generalized AOP
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Daniel L. Villeneuve et al. Toxicol. Sci. 2014;142:312-320
Chemical Property Profile
Receptor/Ligand Interaction
DNA Binding
Protein Oxidation
Gene Activation
Protein Production
Altered Signaling
Protein Depletion
Altered Physiology
Disrupted Homeostasis
Altered Tissue Development or Function
Lethality
Impaired Development
Impaired
Reproduction
Cancer
Toxicant Macro-Molecular
Interactions Cellular
Responses Organ
Responses Individual Responses
Structure
Recruitment
Extinction
Population Responses
Adverse Outcome Pathway
CN
NN
Aromatase inhibition
8
0
2
4
6
E2
(ng
/ml)
*
*
0
10
20
Vtg
(m
g/m
l)
*
**
Control 2 10 50
Fadrozole (µg/l)
8
0
2
4
6
E2
(ng
/ml)
*
*
0
10
20
Vtg
(m
g/m
l)
*
**
Control 2 10 50
Fadrozole (µg/l)
-20 -18 -16-14-12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12 14 16 18 20
Exposure (d)
0
2
4
6
8
10
(Thousands)
Cum
ula
tive N
um
ber
of E
ggs
Control
2
10
50
Fadrozole (ug/L)
**
*
-20 -18 -16-14-12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12 14 16 18 20
Exposure (d)
0
2
4
6
8
10
(Thousands)
Cum
ula
tive N
um
ber
of E
ggs
Control
2
10
50
Fadrozole (ug/L)
**
*
Reduced E2, Vtg synthesis
Impaired vitellogenesis Reduced fecundity
Molecular
initiating event
Key events or predictive
relationships spanning
levels of biological
organization
Adverse outcome relevant to
risk assessment
Adverse Outcome Pathway
Courtesy of G. Ankley 30
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Inhibition
Aromatase
Reduction
E2 levels
Reduction
Vitellogenin levels
Reduction
Fecundity;
filial ELS survival
Decline
Population trajectory
Strong Evidence
Moderate Evidence
Weak Evidence
Comparative Evidence
Direct PPZ evidence
in fish
Comparative PPZ
evidence in mammals
Comparative PPZ
high-throughput
evidence
No evidence,
potential KE
Key Events
Key Event Relationships
Legend
Possible Adverse Outcome Pathways Leading to a Reduction in Fecundity in Fish Exposed to Propiconazole
Activation
CAR/PXR
Increase
E2 biotransformation
Increase
Cytochrome P450
expression
Reduction
E2 synthesis
Decrease
Transcription/Translation
Increase
Oxidative stress
Dysfunction
Mitochondrial
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A Line of Empirical Evidence: The Acute to Chronic Ratio
• The acute endpoint (e.g. LC50) represents non-specific baseline toxicity.
• The chronic endpoint typically reflects a more specific mode of action.
• The more potent an endocrine disrupting agent, the higher will be the acute to chronic ratio (ACR). Ethinyl estradiol ACR = 5,700,000
Most industrial chemical ACR = 10-100
Most aquatic BPA ACRs range from 1.4 to 100 (highest = 290; F2 hatchability in fathead study)
Average = 25, median = 8
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Calculating ACR
• Acute and chronic aquatic toxicity studies were assembled from published studies and government reports
• Scientifically valid studies – data quality important
• Acute LC50 (mortality) and chronic NOEC for survival, growth and development, and reproductive endpoints were recorded
• Acute to chronic ratios were determined • Comparisons made between same species
• All endpoints, endpoints minus chronic survival, and reproduction only endpoints
• All species combined and then separated by fish, invertebrate, and aquatic plant/algae
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ACR Based on Taxa
Fish
Invertebrate
Algae
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Now, Does It Make Sense?
• Propiconazole aromatase inhibiting, E and A activity in the same range (or higher) as other non-endocrine bioactivity
0.0
01
0.0
10.1 1
10
A R
C e ll a d h e s io n m o le c u le s
C e ll c y c le
C y p
C y to k in e
D e v e lo p m e n t
D N A b in d in g
E R
G P C R
G R
M R
N u c le a r re c e p to r
P R
P ro te a s e
P X R
S te ro id h o rm o n e
T ra n s p o rte r
E E 2 in th e T o x C a s t B a tte ry
A C 5 0 v a lu e s (M )
low
er li
mit
o
f c
yto
to
xic
ity
Cy
to
to
xic
ity
p
oin
t
Propiconazole in the ToxCast Battery
• EE2 estrogenic bioactivity much lower than other
bioactivity
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0 1 2 3 4 5
P P AR
M a tr ix M e ta llo p r o te in a s e
G -P r o te in R e c e p to r
C yto k in e
X e n o b io t ic m e ta b o lis m
E s tr o g e n R e c e p to r
T o x c a s t R e s u lts fo r B P A
A C 5 0 v a lu e s ( M )
• Bioactivity
screen indicates
multiple MoA for
BPA in the same
concentration
range
EPA Developing Reference Chemicals
• Tiers 1 and 2 studies being conducted to broaden in vivo ecotox data set
• 5 FSTRAs, 5 AMAs, 3 MEORGTs, 3 LAGDAs
• Additional FSTRAs are planned with fathead minnow, zebrafish, and medaka, to evaluate interspecies responses
• Reference chemicals: bisphenol A, dibutyl phthalate, triclosan, 4-nonylphenol, ethyl paraben
• Data will be used to compare to high-throughput and computational responses and to help build AOPs
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What to Expect in 2016 (and beyond)
Tier 2 test orders o EPA needs to provide OMB with updated ICR to include other possible
tier 2 studies
o OMB needs to approve ICR
o Anticipated sometime in 2016 but will likely slip to 2017
Adoption of draft criteria in Europe o 1st half 2017
ToxCast/ExpoCast prioritization Science Advisory Panel o Scope uncertain but likely focused on androgen and thyroid high-
throughput screens and exposure (ExpoCast).
o 2017
“List 2” screening o Anticipated sometime after the SAP and possibly not until SAP provides
EPA with report (they have 90 days to complete)
o Anticipated first half 2017
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Questions?
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