Aromatase Questions and Next Steps

1. Aromatase Questions and Next Steps Gary Timm EDMVS December 2003

2. Aromatase Questions • Does the EDMVS agree that the prevalidation studies demonstrate that both the placental and recombinant assays are effective in identifying chemicals that inhibit aromatase?

3. 2. Pending the resolution of the issue regarding sources of variability observed in both assays, does EDMVS believe that prevalidation will have been successfully completed for both assays? In not, what additional studies should be performed before beginning interlaboratory studies?

4. Should EPA continue to validate both the placental and recombinant assays?

5. Validation Study Plan • Assumptions: • Need to validate both assays • Select 5 laboratories (RTI +4 labs) • Use 6-8 chemicals in coded study including one negative chemical • Need to include validation of the preparation of placental microsomes

6. Outline of Validation Study Plan • Approach: • Demonstrate estrone production and inhibition of aromatase activity using 8 concentrations of positive control (4-OH ASDN) with recombinant and centrally supplied placental microsomes • 4 replicate studies in each assay

7. Outline of Validation Study Plan • Run coded chemical studies with centrally supplied placental microsomes and recombinant microsomes • 5-6 chemicals including 1 or 2 negatives • Assays run in quadruplicate • 2 independent studies per chemical • Labs 2 and 4 will prepare placental microsomes and supply to labs 3 and 5. The four labs will repeat steps 1 and 2 with these microsomal preparations.

8. Question: Is it necessary to rerun all of the studies using the microsomes prepared in participating laboratories? Proposed Approach:

9. Alternative 1

10. Alternative 2