## ANIMAL PROTECTION COMMENTS ON REVIEW OF EDSP TIER 1 ASSAYS AND BATTERY PERFORMANCE
Patricia L. Bishop, M.S. Research Associate Regulatory Testing Division People for the Ethical Treatment of Animals

FIFRA SAP MEETING, MAY 21
- 23, ARLINGTON, VA

## MAIN CONCERNS FROM ANIMAL PROTECTION PERSPECTIVE

• Large numbers of animals used in the battery

• Additional animals used in range

- finding studies

• More animals killed in culling

• Animals used in binding studies reliant on rat cytosol

• Use of BG1Luc as replacement for STTA ERTA assay

• Elimination of the Hershberger and uterotrophic assays
• Tying results back to mechanistic interpretation

## LARGE NUMBERS OF ANIMALS USED IN THE TIER 1 BATTERY Test Protocols Alone ER Binding: 13 rats AR Binding: 10 rats Uterotrophic : 18 rats Hershberger (agonist and antagonist): 48 rats Male and female pubertals : 45 rats each FSTRA: 96 fish AMA: 320 tadpoles Total of 595 anima ls

## LARGE NUMBERS OF ANIMALS USED IN THE TIER 1 BATTERY

• 52 List 1 chemicals tested would have used approximately 31,000 animals if all assays done

• Use of OSRI to waive some tests saved about 3,300 animals

## RANGE - FINDING STUDIES TO ESTIMATE MTD

• Appears that RF studies were conducted for the same chemical in more than one assay to account for possible age and gender differences

• For example, RF studies conducted for 12 of the 16 chemicals evaluated in male pubertal review

• This used 144 animals before actual assay was even performed (3 animals per dose group times 3 dose groups times 12 chemicals)

## RANGE - FINDING STUDIES TO ESTIMATE MTD

• Dose ranges may vary between assays (3

- week old animals in pubertal vs. 8

- 10 week old adults in OVX uterotrophic ), but it should be possible to develop extrapolation methods to estimate one from the other.

• Recommend development of a standardized method for estimating MTD that draws fully on existing toxicity data from Part 158 studies and reliable literature studies, and requiring its use unless otherwise justified.

## CULLED ANIMALS

• Culling of excess animals may use more than the actual test in some cases.

• Pubertals each use 45 animals in the test: 1 control group and 2 dose groups of 15 each, but cull about 200 animals
.

## CULLED ANIMALS

• To get the 45, 17

- 20 timed
- pregnant dams to produce at least 15 litters of 13

- 14 pups.

• These litters are culled to 6

- 8 pups, discarding about 130 animals.

• Final culling of dams (17

- 20) and another 60 pups to get 45 for test (no more than 1 pup from each litter in dose group).

• Can’t use excess to test another compound because necropsies must be done on same one or two days
– lab personnel issues.

## In Vitro BINDING STUDIES USE ANIMALS

• Information from one lab indicates that 13 rats were needed to run ER binding assay on one chemical
– additional rats needed to test and optimize the method.
• Same lab used about 10 rats per chemical to run AR binding assay with additional animals for optimizing the method.
• Human recombinant ER binding assays that use NO ANIMALS are available and have been used by commercial labs and industry; EPA participating in OECD validation studies with TG expected in 2014.

## In Vitro ERTA ASSAY

• Recommend replacing the current STTA ERTA assay with the recently ICCVAM

- validated BG1Luc ( Lumi
- cell) ERTA assay.
• Performed as well as STTA and has advantage of detecting antagonists.
• Also showed 92% concordance with uterotrophic assay (12 out of 13 reference chemicals). With more comparisons, may provide basis for elimination of uterotrophic
.

## Elimination of Hershberger

• We agree with the preliminary assessment that the Hersberger could be eliminated without compromising identification of chemicals that potentially interact with androgen system.
• Should be verified after analysis of all List 1 results and after determining effects of overt toxicity in FSTRA and male pubertal on positive findings.

## Tie in vivo Results Back to Mechanistic Interpretation

• Cases of apical effects with no supporting binding, TA, or steroidogenesis responses
• Example, Chemical B: + Hersh (OT), +FSTRA (solvent interference), + male pubertal,

- in vitro
• How to interpret?
• Artifice due to experimental conditions?
• Non
- androgen mediated?
• Androgen
- mediated, but not covered in Tier 1 or is metabolically activated?

## Tie in vivo Results Back to Mechanistic Interpretation

• Interpretation could be greatly enhanced by understanding of MoA /AOP

• Explain patterns of complementarity and redundancy within assays

• As well as “non

- concordance” between assays

### Submitted by People for the Ethical Treatment of Animals The Humane Society of the United States Physicians Committee for Responsible Medicine