Background and Rationale for “Extended One-generation” Study

1. Background and Rationale for “Extended One-generation” Study Paul Foster, Ph.D. CIIT Centers for Health Research Email: foster@ciit.org

2. Overview of Presentation • Provide an outline of the basic test protocols that provide information on Endocrine Active Chemicals (EAC’s), with an emphasis on the number of animals examined. • Comment on the strengths and weaknesses of the current multigeneration reproduction study. • Provide some information on how improved design may generate enhanced information for risk assessment of EAC’s.

3. EDSP Tiered Screening and Testing Strategy for Estrogen, Androgen and Thyroid Activity (EAT) • Tier I Screening - Detect Interaction with the Endocrine System • Short term in vitro screens, e.g. receptor binding, transactivation • Short term in vivo pharmacological screens e.g Hershberger, uterotrophic assays • If positive go to Tier II • Tier II Testing - Determine and Characterize Endocrine Disrupting Effects • Multigeneration reproduction study • Data used for Risk Assessment

4. Purpose of Tier 2 test • Provide “definitive” information on hazard characterization of EACs (EPA). • Confirm or refute observations noted in Tier 1 screens/ assays. • Utilize in utero (most sensitive) exposure. • Identify activity vs. end points for which concern has been raised in humans (eg hypospadias, cryptorchidism,sperm); i.d. other endocrine-like activity. • Provide dose-response information to be used in risk assessment.

5. Why is exposure during perinatal development important? • Likely to produce permanent effects • Effects initiated in utero may not be manifest until adulthood. • Timing of exposure may be as important as dose level • Embryo/fetal sensitivity versus dam • Hormonal interplay for normal development can provide unique, sensitive targets for toxicity

6. Development is TOTALLY Hormone-dependent Development is largely hormone- independent Critical Window of Susceptibility:Male or Female? Sexually indifferent fetus Pregnancy Week 6 Testis formation hormones 7-8 Window of hormone susceptibility ~15

7. Prenatal Developmental Toxicology Study • Fetuses examined grossly prior to birth. • No pathology conducted • Expected to detect agents producing major effects on skeletal and visceral structures. • Examination of all offspring increases the probability of detecting low incidence phenomena.

8. Prenatal Developmental Toxicity Study Sexual Maturation Gamete Production & Release Growth & Development Lactation & Postnatal Development Fertilization Assessment Zygote Transport Parturition Sex Differentiation Fetal Development Dose Implantation Embryogenesis

9. EPA Prenatal Developmental Toxicity Study Sexual Maturation Gamete Production & Release Growth & Development Lactation & Postnatal Development Fertilization Assessment Zygote Transport Parturition Extended Dose Period Fetal Development Dose Implantation Embryogenesis

10. General Prenatal Rat Toxicology Protocol GESTATION (days) Sexual Differentiation Critical Gap Dose Dam 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Necropsy dams, examine half of the Fetuses for visceral, other half skeletal Alterations (~3 pups/sex/litter/method) From 20 litters; n= ~ 60/sex/ dose Breed

11. Multigeneration Reproduction Study • Provides substantial information on the effects of agents on reproduction. • Fertility, fecundity, pregnancy, gametes etc • More limited information on (postnatal) development • Pup survival, growth, developmental landmarks etc • Currently considered by EPA the “definitive” Tier 2 test for EAC’s.

12. Multigeneration Reproduction Study Sexual Maturation Gamete Production & Release Growth & Development Lactation & Postnatal Development Fertilization Assessment Zygote Transport Parturition Dose Fetal Development Implantation Embryogenesis

13. General Multigenerational Protocol F0 Dose Parents Gestation Assign to treatment 20/sex/dose 0 1 2 3 Sexual Differentiation Breed F0 F1 Dose F1 Dose Dams 0 3 20 wks Necropsy F1: n= 20/sex/dose, 20 litters Breed F1: 20/sex/dose Wean: Gross Necropsy 3/sex/litter Birth Cull Path= 10/sex/dose

14. How well does the multigeneration study characterize EAC’s? • It covers the critical developmental windows for sexual differentiation. • Should detect potent estrogens and antiandrogens • Only 1/sex/litter of F1examined at adulthood • Limited power to detect reproductive tract malformations (prenatal study examines ALL offspring) ie may produce false negatives. • If an end point in the control population is variable, statistical vs. biological significance can be problematic.

15. How well does the multigeneration study characterize EAC’s? • A number of developmental end points are not included, or are only triggered in the F2 generation, which stops at weaning. • Nipples, AGD • Gross necropsy at weaning is helpful, but not sufficient. • Unlikely to detect subtle malformations (eg epispadias) • Cannot detect effects on organ systems not yet developed (eg sperm production, prostate)

16. Alternate Assay System • EPA is considering an alternate assay systems for potential use. • Transgenerational study design. • Fewer litters, more offspring examined. • More end points related to EAC’s always measured (eg nipple retention, AGD, pathology).

17. Transgenerational Study Sexual Maturation Gamete Production & Release Growth & Development Lactation & Postnatal Development Fertilization Assessment Zygote Transport Parturition Sex Differentiation Fetal Development Dose Implantation Embryogenesis

18. General Transgenerational Protocol Sexual Differentiation Dose Dams Breed GD 0 GD 6 GD 14 Birth GD 22 Dose pups (optional) Dose Dams Postnatal Life (weeks) 0 2 3 4 7 20 Necropsy all offspring (n= ~ 50/sex/dose/ 8 litters) PPS AGD Wean VO Areolae

19. The Multigeneration Study • The multigeneration reproduction study was originally designed to provide significant information on reproductive toxicity and, to a more limited extent, postnatal development. • This study is now being utilized for hazard characterization of Endocrine Active Chemicals, where postnatal development is a key indicator of adverse response. • How could the design of the study be improved to meet this goal?

20. Key Questions to be addressed -1 • Should the same degree of scientific rigor applied in prenatal developmental studies be used in postnatal evaluations? • Increase the number of F1 animals examined at adulthood? • Increase the ability to detect hazard and provide improved dose-response information? • Improve end points routinely examined? • Do the cost, effort, logistics (# of animal rooms etc) justify the potential gains?

21. Key Questions to be addressed - 2 • Should consideration be given to the design of a transgenerational test protocol to specifically meet the needs of hazard characterization of EAC’s ? • Potential use as a substitute for the costly, labor intensive, multigeneration study? • Potential use as an intermediate tier - acting as a bridge to older, multigen studies? • Potential to trigger a “definitive” study containing increased animals/ end points ?

22. Objectives for Proposed “One-Generation Extension” Study • Determine whether some of the effects from perinatal exposure to well characterized antiandrogens (di-n-butyl phthalate, vinclozolin) that can be readily detected after puberty are missed in weanling animals of the F1 generation. • Determine whether some of these effects occur at an incidence that would go undetected if only 1 male per litter is retained past puberty and examined at adulthood.

23. Objectives for Proposed One-Generation Extension Study • Select dose levels of characterized agents that will produce clear effects and approximate a LOAEL. • Use numbers of litters comparable to that used in a standard multigeneration/ prenatal toxicology study. • Since the exposure period is limited, use a route of exposure that maximizes control of administered dose on a mg/kg/d basis.