1. What Information Fulfills EDSP Screening Requirements?
Steve Levine, Ph.D.
Science Fellow
Ecotoxicology & Risk Assessment
Monsanto Company
ISRTP Meeting
September 9, 2009

2. Topics Covered Background on the EDSP
EDSTAC recommendations
Functional equivalence and Other Scientifically Relevant Information (OSRI)
International Regulatory Guideline Studies
TOX
ECOTOX
WoE approaches

3. Tier 1 Screening
The number of substances planned for Tier 1 screening is staggering
Pesticides
Commercial chemicals
Cosmetic ingredients
Food additives
Nutritional supplements
Certain mixtures
Therefore, screening must be efficient, robust, and predictive at identifying potential EACs

4. EDSTAC Framework Priority setting Tier 1 screening Tier 2 testing
Based on potential human exposure
residues in food and drinking water
residential
occupational
Higher priority given to chemicals likely to have human exposure via multiple pathways
Tier 1 screening
In vitro and in vivo assays
Identify chemicals that can potentially interact with the endocrine system
Tier 2 testing
Will establish the relationship between dose of an EAC and the potential effects observed
Assess risks humans and wildlife

5. EDSTAC Recommendations
Recommended a minimal number of screens & tests to make sound decisions, thereby, reducing the time needed to make decisions
T1S screens should be MoA based to identify specific types of endocrine activity
Should be broadly predictive (sensitivity & specificity)
Should produce data that can be interpreted as either positive or negative (minimize Type I & II error rates)
Should be inexpensive and relatively quick and easy to perform

6. Tier 1 Screening Battery
In vitro
Estrogen receptor binding
Estrogen receptor TA
Androgen receptor binding
Steroidogenesis (H295R)
Aromatase
In vivo
Uterotrophic (rat)
Hershberger (rat)
Pubertal female (rat)
Pubertal male (rat)
Amphibian metamorphosis
Fish short-term reproduction
Many of the T1S assays meet the aforementioned criteria but others suffer from lack of specificity for adverse effects by an ED mechanism

7. Alternative Means to Meet
Tier 1 Screening
EDSTAC recommended that it should be permissible to meet T1S requirements by submitting data that are “functionally equivalent” to the data generated by the T1S battery
Functionally equivalent information could be submitted for one or more of the recommended T1S assays or for the entire battery
Pointed out that functionally equivalent information exists for chemicals that have reproductive and developmental toxicity testing
Provisions to cite existing data in policies and procedures document

8. Functional Equivalence & OSRI
Functional equivalence = data of a suitable nature and quality even if different methods were used i.e., provides the same essential predictive information as T1S
OSRI = information that informs the determination as to whether the substance may have a similar effect produced by a substance that interacts with EAT systems
OSRI may either be (1) functionally equivalent to information obtained from the Tier 1 assays or (2) data from assays that perform the same procedure / function as EDSP Tier 1 Screens
3rd bullet – equivalent or identical endpoint

9. EPA’s Approach for Considering OSRI
Overview
Anyone may submit OSRI not just Test Order Recipients
Submitters should provide the information or cite previously submitted information
Information will receive different weights in a WoE - the quality of the information will weigh heavily
Factors will include sensitivity, specificity, confidence in the conclusions and applicability across taxa
Factors will also include route & duration/frequency of administration, dose levels, age at exposure, species, number of test units, variability, and whether the data provides a basis for conclusions for potential interaction with the endocrine system in mammalian and non-mammalian systems
OSRI for Tier 1 or Tier 2: metabolism, carcinogenicity, reproductive and developmental, and toxicogenomic

10. OSRI from OPPTS and OECD Tests
TOXICOLOGY
Sub-chronic (OPPTS )
Chronic (OPPTS )
Developmental/Teratology (OPPTS )
2-gen reproduction (OPPTS )
ECOTOXICOLOGY
Avian reproduction (OPPTS )
Chronic invert reproduction (OPPTS /1350)
Early Life-stage (OPPTS )
Fish full life-cycle (OPPTS )

