What do we learn when we study cytotoxicity? Critical shortcomings in the green chemistry context using imidazolium ionic liquids as an example case†

Cytotoxicity measurements are widely used in chemical research to evaluate the biological effects of chemical compounds, particularly in the context of green chemistry. While these assays appear straightforward, experiments have shown that their outcomes strongly depend on the parameters engaged and the logic applied in data interpretation. In this study, using three common imidazolium ionic liquids tested in two cell lines as an example, we demonstrate how seemingly minor changes in the experimental setup can significantly influence the results, complicating data interpretation and limiting comparability across studies. This work stresses the importance of adopting a systematic approach to cytotoxicity studies, considering cellular responses as part of a complex network of interconnected processes rather than as isolated data points. We aim to raise awareness among chemists about these pitfalls and to provide guidance for more reliable experimental practices, ultimately improving data quality and contributing to safer chemical development in green chemistry.