John C. O’Connor DuPont Haskell Laboratory for Health and Environmental Sciences The 15-Day Intact Adult Male Assay As An Alternative Tier I Screening Assay For Detecting Endocrine-Active Compounds

2 Outline Background Overview of the 15-day intact adult male assay –Study rationale –Study design considerations –Case study with flutamide, ketoconazole, & finasteride Path Forward

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

4 Desirable Attributes of a Screen Reliable (identifies known EACs for EAT activity) 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 Study Design: 15-Day Intact Adult Male Assay Model: –10-Week old intact male rats –n = 15/group –Control + 4 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, DHT, estradiol, prolactin, LH, FSH, T 3, T 4, TSH –Biochemical - preparation of hepatic microsomes Optional Endpoints (if warranted by other findings) –Histopathology – liver –Biochemical Assessment Hepatic UDP-glucuronyltransferase activity Hepatic aromatase activity –Others?

CNS Hypothalamus Anterior Pituitary GnRH (+) FSH (+) Sertoli Cell Leydig Cell Inhibin Testosterone Aromatase Estradiol (-) Testis LH (+) X X X X DHT X 5  -Reductase Target Peripheral Tissues X Hypothalamic-Pituitary-Testis Axis

7 Adult Male Assay: “Expected” Profile for Unknowns

8 Adult 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: ) Adult vs. immature animals –Immature are more sensitive to organ weight changes –Mature are more sensitive to hormonal changes –Mature have greater blood volume for hormonal assessment Duration – 2-week Strain sensitivity differences

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

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

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

12 Data Interpretation High dose level should not exceed MTD, defined as <10% difference in final body weight when compared to the control group Organ Weights –Absolute Testes, epididymis –Relative (% final body weight) Liver, thyroid, prostate, seminal vesicles, ASG unit Histopathology - testis, epididymides, thyroid Control + high dose unless effects are observed Hormonal battery - testosterone, DHT, estradiol, prolactin, LH, FSH, T 3, T 4, TSH Evaluate trends across dose response and overall pattern of hormonal effects

13 Immature vs. Mature Rats Immature Mature

14 Immature vs. Mature Rats Immature Mature

15 Immature vs. Mature Comparison: Study Design Goal: Compare responses of immature and mature rats to 2 positive controls using identical study designs, and evaluate the intrinsic variability of serum hormone and organ weight measurements in mature and immature rats. Model: –Immature intact male rats (age 53 days at necropsy) –n = 15/group –15-Day test (oral) –Control + 4 dose groups Vinclozolin (75 and 150 mg/kg/day) Phenobarbital (5 and 25 mg/kg/day) –Included feed restriction group (targeted to 10% body weight decrement from con. –Included two additional “control” groups (age 49 and 57 days at necropsy) –Second experiment currently being conducted with PTU, flutamide, linuron, & DBP

16 Age – 53 Days Organ Weights in Immature and Mature Rats Gavaged With Vinclozolin for 15 Days Age – 84 Days Note: Organ weight alterations were more pronounced at 300 mg/kg/day in mature rats, and included microscopic alterations of the testes at 150 and 300 mg/kg/day.

17 Serum Hormone Concentrations in Immature and Mature Rats Gavaged With Vinclozolin for 15 Days Age – 53 Days Age – 84 Days Note: Hormonal alterations were more pronounced at 300 mg/kg/day in mature rats.

18 Serum Hormone Concentrations in Immature and Mature Rats Gavaged With Phenobarbital for 15 Days Age – 53 Days Age – 84 Days Note: Thyroid weight was significantly increased at both dose levels in mature rats but was NOT affected in immature rats.

19 Correlation Between Age and Variability in Serum Hormone Concentrations in Rats

20 Correlation Between Age and Variability in Serum Hormone Concentrations in Rats

21 Correlation Between Age and Variability in Serum Hormone Concentrations in Rats

22 Adult Male Assay: Thyroid Timecourse

23 Adult Male Assay: Thyroid Timecourse

24 Detection of p,p’-DDE in the Adult 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, )

25 Effect of p,p’-DDE on Organ Weights in the Adult Male Assay

26 Effect of p,p’-DDE on Serum Hormone Levels in the Adult Male Assay

27 Effect of p,p’-DDE on Thyroid Hormone Levels in the Adult Male Assay

28 Case Study With Flutamide, Ketoconazole, & Finasteride

CNS Hypothalamus Anterior Pituitary GnRH (+) FSH (+) Sertoli Cell Leydig Cell Inhibin Testosterone Aromatase Estradiol (-) Testis LH (+) X X X X DHT X 5  -Reductase Target Peripheral Tissues X Hypothalamic-Pituitary-Testis Axis

30 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, T 3, T 4, TSH

31 Case Study: Organ Weights

32 Case Study: Serum Hormones

33 Effect Of Ketoconazole On Testosterone Biosynthesis From Isolated Rat Leydig Cells

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

35 Case Study: Flutamide, Ketoconazole, & Finasteride Comparison of Organ Weight Data Cannot differentiate mode of action based on organ weight changes

36 Mode of action can be determined based on hormonal changes Case Study: Flutamide, Ketoconazole, & Finasteride Comparison of Serum Hormone Data

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

38 EACs Examined in the Adult Male Assay

39 EACs Examined in the Pubertal Male/Female Assays

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

41 Advantages of Tier I Using Adult 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 –Reduces the number of animals needed for Tier I Intact endocrine system –Design allows integration of new endpoints if desired O’Connor et al., (2002). Evaluation of the Tier I screening options for detecting endocrine-active compounds (EACs). Critical Reviews in Toxicology, 32:

42 Completed Inter-Laboratory Studies Using the 15- Day Intact Adult Male Rat Assay Protocol LaboratoryEACEndocrine ActivityResult* RTI (CRO) MethoxychlorER agonistPositive 1 RTI (CRO) LinuronAR agonistPositive 1 BASF (Industry) NonylphenolER agonistPositive 2 Syngenta (Industry) GenesteinER agonistNegative 2 Dow (Industry) FlutamideAR antagonistPositive 2 WIL (CRO) MethyltestosteroneAR agonistPositive 2 *Data available though the EPA (1) or ACC (2)

43 Proposed Inter-Laboratory Studies Using the 15-Day Intact Male Rat Assay Protocol

44 Rationale for Compound Selection The mode-of-action of the compounds focus on EAT –Linuron – weak anti-androgen –Phenobarbital – weak thyroid-active agent –Ketoconazole – steroid biosynthesis inhibitor All compounds have been previously run in the 15-day intact male assay –Provide a base-line for expected results –Will facilitate inter-laboratory comparisons of the transferability of the 15-day intact male assay All compounds have been previously evaluated in other potential Tier I screening assays (Hershberger, pubertal male and/or pubertal female assays) –Will facilitate future comparisons among assays

45 Issues/Questions for the EDMVAC Is the protocol ready for inter-laboratory validation? –If not, what additional data is needed? Does the EDMVAC agree with the test material selection for inter-laboratory validation studies?