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

Estrogenic: ↑ uterine wt (p < 0.05)

• Positive 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:

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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

• 2nd 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

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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

• 2nd 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 

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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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