Of Lists and Legends: An Endocrine Disruptor Update

Ellen Mihaich, Ph.D., DABT CIR Expert Panel Meeting December 5, 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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What Is An Endocrine Disruptor?

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• 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.

• Resource and regulatory consequences

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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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 not may have

implications for authorization based solely on hazard

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If Only WoE Was This Easy!

Courtesy of Tim Ward 11

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 89 endpoints; if every 4th independent: (0.95)22 = 0.32

Critical Elements in a Weight of Evidence Assessment

• Reliability of information o Quality of the study, transparency of reporting

• Relevance of the information o Appropriate for the question being asked

• Adequacy (or usefulness) of the information o Fit for regulatory decision-making

• Consistent pattern of response o In support of a particular hypothesis

Borgert et al., 2011. Reg Toxicol Pharmacol 61:185-191. Borgert et al., 2014. Birth Defects Res Part B: Develop Reprod Toxicol 101:90-113. de Peyster and Mihaich, 2014. Regul Toxicol Pharmacol 69:348-370.

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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

13 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: 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)

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

Directionality Important in WoE

• Thyroid agonist hypothesis and Triclosan

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Rank Assay Endpoint(s) Response Reference(s)

1 Amphibian

Metamorphosis Assay

Asynchronous development

No effect [1] [2]

Thyroid histopathology

No effect [1] [2]

2

Amphibian Metamorphosis

Assay

Developmental stage No effect [1] [2]

Hind limb length No effect [1] [2]

Pubertal Male Assay

Thyroid weight

Serum T4 ↓ {3}

Serum TSH No effect [3]

Pubertal Female Assay

Serum T4 ↓ {4}

Serum TSH No effect [4]

3

Amphibian Metamorphosis

Assay

Snout-vent length No effect* [1] [2]

Wet weight No effect* [1] [2]

Pubertal Female Assay

Growth No effect [4] Age & weight @ vaginal opening ↓

{4}

Blood chemistry

Pubertal Male Assay

Growth No effect [3] Age & weight @ preputial separation

No effect [3]

Pituitary weight No effect [3]

*Snout-vent length and wet weight were reported to have increased in Fort et al. (2011), however, a thyroid agonist would be expected to decrease these parameters so they are shown as “no effect” for this hypothesis. [1] Fort et al. (2010) [2] Fort et al. (2011) [3] Zorrilla et al. (2009) [4] Stoker et al. (2010)

Mihaich et al. in press, Crit Rev Toxicol – WoE for Triclosan

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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EPA is Shifting Towards Faster Screening 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 .00 1

0 .01

0 .1 1 1 0

A RC e ll a d h e s io n m o le c u le s

C e ll c y c leC y p

C y to k in eD e v e lo p m e n tD N A b in d in g

E RG P C R

G RM R

N u c le a r re c e p to rP R

P ro te a s eP X R

S te ro id h o rm o n eT 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 (µM )

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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

(mg/

ml)

*

* *Control 2 10 50

Fadrozole (µg/l)

8

0

2

4

6

E2

(ng/

ml)

*

*

0

10

20

Vtg

(mg/

ml)

*

* *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 20Exposure (d)

0

2

4

6

8

10

(Tho

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Num

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f Egg

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Control21050

Fadrozole (ug/L)

***

-20 -18 -16-14-12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12 14 16 18 20Exposure (d)

0

2

4

6

8

10

(Tho

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ds)

Cum

ulat

ive

Num

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f Egg

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Control21050

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 23

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

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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 .00 1

0 .01

0 .1 1 1 0

A RC e ll a d h e s io n m o le c u le s

C e ll c y c leC y p

C y to k in eD e v e lo p m e n tD N A b in d in g

E RG P C R

G RM R

N u c le a r re c e p to rP R

P ro te a s eP X R

S te ro id h o rm o n eT 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 (µM )

low

er

lim

it o

f c

yto

tox

icit

y

Cy

toto

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ity

po

int

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 (µ M )

• Bioactivity screen indicates multiple MoA for BPA in the same concentration range

Litany of Lists In 2002, BKH, under contract to DG Environment developed a list of

substances for further evaluation. Other regulatory authorities (e.g., Washington State, Denmark) have used this list to

develop their own lists of endocrine disruptors

UNEP/IPCP “Overview Report I: A Compilation of Lists of Chemicals Recognised as Endocrine Disrupting Chemicals (EDCs) or Suggested as Potential EDCs“ Compilation of lists developed for different purposes Not specific for potentially endocrine active chemicals, Utilizing different methodologies and criteria for designating a chemical as endocrine

active or an endocrine disruptor, No requirement for appropriate data quality or weight of evidence

Endocrine Active Substances Information System (EASIS) Released by the European Commission – DG Environment Searchable database of >500 substances Disclaimer that it has both positive and negative substances No WoE, systematic literature search, or quality criteria apparent https://ec.europa.eu/jrc/en/new/launch-endocrine-active-substances-information-system-easis

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State of the Science of Endocrine Disrupting Chemicals

2002 report

World Health Organization (WHO) and

International Programme on Chemical Safety (IPCS)

2012 report

World Health Organization (WHO) and

United Nations Environment Programme (UNEP)

Critical Review: WHO-UNEP 2012 Report by Lamb et al.

