# EDSP Tier 1 Rodent **In Vivo** Screening Assays

John Liccione, Ph.D. Office of Chemical Safety and Pollution Prevention U.S. Environmental Protection Agency

# Overview

 

# Brief description of each individual assay including:

• Purpose and study design
• Adherence to Performance Criteria
• Technical Issues



#  

# Conclusions

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# Rat **In Vivo** Assays

 

# Uterotrophic (OPPTS 890.1600)

 

# Hershberger (OPPTS 890.1400)

 

# Male Pubertal (OPPTS 890.1500)

 

# Female Pubertal (OPPTS 890.1450)

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# Uterotrophic Assay Study Design

 

## Evaluate ability of chemical to elicit an agonist estrogen response — ↑ uterine wt

 

## Rapid and incorporates first pass metabolism if oral route used

 

## Mechanistic sensitivity and specificity

 

## 2 Models:
 Immature female & Adult ovariectomized (OVX) female rat

• 

### Immature (PND 18): 3 days dosing (oral or sc); necropsy
• 

### Adult OVX (6 - 8 wk): preferred; 3 days dosing (oral or sc); necropsy

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# Uterotrophic Assay

 

### Endpoints (both models)
• Uterine wts (wet & blotted); BW, FC, clinical signs, mortality;
• Optional
— histology of uterus, cervix and vagina

 

### Positive Response
 E strogenic: ↑ uterine wt (p < 0.05)

• 

### P ositive control :17α - ethynyl estradiol (EE)

• 

### ≥4 doses EE (baseline study); dose response for uterine wt; 70 - 80% of effective dose from baseline study

 

### Performance Criteria
• Blotted uterine wts — 0.09% final BW (immature); 0.04% final BW (OVX)

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# Uterotrophic Assay

 

# Summary of Performance
• All 17 chemicals were negative (estrogenic)
• EE (positive control) ↑ uterine wt as expected



##  

# Technical Issues
• No major issues
• Baseline control studies not reported for 2 test chemicals
• Overt toxicity at highest dose tested (HDT) for 1 chemical: (↓ BW; clinical signs)

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# Uterotrophic Assay

 

# Conclusions

• Assay performance was consistent across all 17 test chemicals
• Assay performed as expected
• Dosing considered adequate

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# Hershberger Assay Study Design

 

### Detects androgenic agonists, antagonists and 5α - reductase inhibitors

 

### Rapid and incorporates first pass metabolism if oral route used

 

### Mechanistic sensitivity and specificity

 

### PND42
– 48 male rats (castrated) are dosed for 10 days (oral, sc); HDT
— at or below MTD but not exceed 1000 mg/kg/day

 

### Endpoints based on wts of 5 androgen - dependent tissues in castrated male rats

• Ventral prostate (VP), seminal vesicles (SV), levator ani & bulbocavernosus muscles [LABC], Cowper’s glands [CG], and glans penis [GP]

 

### Other Endpoints: clinical signs, BW, mortality, wts of liver, adrenal & kidney; optional
— testosterone, luteinizing hormone, follicle stimulating hormone

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# Hershberger Assay

 

### Positive Response
• Androgenic: ↑ ≥2 androgen - dependent wts
• Anti - androgenic: ↓ ≥2 androgen - dependent wts
 

### Performance Criteria for organ weights
• Maximum recommended % Coefficient of Variation (CVs) for Sex Accessory Tissues:

9 Tissue Anti - androgenic Androgenic Seminal Vesicle 40% 40% Ventral Prostate 40% 45% LABC 20% 30% Cowper’s gland 35% 55% Glands penis 17% 22%

# Hershberger Assay

 

# Summary of Performance
• None of 17 chemicals showed androgenic response • 7 showed anti - androgenic response

• 

### 4 of the 7 were in presence of overt toxicity (↓BW & FC, clinical signs)


##  

# Technical Issues
• No major issues
• % CV in excess of performance criteria noted in 14 of 17 studies
— but no major impact on study interpretation

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# Hershberger Assay

 

# Conclusions

• Assays conformed to guidelines
• Assay performance consistent across 17 test chemicals
• Dosing considered adequate

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# Female Rat Pubertal Assay Study Design

 

### Purpose
• Identify pubertal development & thyroid function in intact ♀ juvenile rats
• Detects antithyroid, estrogenic, anti - estrogenic chemicals or alterations in hypothalamic - pituitary - gonadal (HPG) axis

 

