## Tier 1 EDSP Uterotrophic and Hershberger Assay Comments Barbara Neal, DABT E x ponent TM 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 pre

pubertal.

• 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 μ g/kg/day EE); excessive doses of EE >10 μ g/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 5 α - 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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