Chad A Blanksma US EPA Great Lakes Toxicology and Ecology Division Histotechnologist

6201 Congdon Blvd. Duluth Minnesota 55804 USA

blanksma.chad@epa.gov 0009-0001-8811-8083 This image is from a region of an adult female fathead minnow ovary. Populations of vitellogenic, cortical alveolar, and perinucleolar oocytes can be observed in this micrograph. Vitellogenic oocyte Cortical alveolar oocytes Perinucleolar oocytes Adult Fathead minnow Germinal vesicle Pigmented macrophage aggregates This information is released under the Creative Commons license - Attribution - CC BY (https://creativecommons.org/licenses/by/4.0/). The consumer of these data ("Data User" herein) is required to cite it appropriately in any publication that results from its use. The Data User should realize that these data may be actively used by others for ongoing research and that coordination may be necessary to prevent duplicate publication. The Data User is urged to contact the authors of these data if any questions about methodology or results occur. Where appropriate, the Data User is encouraged to consider collaboration or co-authorship with the authors. The Data User should realize that misinterpretation of data may occur if used out of context of the original study. While substantial efforts are made to ensure the accuracy of data and associated documentation, complete accuracy of data sets cannot be guaranteed. All data are made available "as is." The Data User should be aware, however, that data are updated periodically and it is the responsibility of the Data User to check for new versions of the data. The data authors and the repository where these data were obtained shall not be liable for damages resulting from any use or misinterpretation of the data. Thank you. 2016 Kingdom Animalia animals 202423 Subkingdom Bilateria triploblasts 914154 Infrakingdom Deuterostomia 914156 Phylum Chordata chordates 158852 Subphylum Vertebrata vertebrates 331030 Infraphylum Gnathostomata 914179 Superclass Actinopterygii ray-finned fishes 161061 Class Teleostei 161105 Superorder Ostariophysi 162845 Order Cypriniformes minnows 162846 Superfamily Cyprinoidea 638796 Family Leuciscidae 1198935 Subfamily Pogonichthyinae 1198937 Genus Pimephales bluntnose minnows 163515 Species Pimephales promelas Fathead Minnow 163517 Chad A Blanksma US EPA Great Lakes Toxicology and Ecology Division Histotechnologist

6201 Congdon Blvd. Duluth Minnesota 55804 USA

blanksma.chad@epa.gov 0009-0001-8811-8083 Image collection device SPOT Insight 4 Mp CCD Scientific Color Digital Camera Image collection software SPOT Advanced Software Image analysis software Image-Pro Premier Graphics Editing Software Corel Photo-Paint Daniel L Villeneuve US EPA Great Lakes Toxicology and Ecology Division villeneuve.dan@epa.gov 0000-0003-2801-0203 Corresponding Author 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. Environ Toxicol Chem 2023;42:100–116. © 2022 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA. 234_5x 234_5x.tif tif histology USEPA_GLTED_PP_Imazalil21d_C2B2 female adult 1 ovary unspecified 1A 4 microns Zeiss Axiovert 35