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 animals
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