# Presentation: 311938

## Next steps in the validation of the pubertal assays

- Issues, questions, plans
- J. Kariya
- OSCP/USEPA
- December 11, 2003

## Initial questions for EDMVS

- Does the EDMVS agree that the pubertal assays show adequate sensitivity over a range of chemicals for use as a Tier 1 assay?  That is, are the pubertal assays ready for an interlaboratory validation study?  If not, what specific areas need further prevalidation work?
- Based on the currently available data, are there endpoints that should be added or dropped from the assay?

## What follows is a proposal...

- ...and the main issues for EDMVS comment are:
- Number and content of studies
- Training of labs
- Number of dosage levels
- Use of positive control
- Dose-setting
- Written comments are particularly helpful!

## Purpose of theinterlab validation study

- 	To determine whether independent laboratories arrive at the same, correct conclusion about the ability of an unknown chemical to interact with the endocrine system when using the pubertal assays, in the absence of other information about the chemical.

## A secondary purpose

**	****To refine the protocol, if necessary, by identifying specific areas of difficulty in performing the assay.**

## Two-part study

- Part A:  Dose-setting done by each lab
- This is the main test of transferability
- Part B:  Dose levels set centrally
- To identify performance problems, if any
- 	The Parts will be done in parallel, to expedite EDSP and to avoid interference with the test of transferability.

## General considerations

| Number of labs | 3 GLP labs, experienced in rat reprotox or developmental tox |
| --- | --- |
| Chemicals | 7 chemicals: 
weak E, anti-A, anti-T; strong positive controls for each; plus 1 “toxic negative” (if one can be agreed upon) |
| Protocol | As used in prevalidation, with clarifications (q.v.) |
| Dosage levels | 2 when set by lab (Part A),
1 when set centrally (Part B) |

## Non-protocol/non-GLP deviations

- Analytical chemistry at one lab only (purity of neat substance, etc.)
- Phytoestrogen in feed recorded (genistein and daidzein only)
- (Positive and “toxic negative” controls)

## Not standardized in protocol*

- Strain
- Feed  (as long as “genistein equivalents < 350 ug/g in each batch)
- Water
- Caging
- Bedding
- etc.
- * beyond what is already in the Critical Reviews in Tox protocol

## Criteria for evaluation of protocol

- Did all labs arrive at the same conclusion about the ability of the weak chemical to interact with the endocrine system?
- Was the conclusion in line with expectations?
- Were any of the endpoints inconsistent across labs?

## Example scheme, Part A

|  | Estrogen |  |  |  | Anti-androgen |  |  |  | Anti-thyroid |  |  |  | Toxic negative |  | Vehicle control |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  | Weak |  | Strong |  | Weak |  | Strong |  | Weak |  | Strong |  |  |  |  |  |
| 
Lab | ♂ | ♀ | ♂ | ♀ | ♂ | ♀ | ♂ | ♀ | ♂ | ♀ | ♂ | ♀ | ♂ | ♀ | ♂ | ♀ |
| 1 |  | A |  | B | C |  | D |  | E |  | F |  | G | H | ✓ | ✓ |
| 2 |  | A |  | B | C |  | D |  | E |  | F |  | G | H | ✓ | ✓ |
| 3 |  | A |  | B | C |  | D |  | E |  | F |  | G | H | ✓ | ✓ |

- Letters denote codes for chemicals.  G and H are identical chemical but coded differently.

**Notes:**
Anti-thyroid could be done in either male or female, as long as it’s the same sex across all labs, the weak chemical and the strong chemical are done in the same sex, blinding is preserved..

Blinding would be maintained.  In this example, instructions to the lab would be:
	“Test A, B, H, and vehicle control in females”
	“Test C, D, E, F, G, and vehicle control in males”
even though G and H are the same chemical.

## Example scheme, Part B

- Same as for Part A, except
- Test strong chemicals only
- Thyroid could be done in different sex from the one in Part A.

## Can this be done more simply by using only one dosage level...

- For the positive control?
- Study would not be blind
- For positive control and toxic negative?
- Study would not be blind for the sex in which only three chemicals are tested
- For all chemicals?
- SAB/SAP recommended two doses

## Simplify? (continued)

- Don’t use positive control?
- ?
- Don’t use “toxic negative”?
- ?

## Clarifications to protocols (1)

- 1.	T4 and TSH will be required endpoints.
- 2.	Thyroid fixative, weighing, and histology to be specified.
- 3.	Removal of LABC complex for weighing to be described.
- 4.	Standardized definitions of estrus stages and “irregular cycling” to be provided.
- 5.	Identification and treatment of outliers to be specified.                                (continued)

## Clarifications to protocols (2)

- 6.	Method for calculating mean and variances for day of VO/PPS when some animals have not reached this endpoint by the time of sacrifice to be included.
- 7.	(dose volume to be specified?)
- 8.	(dose setting procedure?)
- 9.	Reporting format to be specified.

## Optimistic time frame

- Work assignment, including protocol, by Feb. 2004
- Subcontracts
- Individual lab dose-setting
- In-lab main assay
- Audited data available by Feb. 2005?
- EDMVS by July 2005?
- 	Note: last set of data took over a year to go from Work Assignment to EDMVS, without searching for subcontractors, without GLP, without dose-setting, and with a smaller number of labs to compare.

## Issue: train labs?

- Yes
- Otherwise you may be testing the competence of the lab, not the assay
- No
- Training interferes with the test of transferability of the written protocol

## Is there a better sequence of studies consistent with time frame?

- Current sequence

- Alternative?

- Not compatible with time frame

## Dose-setting

- Not realistic in this blinded study: usually some background information will be available.
- Criterion: highest dose causes some decrease in body weight gain, but < 10%  compared to controls, at time of necropsy.

## Final questions for EDMVS (1)

- Is there a better set or sequence of studies to perform for validation?
- Can the number of dose levels be reduced in this study?
- If doses do not reach, or exceed, MTD, should the lab be allowed/required to repeat the study before interlab results are compared?

## Final questions for EDMVS (2)

- Should labs be trained as part of the validation exercise?
- Should positive controls be required in the protocol?  In this study?
- Is there a toxic compound that is known to be endocrine inactive at MTD, which can serve as the “toxic negative” control?