John C. O’Connor DuPont Haskell Laboratory for Health and Environmental Sciences & The American Chemistry Council

1. The Intact Male Assay As An Alternative Tier I Screening Assay For Detecting Endocrine-Active Compounds John C. O’Connor DuPont Haskell Laboratory for Health and Environmental Sciences & The American Chemistry Council

2. Outline • Background • Overview of the 15-day intact male assay • Study rationale • Study design considerations • Case study with flutamide, ketoconazole, & finasteride • Future considerations and final thoughts

3. Comparison of the EDSTAC-Recommended and Alternative Tier I Screening Batteries for Identifying EACs

4. Desirable Attributes of a Screen • Reliable (identified known EACs) • Predictive (known EACs are identified for their mode of action) • Sensitive (low false-negatives) • Quick (i.e., short-term) • Cost effective • Minimize animal usage

5. Proposed Tier I Screening Battery Tier I ER Agonists ER Antagonists Uterotrophic Assay Receptor Binding/ Transactivation Agonist/Antagonist (ER, AR) Thyroid Effects Steroid Biosynthesis ER/AR Agonists ER/AR Antagonists Intact Male Assay

6. 15-Day Intact Male Assay • Model: • 10-Week old intact male rats • n = 15/group • Control + 3 dose groups • 15-Day test (oral) • Required Endpoints: • Organ weights - liver, testes, thyroid, epididymides, prostate, seminal ves., ASG unit • Histopathology - testis, epididymides, thyroid • Hormonal battery - testosterone, estradiol, prolactin, LH, FSH, T3, T4, TSH • Biochemical - preparation of hepatic microsomes • Optional Endpoints (if warranted by other findings) • Histopathology – liver • Hormonal assessment – DHT • Biochemical Assessment • Hepatic UDP-glucuronyltransferase activity • Hepatic aromatase activity

7. Mechanisms that Modify Hormone ActionPositive/Negative Influences • Hormone receptor agonist/antagonist • Alter hormone synthesis (e.g., steroid hormones) • Alter hormone storage or release (e.g., peptide hormones) • Alter hormone metabolism • Alter hormone transport • Alter bioavailability by displacing hormone bound in serum or by altering enterohepatic recirulation • Alter post-receptor activation • Alter endocrine axis centrally (neuroendocrine)

8. Dopamine Agonist (Muselergine) CNS X Hypothalamus (-) (-) Antiandrogens (Flutamide) GnRH (+) X (-) (-) Anterior Pituitary FSH (+) LH (+) Sertoli Cell Leydig Cell Target Peripheral Tissues Steroid Inhibitors (Ketoconazole) Testis X Inhibin Testosterone X Antiandrogens (Flutamide) X X Aromatase 5-Reductase 5a-Reductase Inhibitors (Finasteride) Aromatase Inhibitors (Aminoglutethemide) Estradiol DHT Hypothalamic-Pituitary-Testis Axis

9. Dopamine Agonist (Muselergine) CNS X Hypothalamus (-) (-) Antiestrogens (ICI-182,780) X Pulsatile GnRH (+) (-) (-) Anterior Pituitary LH (+) FSH (+) Theca Cell Granulosa Cell Target Peripheral Tissues X Steroid Inhibitors (Ketoconazole) Ovary X Inhibin X Progesterone + Estradiol Antiestrogens (ICI-182,780) Aromatase Androgens X Aromatase Inhibitors (Aminoglutethemide) Hypothalamic-Pituitary-Ovary Axis

10. Intact Male Assay: “Expected” Profile for Unknowns

11. Intact Male Assay: Study Design Issues • Oral dosing – most relevant route • Dose level selection • Based on range-finder studies • Target ≤ 10% final body weight • Based on dietary restriction studies • O’Connor et al., 2000 (Toxicol. Sci. 54: 338-354) • Adult vs. immature animals • Immature are more sensitive to organ weight changes • Mature are more sensitive to hormonal changes • Duration – 2-week

