Endocrine Disruption: Screening and Testing for Environmental Effects from the US Industry Perspective

Lisa Ortego, PhD, DABT

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Topics: Drivers of the Issue

US Legislative Mandates

Structure of the US EDSP

Tier 1 Screens

OSRI

Weight of Evidence

Tier 2

List 1

List 2

EDSP Future

Environmental Endocrine Disruption Incidences Cited in the WHO-UNEP 2012 Report

• ‘‘endocrine disrupting POPs such as PCBs and organochlorines’’ causing wildlife population declines

• 2012 report links DDT in bird populations and tributyl tin (TBT) in snail populations• The assertion is that these populations are rebounding after the chemical

use was restricted• The WHO-UNEP 2012 report concluded that there was sufficient evidence to

demonstrate that a mixture of PCBs and DDT caused adrenocortical hyperplasia and a Cushing-like condition in Baltic seals

Hypothetical as to whether an endocrine MoA is the cause of these observations and even more so that the chemicals cited may be the cause (Lamb et al., 2014)The literature and news are full of examples of adverse effects on wildlife assumed to be associated with endocrine disease and a chemical causation –but the causation is largely inferred without adequate evidence or a thorough examination of all information and possible explanations

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Events Leading to Regulatory Intervention1962 Rachel Carson’s book “Silent

spring”

1991 Wingspread Conference on chemicals and sexual development

1994 National Academy of Science forms Endocrine Modulators Panel

1996 “Our Stolen Future” published by Theo Colburn and colleagues to sensitize the general public

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Events - continued1996 Endocrine Disrupters Screening and Testing

Advisory Committee (EDSTAC) 1996 Weybridge workshop, report on impact of ED

on human health and the environment

1998 EDSTAC Committee issues its Final Report (August) and validation begins

1999 NAS releases report “Hormonally Active agents in the Environment” (August)

2009 ECETOC publishes guidance for evaluating ED effects and weight of evidence approaches

2009 Initial list of 67 chemicals for screening in US

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• Food Quality Protection Act (FQPA)– Must evaluate pesticides (including inerts) for

estrogenic effects that may affect human health – Must use appropriate validated test systems or

other scientifically relevant information– Can include other endocrine effects

• Safe Drinking Water Act (SDWA)– Screen drinking water contaminants to which

substantial numbers of persons are exposed

US EPA’s Legislative Mandates(August 1996)

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EDSP History: 1996 - Today

•Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC)1996-1998• Expanded remit to test for androgen and thyroid effects in addition to

estrogen, includes impact on wildlife• Proposed a 2 Tier program

•Endocrine Disruptor Screening Program (EDSP)• 1999-Today: EPA adopts EDSTAC recommendations, creates EDSP

and in coordination with OECD standardizes /validates screens and tests

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EDSP Tier 1 Screening AssaysTier 1 Screening

• A battery of 11 screening assays, with deliberate redundancy• In Vitro and In Vivo Assays• Identify chemicals that can potentially interact

with the endocrine system (E, A, and T pathways)• High degree of false positives• Reliability of some endpoints still in question

• As such, an interpretation report (Weight of Evidence or WoE) is necessary for each active describing and interpreting all Tier 1 and OSRI for potential endocrine effects

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Other Scientifically Relevant Information•OSRI is information that informs as to whether the substance may have an effect that is similar to that produced by a substance that interacts with the estrogen, androgen, and/or thyroid hormonal systems.

•OSRI may either be functionally equivalent to information obtained from the Tier 1 assays or may include data that provide information on a potential effect that could be due to interaction at the estrogen, androgen or thyroid systems.

