1. Mammalian Tier I EDSP Screening Assays: What do they tell us?
Sue Marty
The Dow Chemical Company
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. 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

4. Assay to Detect Estrogens
Uterotrophic Assay
Assay to Detect Estrogens
and Antiestrogens

5. Uterotrophic Assay – OECD TG 440
Immature Model:
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

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. 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
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

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. 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

13. Pubertal Assays * * * ♂: Dose, Daily BWt, PPS Exam
Cull on PND 4
♀: Dose, Daily BWt, VO Exam
♂: Dose, Daily BWt, PPS Exam
Female
Necropsy
Male * *
Weaning *
Avg age at VO = 33.4 ( ) – After VO, evaluate estrous cycle
Avg age at PPS = 43.6 ( )
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

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. 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