EDSP Validation

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EDSP Validation

• Gary E.Timm
• Senior Technical Advisor
• Office of Science Coordination and Policy
• U.S. Environmental Protection Agency

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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

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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

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Modes of Action

• Hormone synthesis and clearance
• Hormone storage and release
• Hormone transport
• Receptor binding
• Altered post-receptor activation

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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

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ALTERNATE SCREENING ASSAYS

• Rodent 20-day Pubertal Male Assay with Thyroid
• Placental aromatase
• Rodent in utero through Lactation Assay

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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

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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

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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

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PROPOSED TIER 2 TESTING BATTERY

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

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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

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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

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Validation Process

• 1. Research/Method Development
• 2. Demonstration of Relevance
• 3. Standardization of Protocol
• 4. Validation in multiple laboratories
• 5. Scientific Peer Review

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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

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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

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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

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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

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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

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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

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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

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Status of Assays
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Status of Assays
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Tier I Validation Program Projections
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Tier I Validation Program Projections
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Tier I Validation Program Projections
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Summary
Priority Setting
Tier I Validation
Tier II Validation
Phase I Screening