Towards Animal-Free Toxicology: Establishment of Two Larval Brown Trout Cell Lines for Environmental Risk Assessment.

Advances in cell culture technology have led to fish cell lines being used as cost-effective, reproducible, and ethically favourable instruments in ecotoxicology. The development of new lines contributes to reducing animal experiments and improves model diversity. The brown trout (Salmo trutta), an important bioindicator due to its sensitivity to pollutants, is still underrepresented in cell culture systems. In this study, two novel larvae-derived cell lines, STRlar1 and STRlar2, were established and maintained for over 40 passages. Although derived from sibling larvae of the same parental strain, the two lines showed marked differences in growth dynamics and toxicological responses to ethanol, dimethyl sulfoxide, isopropanol, and acetone. STRlar2 showed greater sensitivity to all chemicals tested, while STRlar1 exhibited longer proliferation and higher impedance, suggesting stronger substrate adhesion. These differences emphasise the importance of cellular heterogeneity, even in closely related lines. Our results underline the need to carefully validate new in vitro models and caution against relying on single cell lines. To improve the robustness and reliability of ecotoxicological assessments, we recommend the use of multiple independently derived lines that better reflect biological variability and reflect in vivo complexity.