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

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 Point out that we are not making a direct comparison to the other tier I screening assays. We are illustrating the utility of the intact male assay for detecting EACs.

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

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

Proposed Tier I Screening Battery Uterotrophic Assay ER Agonists ER Antagonists Receptor Binding/ Transactivation Agonist/Antagonist (ER, AR) Highlight that male is more sensitive than female for detecting thyroid modulators. Thyroid Effects Steroid Biosynthesis ER/AR Agonists ER/AR Antagonists Intact Male Assay

15-Day Intact Male Assay Model: Required Endpoints: 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 Focus discussion on the study design and required endpoints.

Mechanisms that Modify Hormone Action Positive/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)

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

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

Intact Male Assay: “Expected” Profile for Unknowns

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

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

Effect of Diet Restriction on Serum Hormones in Sprague-Dawley Rats One important aspect in the design of endocrine studies is the effect of body weight on circulating hormone concentrations. In the experiment summarized above [O’Connor et al., 2000; Toxicol. Sci. 54, 338-354], male rats were maintained on various levels of dietary restriction in order to produce a range of body weight decrements. The male rats were necropsied after 15 days of dietary restriction and a series of parameters evaluated including reproductive organ weights, reproductive hormone concentrations, and thyroid parameters. When interpreting changes in hormone concentrations, as well as other endpoints, it is important that the effect of body weight loss on the endpoints is evaluated in order to eliminate/minimize potential confounding due to overt toxicity.

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

Immature vs. Mature Rats

Immature vs. Mature Rats

Intact Male Assay: Thyroid Timecourse

Intact Male Assay: Thyroid Timecourse

Case Study With Flutamide, Ketoconazole, & Finasteride

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

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

Case Study: Organ Weights

Case Study: Serum Hormones

Testosterone Biosynthesis (4 Pathway) 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) Pregnenolone Cholesterol Progesterone 17a-Hydroxyprogesterone Androstenedione Testosterone 1a 1b 1c ② ③ ④ Ketoconazole ⑤ ⑥ 5a-Dihydroxytestosterone ⑦ ⑧ Estrone 17b-Estradiol Leydig Cell X Finasteride Target peripheral tissues Flutamide

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

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

Hypothalamic-Pituitary-Testis Axis CNS Hypothalamus Anterior Pituitary GnRH (+) FSH (+) Sertoli Cell Leydig Cell Inhibin Testosterone Aromatase Estradiol (-) Testis LH (+) DHT 5-Reductase Target Peripheral Tissues Antiandrogens (Flutamide) X Steroid Inhibitors (Ketoconazole) 5a-Reductase Inhibitors (Finasteride) Regulation of the hypothalamic-pituitary-testis axis in mammals. Abbreviations: central nervous system (CNS); gonadotropin releasing hormone (GnRH); follicle stimulating hormone (FSH); luteinizing hormone (LH); dihydrotestosterone (DHT). Potential sites of endocrine disruption include: ① Dopamine agonists would act on the CNS to affect GnRH release; ② Steroid biosynthesis inhibitors would inhibit testosterone production, reducing the amount of testosterone and perhaps DHT or estradiol; ③Aromatase inhibitors would inhibit the conversion of testosterone to estradiol; ④ 5α-reductase inhibitors would inhibit the conversion of testosterone to DHT; ⑤Androgen receptor blockers would interfere with the normal androgen feedback to the pituitary and brain, as well as decreasing androgen action peripherally; ⑥Estrogen receptor blockers would interfere with the normal estrogen feedback to the pituitary and brain, as well as decreasing estrogen action peripherally. Inhibin has not yet been rigorously evaluated in environmental toxiciology.

EACs Examined in the 15-Day Intact Male Assay Mention studies were performed under GLPs.

EACs Examined in the Pubertal Assays Point out that for the pubertal assays there is no agreement on how to set and MTD Point out that we did not come prepared to make definitive comparisons between assays. Goal is to return at a later date to discuss differences between the assays.

Profile of Selected EACs Examined in the Intact Male Assay

Profile of Selected EACs Examined in the Pubertal Male Assay

Profile of Selected EACs Examined in the Hershberger Assay

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) Will discuss if time allows.

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

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

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

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

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

Effect of DBP on Organ Weights in the Intact Male Assay

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

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.

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)

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

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.