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

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

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

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

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

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

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

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

EPA’s Approach for Considering OSRI Overview paper @ www.regulations.gov 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

OSRI from OPPTS and OECD Tests TOXICOLOGY Sub-chronic (OPPTS 870.3100) Chronic (OPPTS 870.4100) Developmental/Teratology (OPPTS 870.3700) 2-gen reproduction (OPPTS 870.3800) ECOTOXICOLOGY Avian reproduction (OPPTS 850.2300) Chronic invert reproduction (OPPTS 850.1300/1350) Early Life-stage (OPPTS 850.1400) Fish full life-cycle (OPPTS 850.1500)

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

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

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

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

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

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)

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

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.