• Reviewed described process and methods • Compared to 2002 report • Evaluated all chapters to identify common strengths and

weaknesses • Identified key issues and concerns

• Select examples of unsupported claims • Not a comprehensive assessment or re-evaluation

• Open access manuscript published in peer reviewed journal Reg Tox Pharm: Lamb et al. 2014.Critical comments on the WHO-UNEP State of the Science of Endocrine Disrupting Chemicals – 2012 http://www.ncbi.nlm.nih.gov/pubmed/24530840

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Overall Concerns Identified by Lamb & colleagues in the WHO-UNEP 2012 Report

• Not an objective, state-of-the-science review • Not an update of WHO-IPCS 2002 report • Causation is often inferred – not established • Controversial topics are poorly addressed • Summary for Decision-Makers

• Not a summary of the main report • New “concerns” raised that were

not in full report

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Example: Not an Update Semen/Sperm Quality

WHO-IPCS 2002:

WHO-UNEP 2012:

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Cost of Endocrine Disruptors • In 2015, a series of studies claimed that human

exposure to EDCs is costing the EU hundreds of billions of Euros

• Recently, a similar paper targeted the US, with health costs estimated even higher than in Europe.

• Widely criticized by economists, scientists, and even the European Commission

• Most of the cited chemicals do not meet the WHO-IPCS definition of an ED

• Incomplete and inconsistent correlations between exposure and specific health outcomes

• Lack of transparency in methodology • However, flashy headlines are news!

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Endocrine Assessments in Japan

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• Japan is currently developing assays with which to evaluate endocrine properties

• Many are part of the OECD test guideline development program

• The government is also testing selected materials (some based on effects, some based on detection in monitoring programs)

• SPEED 98 and ExTEND Programs

• Considering two-tiered approach that still includes ecological risk assessment

• Considering in vitro assays used to prioritize to enhance efficiency

Endocrine Assessments in China

• Draft of an agro-industrial standard ‘Evaluation Methods for Pesticide Endocrine Disruptors became available in late 2014

• Final version implemented April 2016 • Focus on in vivo mammalian tests

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Endocrine Assessments in China

• 2 tier system (similar to US-EPA) • Phase 1

• In vitro tests: ER Transcriptional activation and Steroidogenesis assays

• In vivo tests: Uterotrophic, Hershberger and Pubertals • Phase 2

• Extended One-Generation Reproductive Toxicity • Triggered by ‘positives’ at phase 1

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Elsewhere in the Asia Pacific Region

• Most APAC countries are monitoring developments elsewhere in the world

• Australia publically stated that from a regulatory perspective they consider “endocrine disruption” no different than other modes of action suggesting it will be regulated using a risk-based approach

• Some APAC countries are beginning to request additional OECD endocrine studies if reproductive effects are noted in the standard pesticide submissions

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Endocrine Assessments in Latin America

• Brazil and Chile are the only Latin American countries where the endocrine issue is under discussion by the regulators

• Although there is gaining interest on the part of NGOs, particularly in Argentina, concerning pesticides and ED

• Brazil appears to be inclined to a hazard-based approach similar to the EU, although they appear open to other possibilities

• Chile, so far, does not appear to be considering a hazard-based approach

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Quick Quiz Should This Chemical Be Banned?

Assay Result In Vitro ER Transactivation Negative

In Vitro Steroidogenesis Increased Estrogen production at 500 and 1000 µM

Male Rat Pubertal Assay ⬇ body weight. (100 mg/kg/d)

⬇ prostate weight (> 20 mg/kg/d)

⬇ seminal vesicle weight (> 20 mg/kg/d)

⬆ testis weight (> 20 mg/kg/d)

⬇ plasma progesterone (dose-related)

Female Pubertal Assay ⬇ ovarian weight (100 mg/kg/d)

⬇ adrenal gland weight (100 mg/kg/d)

⬆ mRNA levels for FSH (100 mg/kg/d)

⬇ mRNA levels for prolactin (100 mg/kg/d)

⬆ mean length of estrus cycle (100 mg/kg/d)

⬇ (delay) vaginal opening (> 5mg/kg/d)

⬆ age at first estrus by 8 days (> 5 mg/kg/d) From Tinwell H. et al., 2013. Regul Toxicol Pharmacol.66:184-196.

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Thank you.

Questions?