edsp validation gary e.timm senior technical advisor office of science coordination and policy u.s....
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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