# Presentation: 337910

## Male and Female Pubertal Assays

**Endocrine Disruptor Screening Program:**** **

**Ralph L. Cooper**
**Endocrinology Branch**
**Reproductive Toxicology Division**
**NHEERL, U.S. EPA**

**Male and Female Pubertal Assays**

## Puberty

**Puberty is a period of dramatic neuroendocrine development that culminates in reproductive maturation.**** **
**Requires extensive interplay between a variety of hormones, organs and tissues.**** **
**Period of increased sensitivity to environmental agents.**

## Pubertal Assays for Endocrine Disrupting Chemicals

**Many endpoints have been standardized**
**Many endpoints are currently being used in EPA testing**
**Large Toxicology database**
**Female Protocol is recommended**
**Male Pubertal is alternate**

## Objectives

**EDSP Pubertal Protocols**** **
**Review Protocols for Male and Female Rat**
**Discuss data from a variety of sources indicating the ability of these protocols to detect EDCs**
**Discuss advantages and potential problems **

## Assessment of Pubertal Development and Thyroid Function in Juvenile Female Rats

## Assessment of Pubertal Development and Thyroid Function in Juvenile Female Rats

**Purpose**
**   ****The purpose of this protocol is to quantify the effects of environmental compounds on pubertal development and thyroid function in the intact juvenile female rat.**

## Assessment of Pubertal Development and Thyroid Function in Juvenile Female Rats

**Applicability**
**   ****This assay detects agents that display anti-thyroid, estrogenic, anti-estrogenic [estrogen receptor (ER)] or steroid enzyme mediated activity, or alter luteinizing hormone (LH), follicle stimulating hormone (FSH), prolactin, growth hormone (GH) secretion or hypothalamic function.****  **

## Female Pubertal Protocol

## Required Endpoints(Female pubertal assay)

**Growth (body weight)**
**Age and weight at vaginal opening**
**Serum thyroxin (T4) and thyroid stimulating hormone (TSH)**
**Liver, kidney, pituitary and adrenal weight**
**Thyroid histology**
**Uterine and ovarian weights and histology**
**Vaginal cytology**

## Optional Measures (Female pubertal assay)

**Serum estradiol, Prolactin and tri-iodothyronine (T3)**
**Thyroid weight**
**Liver, kidney, pituitary, adrenal & vaginal histology**
***Ex-vivo***** ovary and pituitary hormone production**
**Hypothalamic neurotransmitter concentration**
**Estrous cycle in adulthood**

## Slide 11

## Summary of Significant Effects on Major Endpoints in

**Female  Sprague-Dawley and Long-Evans rats**

| Treatment | Mode of Action | Age at
 VO | Age at
First  E | Histo-pathology | TSH | T4 |
| --- | --- | --- | --- | --- | --- | --- |
| Ethynyl Estradiol
0.005 mg/kd | 
ER agonist |  |  |  | 
= | 
= |
| Tamoxifen
10 mg/kg | ER antagonist Partial agonist |  |  |  | 
  SD |  |
| Propylthiouricil
240 mg/kg | Inhibits T4 Synthesis | 
   SD | 
   SD |  |  |  |
| Ketoconozole
100 mg/kg | Inhibits Steroidogenesis | 
   SD |  |  | 
= |  |
| Pimozide
30 mg/kg | DA receptor blocker |  | 
= |  | 
  LE |  |
| Methoxy-chlor
100 mg/kg | ER agonist |  |  |  | 
  LE | 
= |

## Slide 13

## Gray et al., 1999

## Gray et al., 1999

## Effect of Chlorotriazines on Puberty

**Atrazine Alters Neuroendocrine Control of gonadal Function**** **
**GnRH pulses, LH and prolactin surges decreased**
**Hypothalamic catecholamines decreased, GABA neurotransmission altered****	**
**Hypothesis:  Atrazine will alter onset of puberty in male and female**** **
**Dose response using pubertal protocol**
**Evaluated Wistar strain**