12. Effect of Diet Restriction on Organ Weights in Sprague-Dawley Rats

13. Effect of Diet Restriction on Serum Hormones in Sprague-Dawley Rats

14. Effect of Diet Restriction on Thyroid Hormones in Sprague-Dawley Rats

15. Immature vs. Mature Rats Mature Immature

16. Immature vs. Mature Rats Mature Immature

17. Intact Male Assay: Thyroid Timecourse

18. Intact Male Assay: Thyroid Timecourse

19. Case Study With Flutamide, Ketoconazole, & Finasteride

20. CNS Hypothalamus (-) (-) Antiandrogens (Flutamide) GnRH (+) X (-) (-) Anterior Pituitary FSH (+) LH (+) Sertoli Cell Leydig Cell Target Peripheral Tissues Steroid Inhibitors (Ketoconazole) Testis X Inhibin Testosterone X Antiandrogens (Flutamide) X Aromatase 5-Reductase 5a-Reductase Inhibitors (Finasteride) Estradiol DHT Hypothalamic-Pituitary-Testis Axis

21. Case Study: Study Design • Model: • 10-Week old intact male rats • n = 15/group • 15-Day test (oral) • Control + 4 dose groups • Dose levels selected based on range-finder studies • Flutamide (10 mg/kg/day; high dose) • Ketoconazole (100 mg/kg/day; high dose) • Finasteride (25 mg/kg/day; high dose) • Measured Endpoints: • Organ weights - liver, testes, thyroid, epididymides, prostate, seminal ves., ASG unit • Histopathology - testis, epididymides, thyroid • Hormonal battery - testosterone, DHT, estradiol, prolactin, LH, FSH, T3, T4, TSH

22. Case Study: Organ Weights

23. Case Study: Serum Hormones

24. ① Cholesterol SCC enzyme (CP-450) a. 20-hydroxylase b. 22-hydroxylase c. 20,22-lyase ② 3b-Hydroxysteroid Dehydrogenase ③ 4,5-Ketosteroid Isomerase ④ 17-Hydroxylase (CP-450) ⑤ C-17,20-Lyase (CP-450) ⑥ 17b-Hydroxysteroid Dehydrogenase ⑦ 5a-Reductase ⑧ Aromatase (CP-450) Cholesterol Leydig Cell 1a 1b Pregnenolone 1c ② ③ Progesterone X ④ Ketoconazole 17a-Hydroxyprogesterone ⑤ ⑧ Androstenedione Estrone ⑥ ⑧ Testosterone 17b-Estradiol X ⑦ Finasteride X 5a-Dihydroxytestosterone Target peripheral tissues Flutamide Testosterone Biosynthesis (4 Pathway)

25. Case Study: Flutamide, Ketoconazole, & FinasterideComparison of Organ Weight Data Cannot differentiate mode of action based on organ weight changes

26. Case Study: Flutamide, Ketoconazole, & FinasterideComparison of Serum Hormone Data Mode of action can be determined based on hormonal changes

27. CNS Hypothalamus (-) (-) Antiandrogens (Flutamide) GnRH (+) X (-) (-) Anterior Pituitary FSH (+) LH (+) Sertoli Cell Leydig Cell Target Peripheral Tissues Steroid Inhibitors (Ketoconazole) Testis X Inhibin Testosterone X Antiandrogens (Flutamide) X Aromatase 5-Reductase 5a-Reductase Inhibitors (Finasteride) Estradiol DHT Hypothalamic-Pituitary-Testis Axis

28. EACs Examined in the 15-Day Intact Male Assay

29. EACs Examined in the Pubertal Assays

30. Profile of Selected EACs Examined in the Intact Male Assay

31. Profile of Selected EACs Examined in the Pubertal Male Assay

32. Profile of Selected EACs Examined in the Hershberger Assay

33. Detection of p,p’-DDE in the Intact Male Assay • Weak AR antagonist • Strain differences observed in 15-day intact male assay • O’Connor et al., 1999 (Toxicol. Sci. 51: 44-53) • CD rats – not clearly identified as an AR antagonist • LE rats – identified as an AR antagonist • Consistent with strain differences observed in studies of You et al., 1998 (Toxicol. Sci. 45, 162-173)

34. Effect of p,p’-DDE on Organ Weights in the Intact Male Assay

35. Effect of p,p’-DDE on Serum Hormone Levels in the Intact Male Assay

36. Effect of p,p’-DDE on Thyroid Hormone Levels in the Intact Male Assay

37. Detection of Di-n-Butyl Phthalate (DBP) in the Intact Male Assay • Antiandrogen-like mode of action • Inhibition of steroidogenesis? • Mylchreest et al., 2002 (Reprod. Toxicol. 16, 19-28) • Shultz et al., 2001 (Toxicol. Sci. 64, 233-242) • Results from intact male assay are consistent with steroid biosynthesis inhibition as the mode of action of DBP

