Citation:

Villeneuve, D., B. Blackwell, C. Blanksma, J. Cavallin, W. Cheng, R. Conolly, K. Conrow, D. Feifarek, L. Heinis, K. Jensen, M. Kahl, R. Milsk, S. Poole, E. Randolph, T. Saari, K. Watanabe, AND G. Ankley. Case Study in 21st-Century Ecotoxicology: Using In Vitro Aromatase Inhibition Data to Predict Reproductive Outcomes in Fish In Vivo. ENVIRONMENTAL TOXICOLOGY AND CHEMISTRY. Society of Environmental Toxicology and Chemistry, Pensacola, FL, 42(1):100-116, (2023). https://doi.org/10.1002/etc.5504

Impact/Purpose:

Under the Frank R. Lautenberg Chemical Safety for the 21st Century Act and a September 2019 directive by EPA Administrator Wheeler, EPA has committed to reducing, refining, or replacing the use of vertebrate animals in chemical safety testing. In support of these goals, ORD's Chemical Safety for Sustainability program has been developing new approach methodologies (NAMs) that intend to provide information to support chemical hazard and risk assessment without the use of intact animal testing. The case study examined the use of a in vitro bioactivity screening assays, coupled with scientific understanding and evidence organized according to the adverse outcome pathway (AOP) framework to predict outcomes traditionally observed in long-term, resource intensive, fish endocrine disruptor screening assays. The study demonstrates that the in vitro assay along with the AOP were accurate in predicting in vivo hazards elicited by compound (imazalil) demonstrated to inhibit aromatase, a key enzyme involved in estrogen biosynthesis, to fish reproduction. Likewise, a set of computational models based on the AOP were effective in guiding experimental design and predicting both the magnitude and potency of the compound with an accuracy similar to that which may be expected by repeating an intact animal experiment. While the methods may not be suited for full replacement of intact animal testing in higher tier risk assessments, the results demonstrate capacity to reduce and refine animal use in testing. This research supports the efforts of program offices (e.g., OCSPP/OPPT; OPP) seeking to implement greater use of NAMs as alternatives to intact animal testing.

Description:

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 µg/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 17β-estradiol (E2) by ovary tissue (≥165 µg imazalil/L), plasma E2 concentrations (≥74 µg imazalil/L), vitellogenin (Vtg) messenger RNA expression (≥165 µg imazalil/L), Vtg plasma concentrations (≥74 µg imazalil/L), uptake of Vtg into oocytes (≥260 µg imazalil/L), and overall reproductive output in terms of cumulative fecundity, number of spawning events, and eggs per spawning event (≥24 µg 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.

URLs/Downloads:

Record Details: