Case Study in 21st-Century Ecotoxicology: Using In Vitro Aromatase Inhibition Data to Predict Reproductive Outcomes in Fish In Vivo

To reduce the use of intact animals for chemical safety testing, while ensuring protection of ecosystems and human health, there is a demand for new approach methodologies (NAMs) that provide relevant scientific information at a quality equivalent to or better than traditional approaches. The present case study examined whether bioactivity and associated potency measured in an in vitro screening assay for aromatase inhibition could be used together with an adverse outcome pathway (AOP) and mechanistically based computational models to predict previously uncharacterized in vivo effects. Model simulations were used to inform designs of 60-h and 10-21-day in vivo exposures of adult fathead minnows (Pimephales promelas) to three or four test concentrations of the in vitro aromatase inhibitor imazalil ranging from 0.12 to 260 mu g/L water. Consistent with an AOP linking aromatase inhibition to reproductive impairment in fish, exposure to the fungicide resulted in significant reductions in ex vivo production of 17 beta-estradiol (E2) by ovary tissue (>= 165 mu g imazalil/L), plasma E2 concentrations (>= 74 mu g imazalil/L), vitellogenin (Vtg) messenger RNA expression (>= 165 mu g imazalil/L), Vtg plasma concentrations (>= 74 mu g imazalil/L), uptake of Vtg into oocytes (>= 260 mu g imazalil/L), and overall reproductive output in terms of cumulative fecundity, number of spawning events, and eggs per spawning event (>= 24 mu g imazalil/L). Despite many potential sources of uncertainty in potency and efficacy estimates based on model simulations, observed magnitudes of apical effects were quite consistent with model predictions, and in vivo potency was within an order of magnitude of that predicted based on in vitro relative potency. Overall, our study suggests that NAMs and AOP-based approaches can support meaningful reduction and refinement of animal testing. Environ Toxicol Chem 2022;00:1-17. (c) 2022 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Automated summary

In order to reduce the use of intact animals for chemical safety testing, new approach methodologies are needed to provide relevant scientific information equivalent to or better than traditional approaches. A case study was conducted to examine whether bioactivity measured in an in vitro screening assay for aromatase inhibition can predict previously uncharacterized in vivo effects. The results suggest that the use of new approach methodologies and adverse outcome pathway-based approaches can support significant reduction and refinement of animal testing.