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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