Tier 1 EDSP Uterotrophic and 

Hershberger Assay Comments

Barbara Neal, DABT

ExponentTM

On Behalf of the Endocrine Policy 

Forum
Introduction

• Provide comments on the technical 

performance and reliability of:

– The uterotrophic assay (OCSPP 890.1600)

• Assay for potential estrogenicity

– The Hershberger assay (OCSPP 890.1400)

• Phase I assay for potential androgenicity

• Phase II assay for potential anti-androgenicity

– Comment on how these assays may add to the 

Tier I test battery

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Uterotrophic Assay

• The uterotrophic assay for estrogenicity is 

well-validated, scientifically sound and easy to 

interpret. 

– The agonist option (currently required in Tier I 

screening) is mechanistically based and reliably 

detects potential estrogen agonists. 

– (The antagonist option is not recommended at the 

present time).

– Both the ovariectomized model (OVX) and the 

immature model appears reliable.

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Uterotrophic Assay Difficulties

• Range-finding studies generally required for 

dose selection; MTD criteria are hard to meet. 

• OVX model: external supplier may not 

provide age of animals and dates for 

ovariectomy; ovariectomies may not be 

complete.

• Immature model: animals must be prepubertal.

• EPA identified no difficulties sufficient to 

compromise interpretation of the assay.

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Uterotrophic Assay-Performance Criteria

• Positive control data met criteria for all assays

• Both models showed statistically significant 

increases in positive control blotted uterine 

weights (generally dose-related up to 10 

μg/kg/day EE); excessive doses of EE >10 

μg/kg/day may lead to non-dose-related 

responses.

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Utility of the Uterotrophic Assay

• Gold standard assay-specific, reliable and 

sensitive (in vivo so includes ADME).

• Premature to consider its removal from Tier 1 

battery.

• It can distinguish compounds that potentially 

interact via the ER receptor from compounds that 

cause sporadic or non-specific findings (e.g., fish 

fecundity) in other Tier 1 assays or in OSRI. 

• Animal welfare: Data from reproductive toxicity 

studies may also be used in lieu of this assay 

using weanling uterine weights prior to puberty 

onset.

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Hershberger Assay

• The Hershberger assay reliably detects chemicals 

that potentially interact with the androgen 

pathway, either as androgen receptor agonists, 

antagonists, or 5α-reductase inhibitors. 

• Can be confounded in Phase II of the assay by 

hepatic enzyme inducers-increased clearance of 

exogenous testosterone.

• With appropriate dose level selection (absence 

of overt toxicity) the assay is specific for those 

activities. 

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Difficulties With the Hershberger Assay

• The assay guideline is very complex; it should 

be simplified to include only information 

critical to assay conduct.

• A dose range-finding study(s) is generally 

necessary.

• The number of animals per group (6) is too 

small to develop reliable serum hormone 

data.

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Difficulties with the Hershberger Assay

(Continued)

• The assay is also potentially subject to artifacts 

from high systemically toxic doses which may 

impact some accessory sex organ weights.

• Multivariate analyses, when there are 

differences in only one androgen-sensitive 

tissue weight, may lead to an increased number 

of false positives.

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Utility of the Hershberger Assay

• The Hershberger assay is the “gold standard” 

for detection of potential androgen pathway 

interactions. 

• It is both sensitive and relatively specific for 

this purpose provided excessively toxic doses 

are not used. 

• This assay provides additional information on 

activity and mode of action for compounds 

that show sporadic or less specific indications 

of potential androgen pathway interactions in 

the male pubertal or FSTRA, or in OSRI. 

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Utility of the Hershberger Assay 

(continued)

• Premature to consider its removal from the 

Tier 1 battery.

• Similar to the uterotrophic assay, however, the 

anti-androgenic phase of the Hershberger in 

particular could provide an important assay to 

help inform Tier 2 testing decisions.

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