# Presentation: 056431

## 
John C. O’Connor

DuPont Haskell Laboratory for Health and 
Environmental Sciences

**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**

## Outline

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

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

## 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**

## 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?**

## Hypothalamic-Pituitary-Testis Axis

**Hypothalamic-Pituitary-Testis Axis**

## Adult Male Assay:  “Expected” Profile for Unknowns

## 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: 338-354)**
**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**

## 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

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

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

## 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**

## Immature vs. Mature Rats

**Immature vs. Mature Rats**

**Immature**

**Mature**

## Immature vs. Mature Rats

**Immature vs. Mature Rats**

**Immature**

**Mature**

## 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**

## Organ Weights in Immature and Mature Rats Gavaged With Vinclozolin for 15 Days

_**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.

## Serum Hormone Concentrations in Immature and Mature Rats Gavaged With Vinclozolin for 15 Days

**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.

## 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.

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

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

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

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

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

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

## Adult Male Assay:  Thyroid Timecourse

## Adult Male Assay:  Thyroid Timecourse

## 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, 162-173****)**

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

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

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

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

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

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

## Case Study With Flutamide, Ketoconazole, & Finasteride

## Hypothalamic-Pituitary-Testis Axis

**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**

## Effect Of Ketoconazole On Testosterone 

**Biosynthesis From Isolated Rat Leydig Cells**

## 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**

**2**

**3**

**4 ****Ketoconazole**

**5**

**6**

**5********-Dihydroxytestosterone**

**7**

**8**

**8**

**Estrone**

**17********-Estradiol**

**Leydig Cell**

**X**

**Finasteride**

**X**

- Target peripheral tissues

**X**

**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

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

## Hypothalamic-Pituitary-Testis Axis

## EACs Examined in the Adult Male Assay

**EACs Examined in the Adult Male Assay**

## EACs Examined in the Pubertal Male/Female Assays

## 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)**

## 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: 521-549.**

## Completed Inter-Laboratory Studies Using the 15-Day Intact Adult Male Rat Assay Protocol

| Laboratory | EAC | Endocrine Activity | Result* |
| --- | --- | --- | --- |
| RTI
(CRO) | Methoxychlor | ER agonist | Positive1 |
| RTI
(CRO) | Linuron | AR agonist | Positive1 |
| BASF
(Industry) | Nonylphenol | ER agonist | Positive2 |
| Syngenta
(Industry) | Genestein | ER agonist | Negative2 |
| Dow
(Industry) | Flutamide | AR antagonist | Positive2 |
| WIL
(CRO) | Methyltestosterone | AR agonist | Positive2 |

- *Data available though the EPA (1) or ACC (2)

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

## 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**

## 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?**