38. Effect of DBP on Organ Weights & Histopathology in the Intact Male Assay

39. Effect of DBP on Organ Weights in the Intact Male Assay

40. Comparison of the EDSTAC-Recommended and Alternative Tier I Screening Batteries for Identifying EACs

41. Advantages of Tier I Using Intact Male Assay • Comprehensive mode-of-action screen • Capable of evaluating several different modes of action in a single assay -- by measuring mechanistic endpoints (androgen, estrogen and thyroid agonists/antagonists; steroid hormone synthesis (aromatase & steroidogenesis) • Tier I with intact male provides mode of action “profile” to focus direction of any further testing • Intact endocrine system • Design allows integration of new endpoints if desired • Consider value of using Intact male in Tier 1 • Need a more in-depth analysis – side by side comparison (apples to apples) of Tier 1 in vivo assays (Hershberger/pubertals/intact male) • Specificity and sensitivity of the alternative approaches should be directly assessed with common set of substances across different modes of action • O’Connor et al., (2002). Evaluation of the Tier I screening options for detecting endocrine-active compounds (EACs). Critical Reviews in Toxicology, 32: 521-549.

42. Inter-Laboratory Studies Using the Intact Male Assay • EPA • Linuron • Methoxychlor • 3 to 4 more in 2004? • CTL • Genistein • Dow • Flutamide • Bayer • Nonylphenol • WIL • Methyltestosterone • Exxon-Mobil • p,p’-DDE (2004)

43. Identification of EACs Tier I ER Agonists ER Antagonists Uterotrophic Assay Receptor Binding/ Transactivation Agonist/Antagonist (ER, AR) Thyroid Effects Steroid Biosynthesis ER/AR Agonists ER/AR Antagonists Intact Male Assay

44. Publications: 15-Day Intact Male Assay O’Connor, J.C., Cook, J.C., Marty, M.S., Davis, L.G., Kaplan, A.M., and Carney, E.W. (2002). Evaluation of the Tier I screening options for detecting endocrine-active compounds (EACs). Critical Reviews in Toxicology, 32: 521-549. O’Connor, J.C., Frame, S.R., and Ladics, G.S. (2002). Evaluation of a 15-day screening assay using intact male rats for identifying antiandrogens. Toxicological Sciences, 69: 92-108. O’Connor, J.C., Frame, S.R., and Ladics, G.S. (2002). Evaluation of a 15-day screening assay using intact male rats for identifying steroid biosynthesis inhibitors and thyroid modulators. Toxicological Sciences, 69: 79-91. O’Connor, J.C., Davis, L.G., Frame, S.R., and Cook, J.C. (2000). Detection of dopaminergic modulators in a Tier I screening battery for identifying endocrine-active compounds (EACs). Reproductive Toxicology, 14: 193-205. O’Connor, J.C., Davis, L.G., Frame, S.R., and Cook, J.C. (2000). Evaluation of a Tier I screening battery for detecting endocrine-active compounds (EACs) using the positive controls testosterone, coumestrol, progesterone, and RU486. Toxicological Sciences, 54: 338-354. O’Connor, J.C., Cook, J.C., Frame, S.R., and Davis, L.G. (1999). Detection of the environmental antiandrogen p,p’-DDE in Sprague-Dawley and Long-Evans rats using a Tier I screening battery and a Hershberger Assay. Toxicological Sciences, 51: 44-53. O’Connor, J.C., Frame, S.R., and Cook, J.C. (1999). Detection of thyroid toxicants in a Tier I screening battery and alterations in thyroid endpoints over 28 days of exposure. Toxicological Sciences, 51: 54-70. O’Connor, J.C., Cook, J.C., Slone, T.W., Frame, S.R., and Davis, L.G. (1998). An ongoing validation of a Tier I screening battery for detecting endocrine-active compounds (EACs). Toxicological Sciences, 46: 45-60. O’Connor, J.C., Frame, S.R., Biegel, L.B., Cook, J.C., and Davis, L.G. (1998). Sensitivity of a tier I screening battery compared to an in utero exposure for detecting the estrogen receptor agonist 17-estradiol. Toxicological Sciences 44: 169-184. Cook, J.C., Kaplan, A.M., Davis, L.G., and O’Connor, J.C. (1997). Development of a tier I screening battery for detecting endocrine active compounds (EACs). Regulatory Toxicology and Pharmacology 26: 60-68.