EDSP Validation

Gary E.Timm

Senior Technical Advisor

Office of Science Coordination and Policy

U.S. Environmental Protection Agency

Framework

EPA proposed a Two Tier screening process:

• Screening (Tier 1)– Identifies substances for further testing

• Testing (Tier 2)– Identifies adverse effects and establishes

dose-response relationship for hazard assessment

TIER 1 SCREENING Detect potential for endocrine disruption

• Screens should be– inexpensive, quick, simple

– standardized and validated

– more sensitive than specific

– able to detect multiple endpoints

– readily interpretable

• Use information to – make initial judgments– direct and focus tier 2 tests

Modes of Action

• Hormone synthesis and clearance

• Hormone storage and release

• Hormone transport

• Receptor binding

• Altered post-receptor activation

PROPOSED SCREENING BATTERY (Tier 1)

• In vitro Screens– ER Binding / Reporter Gene Assay*

– AR Binding / Reporter Gene Assay*

– Steroidogenesis Assay with minced testis

• In vivo Screens– Rodent 3-day Uterotrophic Assay (sc)

– Rodent 20-day Pubertal Female Assay with Thyroid

– Rodent 5-7 day Hershberger Assay

– Frog Metamorphosis Assay

– Fish Reproduction Screening Assay

* These assays are in the HTPS

ALTERNATE SCREENING ASSAYS

• Rodent 20-day Pubertal Male Assay with Thyroid

• Placental aromatase• Rodent in utero through Lactation Assay

Screening Battery Example 1

• In vitro Screens

– ER Binding or Reporter Gene Assay*

– AR Binding or Reporter Gene Assay*

– Placental aromatase +

– Steroidogenesis Assay with minced testis #

• In vivo Screens

– Rodent 3-day Uterotrophic Assay (sc)

– Rodent 20-day Pubertal Male Assay with Thyroid +

– Rodent 20-day Pubertal Female Assay w/ Thyroid #

– Rodent 5-7 day Hershberger Assay #

– Frog Metamorphosis Assay

– Fish Gonadal Recrudescence Assay

+ Assays in yellow replace assays in red #

Screening Battery Example 2

• In vitro Screens– ER Binding or Reporter Gene Assay*– AR Binding or Reporter Gene Assay*– Steroidogenesis Assay with minced testis #

• In vivo Screens– Rodent 3-day Uterotrophic Assay (sc) #– In-utero Assay +– Rodent 20-day Pubertal Female Assay w/ Thyroid #– Rodent 5-7 day Hershberger Assay #– Frog Metamorphosis Assay– Fish Gonadal Recrudescence Assay

+ Assays in yellow relace assays in red #

TIER 2 TESTING Confirm and characterize endocrine effects

• Tests should – determine if effects are a primary or secondary disturbance of

endocrine function

– establish exposure/concentrations/timing and effects relationships

– be sensitive and specific

– assess relevant endpoints

– include life cycle of live-bearing and egg-laying species

– include a dose range for full characterization of effects

– be conducted in accordance with GLP

– be validated

PROPOSED TIER 2 TESTING BATTERY

• Multigeneration reproduction and development studies– Rodents– Birds– Frogs– Fish – Shrimp

Validation ProcessStakeholder Involvement

• Standardization and Validation Task Force– Technical group made up of major stakeholders– 3 expert workgroups: in vitro, mammalian,

ecotoxicity– To be replaced by FACA

• EDSPVAC– To be chartered by September 2000– Will advise EPA on Standardization and validation

issues

Validation of Test MethodsICCVAM

• What is it? Interagency Coordinating Committee for the Validation of Alternative Methods

• What does it do? – Establises criteria for validation and regulatory

acceptance of alternative test methods– Set up process for regulatory acceptance of

alternative methods

Validation Process

1. Research/Method Development

2. Demonstration of Relevance

3. Standardization of Protocol

4. Validation in multiple laboratories

5. Scientific Peer Review

Validation ProcessStatutory Requirements

• All screens and tests in the EDSP must be validated

• The FIFRA Scienctific Advisory Panel (SAP) must review the EDSP

• The SAP must peer review all test guidelines to be used under FIFRA

Validation ProcessICCVAM Role

• ER and AR binding assays to be validated by ICCVAM– no laboratory work required

– generic performance criteria to be established rather than specific methods

• All other assays to be validated in EPA process with extensive ICCVAM coordination

EPAValidation ProcessICCVAM Role

• Method development and preparation of Background Review Document (BRD)

• Pre-validation--demonstration of relevance and development of standardized protocol

• Determination of readiness for validation in consultation with ICCVAM

• Validation• Peer review by SAP/SAB and review by ICCVAM

VALIDATION APPROACH

• Human health related assays are highest EPA priority (2- 3 year time frame ) – in vitro assays – Uterotrophic, Hershberger

– Female and male pubertal assays

– Rodent in utero screen

– Rodent multigeneration demonstration

• Ecotoxicological assays EPA second order priority(3 -5 year time frame )– frog metamorphosis assay

– fish reproductive screen

– fish, frog, and invertebrate reproduction and development studies

VALIDATION APPROACH

• Select chemicals specific to each assay, validate assay rather than battery

• More chemicals in pre-validation to establish relevance of assay and standardize the protocol than in validation phase

• Battery validation will be a paper exercise

Ongoing EPA Work

• Completed initial demonstration of relevance– Female Pubertal Development Assay (4/2000)

– Male Pubertal Development Assay (4/2000)

• Need to refine and standardize protocols• Initial developmental protocol trials

– Frog metamorphosis assay (8/99)

– Fish Reproduction Screening Assay (8/99)

• Additional effort needed on frog assay before a determination of relvance/usefulness can be made

OECD ENDOCRINE DISRUPTOR TESTING AND ASSESSMENT WORKGROUP

• Framework for Screening and Testing

• Standardizing/validating three screens:

– Uterotrophic

– Hershberger

– 28-Day repeat dose study (OECD Method 407)

• Updating existing guidelines

– Example: Mammalian multi-generation reproduction study (OECD Method 416)

• Developing new guidelines

– Avian multi-generation reproduction study

– Fish partial life-cycle reproduction research

Status of AssaysAssay Research/ Devel Demonstration Standardization Validation

ER/AR Binding

Steroidogenesis

Aromatase

Uterotrophic

Hershberger

Pubertal female

Pubertal male

In utero/lactat

Frog thyroid

Fish repro

Status of Assays

Assay ResearchDevelopment

Demonstration Standrdization Validation

Mammalian2-gen

Avian 2-gen

Fish Life cycle

Amphibian 2-gen

Shrimp 2-gen

Tier I Validation Program Projections

Assay Pre-validation Validation

ER/AR Binding 2001

Steroidogenesis 2000 2001

Aromatase 2000 2001

Uterotrophic 2000 2001

Hershberger 2000 2001

Tier I Validation Program Projections

Assay Pre-validation Validation

Pubertal Female 2000 2002

Pubertal Male 2000 2002

In Utero/Lactation 2001 2003

Frog Thyroid 2000 2002

Fish Repro Screen 2000 2002

Tier I Validation Program Projections

Assay Pre-validation Validation

Pubertal Female 2000 2002

Pubertal Male 2000 2002

In Utero/Lactation 2001 2003

Frog Thyroid 2000 2002

Fish Repro Screen 2000 2002

Summary

2000 2001 2002 2003 2004 2005

Priority Setting

Tier I Validation

Tier II Validation

Phase I Screening