11. OSRI Will Include
Toxicity tests following international regulatory guidelines where full histopathology is carried out on:
Endocrine tissues (pituitary, thyroid, parathyroid, pancreas adrenal, ovary and testis)
Hormone sensitive male tissues (prostate, epididymides, seminal vesicles)
Hormone sensitive female tissues (mammary, uterus and reproductive tract)
And toxicity protocols that focus on reproduction, fertility and development for mammalian species and other taxa (birds, fish)
These studies provide the strongest evidence of potential endocrine effects and should be weighted above less comprehensive data

12. Rat Multi-Gen
The 1996 USEPA guideline for a multiple generation reproductive and developmental toxicity assay was revised to include developmental benchmarks predictive of ED potential
A diverse set of female endpoints (ovaries, uterus, vagina, and mammary glands)
Day of vaginal opening and first estrus
Mating and fertility indices
number of implantation sites, estrous cyclicity
Male tissue weights and histopathology (testes, epididymis, prostate, and seminal vesicles) and other male parameters (sperm number and analyses)
FIX

13. Bridging OSRI to T1S Assays
High concordance between the results of rat multi-gen and the rat Uterotrophic assay
Consistency between MEDs for the Uterotrophic assay and estrogen-related LOEL/LOAEL in multi-generation testing
High concordance between the Uterotrophic assay and the ER binding assay & the stably transfected TA for a large number of chemicals
2-gen data should meet the requirement for the pubertal assays:
Pubertal assays largely have redundant endpoints with the 2-gen study
Building the bridge - Similar comments can be made for the Hershberger assay – rationale for pubertals

14. OSRI to T1S Assays - ECOTOX
Avian reproduction studies
Gold standard or highest tier of avian testing
Bobwhite quail and mallard duck
Studies do not include exposure during all relevant stages of development or organ histopathology
However, provides valuable information in a WoE context to assess potential effects of endocrine active substances
Sensitivity of 1-gen vs 2-gen Japanese quail?
Reproduction study design
Dietary exposure for ~10 weeks prior egg-laying
Photoperiod change initiates egg-laying
Exposure continues for ≥8 weeks
Endpoints: eggs laid, eggs damaged, eggs set, egg shell thickness, viable embryos, live embryos, hatchlings, 14-day survivors, eggs laid/female, eggs laid/female/day, 14-day survivors/female
Make point regarding why these studies were developed and embryo toxicity

15. OSRI to T1S Assays - ECOTOX
Fish full life cycle (FFLC) study
Gold standard for aquatic vertebrate ecotox testing
Entire LC is exposed = most sensitive life-stage is tested
~260 to 300 days for FTHD minnows = T1S spp.
FFLC study design
ELS → growth & repro → ELS
First gen: hatching success, time-to-hatch, survival, growth and development, reproductive success (# of spawns, # of eggs)
Second-gen: time-to-hatch, survival, growth and development
Compare with 21-day screening assay

16. Principles for Evaluating Data in a WoE .
Consistent pattern of responses for a MoA (+ or -)
The nature and range of the biological effects observed
The shape of DRCs curves when available
The severity and magnitude of the effects
Interpretation made in the context of biological significance & biological plausibility
The presence or absence of responses in multiple taxa
Evaluate results in the context of natural variability using control data (historical and concurrent)

17. Principles for Evaluating Data in a WoE
Results from apical assays (MTD dose) need to be weighted appropriately when accompanied by decreases in BW or other potential confounders
In vivo results generally are considered to have more weight than in vitro results
Available in vitro assays should not be used as yes/no determinants to proceed to Tier 2 (ER binding example)
SARs = critical step in the WoE process
A WoE approach for OSRI must be developed along with the WoE framework for the T1S

18. Closing Thoughts SARs are OSRI and weighted appropriately
There will be instances where OSRI will meet all or some of the T1S requirements, particularly for food use pesticides.
Development of a transparent WoE approach is desperately needed not only in the interpretation of OSRI but for the T1S data before testing is required.