<i>In Vitro</i> Liver Models for Studying Pharmaceutical Metabolism in Fish – A Critical Analysis of Their Applications and Limitations

Bioaccumulation studies performed as part of the environmental risk assessment of human pharmaceuticals are time- and resource-intensive, prompting interest in alternative nonanimal screening methods. For xenobiotics, including active pharmaceutical ingredients (APIs), biotransformation and metabolic clearance are key determinants of bioaccumulation and, consequently, effects in nontarget organisms like fish. Various in vitro liver models are available for studying pharmaceutical metabolism in fish, including microsomes, S9 fractions, and primary hepatocyte cultures maintained as suspensions, monolayers, or three-dimensional spheroids. However, broader application of these models is limited by a lack of robust in vitro-in vivo correlation data, as well as uncertainty regarding the most appropriate systems for different modeling purposes. This study critically evaluates in vitro liver models commonly used for assessing API clearance in fish, comparing their functionality, resource requirements, and informative value. Drawing on published data for selected APIs and on the strategies used to select suitable mammalian model systems, we propose the use of subcellular fractions for high-throughput screening of hepatic clearance (metabolic stability) and enzyme inhibition, hepatocyte suspensions for assessing intrinsic clearance while accounting for the impact of active drug transport, and hepatocyte monolayers or three-dimensional cultures for longer-term clearance and targeted effect studies. Key knowledge gaps are identified, and recommendations for future research are presented.