- Cooper et al., 2000

## Effect of Atrazine on Puberty in Female Wistar Rats

- Laws et al., 2000

## Effect of Atrazine on Puberty in Female Wistar Rats

- Laws et al., 2000

## Assessment of Pubertal Development and Thyroid Function in Immature Male Rats

## Assessment of Pubertal Development and Thyroid Function in Immature Male Rats

**Purpose**
**   ****The purpose of this protocol is to quantify the effect of environmental compounds on pubertal development and thyroid function in the intact juvenile/peripubertal male rat**

## Assessment of Pubertal Development and Thyroid Function in Immature Male Rats

**Applicability**
**   ****This assay detects compounds that display anti-thyroid, estrogenic, androgenic, anti-androgenic [androgen receptor (AR)] or steroid enzyme mediated activity or alters puberty via changes in follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin, growth hormone (GH) or hypothalamic function.  **

## Slide 22

## Required Endpoints(Male pubertal assay)

**Growth (body weight)**
**Age and weight at preputial separation**
**Serum thyroxin (T4) and thyroid stimulating hormone (TSH)**
**Thyroid histology**
**Seminal vesicle plus coagulating gland ****	****(with and without fluid) **
**Liver, kidney, adrenal, pituitary and ventral Prostate weight**
**Levator ani plus bulbocavernosus weight**
**Epididymal and testis weight & histology**

## Optional Measures (Male pubertal assay)

**Serum testosterone, estradiol, LH, prolactin and tri-iodothyronine (T3)**
**Liver, kidney, adrenal, pituitary histology**
***Ex-vivo***** testis and pituitary hormone production**
**Hypothalamic neurotransmitter concentrations**

## Slide 25

## Slide 26

## Summary of Significant Effects on Major Endpoints in

| Treatment | Mode of Action | Age at Preputial Separation | Histo-
pathology | TSH | T4 |
| --- | --- | --- | --- | --- | --- |
| Fluatamide
(50 mg/kg/d) | AR
Antagonist |  |  | = | = |
| Methyl
Testosterone
80 mg/kg/d | AR
Agonist |  |  | 
= | 
= |
| Propylthiouracil
240 mg/kg/d | Inhibits 
T4 synthesis |  |  |  |  |
| Ketoconozole
100 mg/kg/d | Inhibits 
Steroido-    genesis |  | 
= | 
= | 
= |
| Pimozide
30 mg/kg/d | Dopamine
Receptor 
Blocker |  | LE | 
= | 
= |
| Dibutylphthalate
1000 mg/kg/d | Anti-androgenic
 | 
  LE | LE | =
 | 
  LE |

**Summary of Significant Effects on Major Endpoints in**
**Male  Sprague-Dawley and Long-Evans rats**

## Monosson et al., 1999

## Monosson

-  et al., 1999

## Stoker et al., 2000

## Slide 31

## Slide 32

## Summary

**Pubertal protocols detect a wide variety of EDCs**
**Advantages **
**Tests are apical**
**Dose response information, metabolism, dose individual rats**
**Provide information for MOA and mechanistic studies**
**Appear to be robust across strains**
**Protocols involve relatively simply procedures**

## Summary

**Pubertal protocols detect a wide variety of EDCs**
**Difficulties/drawbacks **
**Precise measures of hormones**
**Body weight issues**
**Dosing not done during organogenesis (i.e., not a transplacental assay)**
**Length of assay**
**Cost**

## Single Dose Study

**Discrepancy between the ages of preputial separation identified in the two strains of rats.**
**Large degree of variation associated with the means of the fluid-filled and small tissue weights**

## Single Dose Study

**Improve descriptive text in protocols such that every key step is clear.**
**Establish performance criteria for inclusion into the protocols**
**Evaluate lower limits of detection of protocols by examining dose response for weaker EDCs**
**Should strain be recommended?**

## Collaborators

**Endocrinology Branch, Reproductive Toxicology Division, NHEERL**
**L. Earl Gray Jr., Ph.D.**
**Susan C. Laws, Ph.D.**
**Tammy Stoker, Ph.D.**
**Jerome M. Goldman, Ph.D.**
**Robert J. Kavlock, Ph.D. **

**Office of Science Coordination and Policy**
**	****OPPTS**
**Jim Kariya**
**Gary Timm**