# Presentation: 522635

## Validation Plan for the EDSP MammalianTier 2 Assay

- Presentation to the
- Endocrine Disruptor Methods Validation Subcommittee
- June 6, 2003
- J. Kariya

## Overview of presentation

- Validation plan
- Concerns
- Questions for EDMVS

## Validation plan

- Accept the OPPTS Guideline for Reproductive Toxicity, with additional thyroid endpoints, as valid for EDSP Tier 2 purposes.
- Include clarifications of certain procedures and endpoints already in the Guideline as part of the EDSP assay.
- Ask for independent peer review of the validity of the Guideline and clarifications for EDSP purposes.

## Additional plans

- Encourage one-generation extension of F1 when indicated by Tier 1, and in all Tier 1 bypasses.
- Develop additional information on the relevance and reliability of the one-generation extension, for possible future inclusion in the EDSP assay.

## The 1998 Reprotox Guideline is generally accepted as valid...

- ... for regulatory risk assessment of reproductive toxicity
- Reproductive toxicity can be “an effect produced by a naturally occurring estrogen” and can be an indicator of endocrine effects
- ˆ the Guideline is valid for EDSP purposes.

## Additional endpoints

- EDSTAC:  consider specific additional endpoints, for non-reprotox endpoints
- SVTF:  adding all of these would be impractical and in some cases not useful;  focus on thyroid-specific endpoints
- Relevance of most of the thyroid-specific endpoints was shown in the PTU study which EDMVS reviewed (but drop T3)

## Table 1: Additional endpoints proposed for EDSP purposes

| TSH, T4, thyroid weight, thyroid histology, all at necropsy | Not in current Guideline | Declined to add T3 on the basis of PTU study results.

Add – or change – T4/TSH measurement to PND 21? |
| --- | --- | --- |
| Areola/nipples: what, where, how many, in both males and females, F1 and F2, PND 13.  Also at necropsy for F1 only. | “At the time of termination or death during the study, all parental animals (P and F1) and when litter size permits at least three pups per sex per litter from the unselected F1 weanlings and the F2 weanlings should be examined macroscopically for any structural abnormalities or pathological changes.  Special attention should be paid to the organs of the reproductive system.” | Differences are: addition of a new timepoint for examination (pnd 13); and number of animals (all, vs. 3/sex/litter). |
| Anogenital distance, all animals in both F1 and F2, at birth (PND 2) | AGD only for F2 and only when triggered by a treatment-related effect in F1 sex ratio or sexual maturation.   At birth (PND 0)
 |  |
| Whole-mount histology of mammary tissue in males, triggered if abnormalities are seen in gross examination. | “For F1 and F2 weanlings, histopathological examination of treatment-related abnormalities noted at macroscopic examination should be considered, if such evaluation were deemed appropriate and would contribute to the interpretation of the study data.”
(Whole-mount not specified) |  |

## Clarifications tocurrent Guideline

- Table 2: Specification of endpoints for mammalian two-generation study” (in handout)

## Concerns

- Strain/species White Paper (August)
- Interlab variability
- Thyroid: other mechanisms; sensitivity
- Extension of F1: additional studies
- Timing, if other studies are needed

## Questions for EDMVS#1 of 5

- Does the EDMVS agree that the additional endpoints/clarifications proposed for the 2-generation assay (Table 1) are well-characterized and that further validation of this set of endpoints for use in EDSP Tier 2 is unnecessary?

## Questions for EDMVS#2 of 5

- Does the EDMVS agree that the endpoints in the Tier 2 assay will allow a compound to be identified as possibly having “an effect in humans that is similar to an effect produced by a naturally occurring estrogen” (or androgen/anti-androgen or thyroid mimic/inhibitor) *in the absence of Tier 1 data*? If not, what other endpoints should be included, or what supplemental testing would be appropriate?

## Questions for EDMVS#3 of 5

- Does the EDMVS agree that the procedures and endpoints in Table 2 of the handout should be listed explicitly, to ensure adequate examination?

## Questions for EDMVS#4 of 5

- If EDMVS advises EPA to validate additional endpoints,
- can “new” endpoints be validated separately from endpoints already in the  reproductive toxicity assay?
- is it necessary to validate all new endpoints in a *two*-generation study, or can relevance and reliability be established in a shorter assay
- how many laboratories should be required for interlaboratory comparability?
- how many chemicals per mode of endocrine activity should be tested in validation?  (e.g., ER/AR binding, each step of steroidogenesis, thyroid hormone transport protein binding, thyroid hormone metabolism, etc.)

**Notes:**
Question 1: I.e., is it scientifically acceptable to examine the relevance and reliability of endpoints or must we validate the entire assay?

Question 2:  e.g., one-gen or in-utero-through-lactation study

## Questions for EDMVS#5 of 5

- Does the EDMVS agree that the one-generation extension study shows increased sensitivity and provides greater precision in dose/response assessment, which will be of use in risk assessment, when the F1 animals are allowed to mature to PND 95 than when they are sacrificed at PND 21?

## Additional endpoints recommended by EDSTAC for (anti/)estrogenic activity

- Accessory sex organ function (secretory products)
- Sexual development and maturation (nipple development and retention)
- Androgen and estrogen levels
- LH and FSH levels
- Testis descent

## Additional endpoints recommended by EDSTAC for (anti/)androgenic activity

- (None)

## Additional endpoints recommended by EDSTAC for thyroid activity 

- Neurobehavioral deficits (see developmental landmarks below)
- TSH, T4, thyroid weight and histology
- Developmental landmarks:
- Prewean includes pinna detachment, surface righting reflex, eye opening, acquisition of auditory startle, negative geotaxis, mid-air righting reflex, motor activity on PND 13, 21, etc.
- Postwean includes motor activity PND 21 and postpuberty ages (sex difference); learning and memory PND 60 – active avoidance/water maze
- Brain weight (absolute), whole and cerebellum
- Brain histology