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•An evaluation of all the data that exists; across both TOX and ECOTOX, including published literature, and key PHYSCHEM properties •In the context of determining what compounds need Tier 2 testing, it encompasses the Tier 1 screening data and OSRI

Tier 1 Weight of Evidence (WoE)

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US EPA Tier 1 Screening Battery

Tier 1 Assay GuidelineEstrogen Receptor Binding 890.1250

Estrogen Receptor Transactivation

890.1300/OECD 455

Androgen Receptor Binding 890.1150

Aromatase 890.1200

Steroidogenesis 890.1550

Uterotrophic 890.1600/OECD 440

Hershberger 890.1400/OECD 441

Male Pubertal 890.1500

Female Pubertal 890.1450

Fish Short-Term Reproduction 890.1350/OECD 229

Amphibian Metamorphosis 890.1100/OECD 231

EPA’s EDSP Tier 1•The 890 series of test guidelines is a battery with deliberate redundancy

• Identify substances that have the potential to interact with the estrogen, androgen, or thyroid hormone system

•Interaction does not necessarily equate to adverse effects in humans or ecological systems

•Not intended for risk assessment; prioritization for further testing

•Minimize false negatives

•Some endpoints are apical in nature, making it difficult to distinguish between endocrine and non-endocrine responses

http://www.epa.gov/ocspp/pubs/frs/publications/Test_Guidelines/series890.htm

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US EPA’s Tier 2 TestsLonger term studies using several species• Confirm endocrine activity detected in Tier 1 and characterize adverse

effects and • Establish NOAELs and LOAELs for risk assessment

Tier 2 studies (guidelines for the last 3 have issued in 2015)• 2-generation rodent (~EPA 870-4100) or extended 1-generation rodent• Multi-generation medaka• Amphibian larval growth and development• 2-generation avian

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Medaka Extended 1-Generation TestFavored (EPA) fish chronic test design

• Species not previously used in US or Europe regulatory testing

• Shorter in duration than previously proposed assay (19 weeks versus 35 weeks)

Guideline Elements (OCSPP 890.2200)• Exposure begins with young actively reproducing fish and evaluation

continues through F1 reproduction• 5 treatments plus control• Replication varies 6-12 depending on phase• Traditional endpoints – hatch, survival, growth, fecundity and fertility plus

• Genetic sex, VTG, secondary sex characteristics, gonad phenotype, histopathology of gonad, kidney, and liver

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Larval Amphibian Growth & Development Assay (LAGDA)Limited experience with regulatory amphibian testingGuideline Elements (OCSPP 890.2300)• Xenopus laevis• Exposure NF stage 8 to 10 for 10 weeks after the median time to NF

62 (typically 16 -18 week test)• 4 test concentrations plus control; Replication – 4 per test

concentration, 8 for control• Endpoints – survival, time to NF stage 62, growth, geno-, phenotypic

sex ratios, VTG (optional), liver somatic index, histopathology of thyroid, gonad, liver, kidney

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Avian 2-Generation TestCurrent regulatory testing utilizes bobwhite quail and mallard for avian reproduction testing• 1 generation design (22 weeks, Bobwhite quail)

New EPA guideline utilizing 2 generation design in Japanese quail (OCSPP 890.2100)• Exposure from young adults (F0) through F2 (14 days), 38 weeks

• 4 test concentrations and a control; 12 breeding pairs per concentration

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Avian 2-Generation EndpointsGuideline elements not included in 1-generation test guideline• Secondary sex characteristics through documentation of plumage

color and length, cloacal size, and date of first egg or first foam (F1) • Egg weight at 8 days and 24 hours prior to hatch• Genetic sex of F1/F2 chicks• Histopathology of brain, thyroid gland, liver, adrenal glands and

gonads• Glandular T4 content, thyroid hormone, sex steroids and VTG• Sex ratio (phenotypic and/or genotypic)• Time to first egg laying• Sexual behavior

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EDSP ProcessListing – based on exposure so far – process will changeTier 1 Test Order Issued by EPA – allowed 2 yearsRecipients must respond• Withdraw from market and not test?• Form testing consortium, if necessary, develop and submit OSRI EPA evaluates OSRI (Other Scientifically Relevant Information) and issues decision on which assays are neededRecipients perform assays and submit them with interpretation document (WoEor Weight of Evidence)EPA to evaluate Tier 1 data in a weight of evidence process along with all other existing data including the literature and determine if Tier 2 testing is necessaryTier 2 process will culminate in a risk assessment

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• List 1 selection process started in 2005; NOT a list of suspected EDs, based on exposure potential• For pesticides, 3 of 4 exposure routes: food, drinking water,

residential use, occupational exposure

• Finalized in 2008, official List 1 issued April 2009; predominately pesticides

• Test orders issued between Oct 2009 and April 2010, waivers based on OSRI arguments granted in late 2010

