# Presentation: 685975

## 
John C. O’Connor

DuPont Haskell Laboratory for Health and 
Environmental Sciences
&
The American Chemistry Council

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

**The Intact Male Assay As An Alternative Tier I Screening ****Assay For Detecting ****Endocrine-Active Compounds**

## 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**

**Notes:**
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

***Tier I***

**Uterotrophic**
**Assay**

**Receptor Binding/**
**Transactivation**

**Intact Male**
**Assay**

**Thyroid Effects**
**Steroid Biosynthesis**
**ER/AR Agonists**
**ER/AR Antagonists**

**ER Agonists**
**ER Antagonists**

**Agonist/Antagonist**
**(ER, AR)**

**Notes:**
Highlight that male is more sensitive than female for detecting thyroid modulators.

## 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, T****3****, T****4****, 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**

**Notes:**
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

## Hypothalamic-Pituitary-Ovary Axis

## 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 Organ Weights in Sprague-Dawley Rats**

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

**Notes:**
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

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

## Immature vs. Mature Rats

**Immature**

**Mature**

## Immature vs. Mature Rats

**Immature vs. Mature Rats**

**Immature**

**Mature**

## Intact Male Assay:  Thyroid Timecourse

## Intact Male Assay:  Thyroid Timecourse

## Case Study With Flutamide, Ketoconazole, & Finasteride

## Hypothalamic-Pituitary-Testis Axis

## 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**

## Case Study:  Organ Weights

## Case Study:  Serum Hormones

**Case Study:  Serum Hormones**

## Testosterone Biosynthesis (4 Pathway)

## 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

**Case Study:  Flutamide, Ketoconazole, & Finasteride****Comparison of Serum Hormone Data**

## Hypothalamic-Pituitary-Testis Axis

**Notes:**
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

**EACs Examined in the 15-Day Intact Male Assay**

**Notes:**
Mention studies were performed under GLPs.

## EACs Examined in the Pubertal Assays

**Notes:**
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 Intact Male Assay**

## Profile of Selected EACs Examined in the Pubertal Male Assay

**Profile of Selected EACs Examined in the Pubertal Male Assay**

## Profile of Selected EACs Examined in the Hershberger 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****)**

**Notes:**
Will discuss if time allows.

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

**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 Serum Hormone Levels in the Intact Male Assay**

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

**Effect of  *****p,p’*****-DDE on Thyroid Hormone Levels 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

**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**

**Detection of Di-n-Butyl Phthalate (DBP) in the Intact Male Assay**

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

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

## Effect of  DBP on Organ Weights 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**

**Receptor Binding/**
**Transactivation**

**Intact Male**
**Assay**

**Thyroid Effects**
**Steroid Biosynthesis**
**ER/AR Agonists**
**ER/AR Antagonists**

**ER Agonists**
**ER Antagonists**

**Agonist/Antagonist**
**(ER, AR)**

## Publications:  15-Day Intact Male Assay

- 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.

**Publications:  15-Day Intact Male Assay**