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

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

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

Assay to Detect Estrogens Uterotrophic Assay Assay to Detect Estrogens and Antiestrogens

Uterotrophic Assay – OECD TG 440 Immature Model: 18 21 25 Daily BWt, Dose 3 d; VO Exam Cull on PND 4 Weaning Endpoints: Wet & Blotted Uterine Wts Histology (optional) VO at necropsy (immature) Ovariectomized (OVX) Model: OVX at 6-8 wks Dosing > 3 days Exam for ovary remnants at necropsy 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

Laboratory Proficiency for the Uterotrophic Assay

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

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

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

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

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

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

Pubertal Assays * * * ♂: Dose, Daily BWt, PPS Exam Cull on PND 4 21 25 30 35 40 42 45 50 53 ♀: Dose, Daily BWt, VO Exam ♂: Dose, Daily BWt, PPS Exam Female Necropsy Male * * 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) Necropsy = Tissue wts, Blood collection, Histo N > 15/group; Minimum of 2 treated groups; animals necropsied ~2 h after last dose Dosing: Oral; MTD < 10% Δ terminal BWt; Limit dose 1000 mkd

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 (♂)

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”.

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”.

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

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

SAP: EDSP Tier I Assay Redundancy

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

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