Mammalian Tier I EDSP Screening Assays: What do they tell us?

1. Mammalian Tier I EDSP Screening Assays: What do they tell us? Sue Marty The Dow Chemical Company mmarty@dow.com ISRTP Workshop September 9, 2009

2. Purpose of Tier I Screening Assays • Screening assays should: • Identify Potential Hazard (Info on MOA) • Identify all compounds affecting the EAT systems (minimize false negatives) • Info on D-R for subsequent studies • Tier I should be quick and relatively inexpensive

3. In Vitro Assays ER binding/ transactivation AR binding Steroidogenesis (H295R) Aromatase In Vivo Assays Uterotrophic Hershberger Pubertal Female Pubertal Male 15-day Intact Male Modified OECD 407 Fish Short-term Reproduction Amphibian Metamorphosis Tier I Assays

4. Uterotrophic Assay Assay to Detect Estrogens and Antiestrogens

5. Daily BWt, Dose 3 d; VO Exam Cull on PND 4 18 21 25 Endpoints: Wet & Blotted Uterine Wts Histology (optional) VO at necropsy (immature) Weaning N > 6/group; >2 treated groups, optional ED70-80 pos. control; necropsy 24 h after last dose Dosing: Oral or sc; MTD: “without significant toxicity or distress”; Limit dose 1000 mkd Uterotrophic Assay – OECD TG 440 Immature Model: Ovariectomized (OVX) Model: OVX at 6-8 wks Dosing > 3 days Exam for ovary remnants at necropsy

6. Laboratory Proficiency for the Uterotrophic Assay

7. Uterotrophic Assay: Points to Consider • Assay primarily detects ERα agonists: • Not ERβ agonists (Piu et al., 2008; Jazbutyte et al., 2008; Jung, 2009) • Comparable sensitivity between adult and immature models • Immature model somewhat less specific: • Aromatizable androgens – positive response • More sensitive to dietary phytoE2 (< 350 µg gen equiv/g lab diet) • Body wt impacts uterine wt (<+20% of mean wt) • Route of exposure • TG: Relevance to human exposure (gavage - ingestion; sc - inhalation or dermal) • Data on metabolism (avoid first pass metabolism) • SAP: Relevant route is not necessary for screening ED potential • If little know about metabolic disposition, use the sc route • Interpretation: Supporting evidence • in vitro: ER binding/transactivation • in vivo: pubertal female, fish short-term reproduction • Route may complicate weight of evidence

8. Hershberger Assay Assay to Detect Androgens, Antiandrogens and 5α-Reductase Inhibitors

9. Endpoints: Wts: Ventral Prostate (VP), Cowpers Glands (CG), Seminal Vesicles with Coagulating Glands (SV, Glans Penis (GP), Levator Ani-Bulbocavernosus Muscle (LABC) N > 6/group; > 2 treated groups; animals necropsied 24 h after last dose Dosing: Oral or sc; MTD < 10% Δterminal BWt; Limit dose 1000 mkd Hershberger Assay (+/- 0.2 or 0.4 mg/kg/day TP - sc) Daily BWt, Dose 10 d Castrated on > PND 42 49 59 60 70 Necropsy Weanling Model: Include testes & epididymal wts

10. Hershberger Assay • Extensive OECD validation • Positive assay results: • Androgens: Increase (decrease for testes) in >2 target organ wts • Anti-androgens: Decrease (increase for testes) in >2 target organ wts • Interpretation: Supporting evidence • In vitro: AR binding assay • In vivo: male pubertal assay, fish short-term reproduction assay

11. Hershberger Assay: Points to Consider • Castrated adult vs. weanling models • Juvenile animals somewhat less sensitive than castrated model • Testes wt changes are variable; not reliable for anti-androgens • Treatment-related decreases in body wt may affect AST wts • Differential effects on tissue weights • 5α-reductase inhibitors: • Conserved/increased LABC and GP wts (testosterone sensitive) • Greater decreases in VP wts (DHT sensitive) • Positive results not always due to androgenicity • Potent estrogens can increase SV wts • Increased adrenal steroidogenesis can increase AST wts • Enhanced steroid metabolism can lower serum T levels, even for exogenously administered TP

12. Male & Female Pubertal Assays: (Anti)estrogens/(anti)androgens Steroid biosynthesis inhibitors Agent that alter pubertal devt via: HPG axis HPT axis Tier I in vivo Assays:Multimodal Assays with Intact Animal Models