• Studies and company WoE documents provided to EPA in 2012; were in review for >3 years

List 1 Tier 1

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EPA Weight of Evidence Status• 6/30/2015 public release of 52 List 1 WoEs via EPA website• Tier 1 Data Evaluation Records (DERs) now available

• EPA included a disclaimer on each DER to keep the battery concept clear• DERs should not include any conclusion about interaction with endocrine

system-that is the purpose of the WoE documents

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EPA Weight of Evidence Findings• 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 ConclusionsFor several of the chemicals displaying bioactivity in the screening tests, EPA determined they have enough information to conclude that they do not pose risks.EPA recommended the following higher tier studies:• A comparative thyroid assay for 4 chemicals that showed interaction

with the thyroid pathway in mammals, • A medaka one-generation reproductive test (MEOGRT) for 13

chemicals that showed interaction with the estrogen or androgen pathways in wildlife,

• A larval amphibian growth and development assay (LAGDA) for 5 chemicals that showed interaction with the thyroid pathway in wildlife.

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The ICR proposes a process similar to Tier 1• A test order will be issued for Tier 2 testing• Registrants have 90-120 days to respond (120 days for those materials

with more than one registrant)• During this time, any argumentation or proposed modification to the

testing requirements may be submitted• 2 years from the date of the test order will be allowed for completion of

the testing• Will take longer than 2 years for all testing by all registrants to be completed

• Tier 2 Test Orders could be issued by end 2015

Tier 2 Process

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Draft EDSP List 2• Released in 2010 – 134 substances (51 pesticide actives, including

those on the 2007 and 2008 registration review schedule, 83 chemicals)

• Revised List 2 released in 2013-109 substances (41 pesticide actives, 68 chemicals identified under SDWA)

• Amended in 2014 (2 chemical substances removed due to instability/phys chem properties)

• List 2 Test Orders not anticipated until 2017

List 2 Tier 1

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• EPA will replace EAT Tier 1 with HTS• EPA will use a combination of HTS and computational

models to prioritize chemicals for EAT human and wildlife testing

• Registrants will assume the responsibility for HTS-could become required data for pre-manufacturing notices

• In the longer term, additional pathways will be investigated and added

EDSP is Evolving

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US EPA Goals for Endocrine Program (EDSP21/Comprehensive Management Plan)

EDSTAC report originally included a prioritization step• Early attempts using high throughput assays were not successful• The program then focused on the use of Tier 1 screens

With the thousands of chemicals to be evaluated, a multi-level and integrated approach is needed to determine the potential to interact with E, A or T The first step…• Using existing data, in silico models, and individual or suites of in vitro

HTP assays• Employ exposure metrics (ExpoCast)

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PrioritizationToxCast• Expanding use of CompTox

(PhysChem properties, QSARS, etc.) to support screening and prioritization

• Transparent and collaborative

ExpoCast• Rapid exposure estimation based on

readily available chemical use and production data

• Use toxicokinetics to bridge in vitro, concentration-based ToxCast data to in vivo, dose-based Exposures from ExpoCast

ToxCast

ExpoCast

High Throughput Prioritization

26Slide courtesy of D. Dix, US EPA

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Use of High-Throughput ScienceThree main objectives: • Prioritization - The near-term goal (<2 years) - ongoing

• Screening (Tier 1)- The intermediate-term goal (2-5 years)• EPA recent position is that it can replace ER, ERTA, and uterotrophic assays

• Replacement – The long-term goal (>5 years)

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mg/kg BW/day

LowPriority

MedPriority

NoPriority  (Hold)

NoPriority  (Hold)

Bioactivity

ExposureHigherPriority

Figure courtesy of D. Dix, US EPA

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• EPA leads the screening and testing effort globally and as such will continue making progress to demonstrate case examples of success in the regulatory science arena

• There are challenges on the adequacy of EPA’s program approach

• We hope that EPA can gain buy-in and support of the approach from some “regulatory science” authorities

The Future for EDSP

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