### Design
• ♀ rats treated orally (daily) from PND22
– 42 (21 days)
• Weight and age of attainment of VO recorded
• ANCOVA with body weight at weaning as covariate: age & BW at VO and organ weights
 

### Endpoints
• Endocrine - related: age at VO and first estrus; changes in estrus cyclicity ; wts and histology of ovary, uterus, thyroid; serum T4, TSH
• Other: clinical signs, mortality, BW, clinical chemistry, wts of liver, pituitary, adrenal, kidney

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# Female Rat Pubertal Assay

 

### Dose Selection
• HDT: at or just below the MTD but not exceed 1000 mg/kg/day; toxicity profile of the chemical considered
• 2 nd dose level spaced to produce a lesser degree of toxicity relative to HDT
 

### Strengths & Limitations
• Involves pubertal period of development
• Well - established knowledge base of estrogen and thyroid dependent endpoints
• Variability of organ wts (associated with ovarian cycle)
• Inability to identify mechanisms of action

 

### Critical Evaluation
• Alterations in VO, first estrus, estrus cyclicity ; wt and histology of ovaries and uterus; changes in thyroid wt and histology; T4 and TSH

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# Female Rat Pubertal Assay

 

### Performance Criteria
• For vehicle - control: mean, 2 SD, CV, 1.5 CV, and range of values

• Endpoints: weights of uterus, ovaries, thyroid, adrenals, kidneys & pituitary; T4 levels; final BW; and age and weight at VO

 

### Summary of Performance
• 6 of 17 chemicals showed response in at least 1 estrogen - dependent endpoint

• 4 of the 6
— in presence of overt toxicity (↓BW & FC, clinical signs)
• 1 of 17 chemicals showed response in thyroid - related endpoints

• 4 of the 11
— in presence of overt toxicity (↓BW & FC, clinical signs)

 

### Technical Issues
• No issues that compromised conduct and/or interpretation
• Minor deviations in %CV outside limits of performance criteria

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# Female Rat Pubertal Assay

 

# Conclusions

• Assays conducted in accordance with guidelines
• Assay performance was generally consistent across all 17 test chemicals
• Dosing considered adequate

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# Male Rat Pubertal Assay Study Design

 

### Purpose
• Identify effects on pubertal development & thyroid function in intact rat
• Detects chemicals with antithyroid, androgenic, or anti - androgenic activity, or perturbations in HPG axis

 

### Design
• Rats treated orally (daily) from PND23
– 53 (30 days)
• Examine for preputial separation (PPS) at PND30
• ANCOVA with body weight at weaning as covariate: age & BW at PPS and organ weights

 

### Endpoints
• Endocrine - related: age & wt at PPS ; wts of VP, DP, LABC, SV, epididymides, testes, thyroid; histology of epididymis, testes, thyroid; T4, TSH; testosterone
• Other: clinical signs, mortality, BW, wts of liver, kidneys, pituitary, adrenals; histology of the kidney; clinical chemistry

16

# Male Rat Pubertal Assay

 

### Dose Selection
• HDT: at or just below the MTD but not exceed 1000 mg/kg/day; toxicity profile of the chemical considered
• 2 nd dose level spaced to produce a lesser degree of toxicity relative to HDT
 

### Strengths & Limitations
• Involves pubertal period of development and includes metabolism
• Well - established knowledge base of androgen and thyroid dependent endpoints
• Inability to identify mechanisms of action

 

### Critical Evaluation
• Alterations in PPS, changes in androgen - sensitive organ wts , testosterone, thyroid wt and histology; T4 and TSH

17

# Male Rat Pubertal Assay

 

### Performance Criteria
• For vehicle - control: mean, 2 SD, CV, 1.5 CV, and range of values

• Endpoints: wts of VP, LABC, SV, epididymides, testes, thyroid, adrenal, kidneys, liver, pituitary; testosterone; age at PPS; T4 & TSH; weaning wt; final wt

 

### Summary of Performance
• 9 of 16 chemicals showed response in at least 1 androgen - dependent endpoint

• 2 of the 9
— in presence of overt toxicity (↓ BW & FC; salivation)
• 7 of 16 chemicals showed response in thyroid - related endpoints

• 2 of the 7
— in presence of overt toxicity (↓ BW & FC; salivation)

 

### Technical Issues
• No deficiencies that compromised conduct and/or interpretation

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# Male Rat Pubertal Assay

 

# Conclusions

• Assays conducted in accordance with guidelines
• Assay performance was generally consistent across all 16 test chemicals
• Dosing considered adequate

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