13. ♂: Dose, Daily BWt, PPS Exam ♀: Dose, Daily BWt, VO Exam Cull on PND 4 * * 21 25 30 35 40 42 45 50 53 Male Necropsy Female Necropsy Weaning * Avg age at VO = 33.4 (31.6-35.1) – After VO, evaluate estrous cycle Avg age at PPS = 43.6 (41.8-45.9) N > 15/group; Minimum of 2 treated groups; animals necropsied ~2 h after last dose Dosing: Oral; MTD < 10% Δterminal BWt; Limit dose 1000 mkd Pubertal Assays Necropsy = Tissue wts, Blood collection, Histo

14. Pubertal Assay Endpoints • Dose levels: MTD < 10% BWt change, clinical signs • Age and body wt at VO/PPS • Age at first estrus (♀) • Regularity of estrous cycle (♀) • Necropsy: (consider estrous cycle stage - ♀) • Liver, kidneys, pituitary and adrenal weights • Ovarian and uterine (wet & blotted) weights (♀) • Testes, epididymides, ventral prostate, dorsolateral prostate, seminal vesicles with coagulating glands and levator ani/bulbocavernosus muscle complex weights (♂) • Thyroid wt after fixation • Serum T4 and TSH levels • Serum T (♂) • Thyroid histopath • Ovarian and uterine histopath (♀) • Testicular and epididymal histopath (♂)

15. Interpreting Pubertal Assays • Inherent variability in age at VO and PPS (apical endpts) • Assay Specificity • Female pubertal assay: • Insufficient monitoring period for estrous cycling • Ovarian and uterine wts complicated by estrous cycling • Male pubertal assay: • Phenobarbital (< 100 mkd) - not detected for thyroid effects • Male and Female Pubertal Assays: • Negative control data is lacking (2-chloronitrobenzene) • EPA is in the process of conducting negative control studies • SAP: “…that a negative control substance has not been identified (in the pubertal assays)…is a major limitation to the Tier I battery. Lacking demonstration of expected negative results remains an issue for the validity of these assays”.

16. Pubertal Assay Specificity & Body Weights • Feed restriction studies with the pubertal assay designs: • Laws et al. (2007) and Marty et al. (2003) • 9-12% change in terminal body wt • Decreased abs. adrenal & pituitary (♀♂) & ovarian wts • Decreased abs. epididymal, VP and SV wts • T3 and T4 are sensitive to body wt changes • 9% bwt change altered thyroid endpts (♂) • SAP: “Body weight reductions were closely associated with perturbations in the onset of puberty and/or normal cycling. Therefore the specificity of the pubertal assays for detecting alterations in the HPG axis due to purely endocrine-related disruption is currently unclear”.

17. Statistical Analysis and Body Wt Effects • EPA recommended a covariate analysis • Covariate = body wt at weaning • Covariate not affected by treatment, but doesn’t account for body wt effects on organ wts • SAP: Difficult to distinguish effects attributable to body weight loss from endocrine disruptor effects. • Covariate analysis is warranted, but form of the analysis may not be straightforward • Further consultation with EPA/ORD statisticians

18. SAP: Interpreting Assays & Weight of Evidence • Substances should not be administered near the MTD • Increased potential for false positive results • D-R is advantageous - Caution should be used when interpreting endocrine effects observed only at the MTD • False positives can be eliminated by weight of evidence requiring positive results across >2 in vivo assays • Based on redundancy and complementarity of assays, false negatives would be extremely rare

19. SAP: EDSP Tier I Assay Redundancy

20. Closing Thoughts • Minimizing “false negatives” has a cost • Tier II testing includes: • One- or two-generation rat reproductive toxicity study • Avian reproduction study • Fish life cycle • Amphibian development and reproduction • Mysid (invertebrate) life cycle • Relevance for assessing human risk? • MTDs for some assays should be reconsidered • Use caution in interpreting data confounded by bwt effects • Compounds administered at high doses • Gavage typically results in unrealistic Cmax values (nonlinear PK) • EPA needs to offer guidance on interpreting the Tier I battery • Weight of evidence will be difficult to apply in some cases

21. Dow Ed Carney Lynn Kan Melissa Schisler Bhaskar Gollapudi Keith Brooks Sonya Parshall Collaborators Jim Crissman Grantley Charles Keith Johnson John O’Connor Mike Kaplan Acknowledgements