Towards safer deep eutectic solvents: Insights from an in vivo study of reline toxicity under different preparation methods

Deep eutectic solvents (DESs), regarded as green alternatives to volatile organic solvents, are commonly prepared by heating; however, despite the possible formation of byproducts during heating, the toxicological relevance of their preparation conditions remains unresolved. The aim of this study was to disentangle preparation-dependent in vivo toxicity from the intrinsic effects of DESs through mechanistic insights using reline—a widely used DES comprising choline chloride and urea (1:2)—as a model solvent. Two forms of reline, prepared by either heating (reline_H) or freeze-drying (reline_FD), were identical in composition and eutectic structure, except for thermally generated byproducts such as ammonia. At 1.8 g/kg (p.o.), reline_H induced corrosive symptoms and gastric injury to ICR mice, irrespective of sex, whereas reline_FD was well-tolerated. Mechanistic investigations suggested that reline_FD induces a metabolic burden inherent to the reline components. Contrastingly, reline_H caused additional perturbations, including a pronounced redox imbalance, accelerated uptake of reline, and delayed ammonia clearance, consistent with alkaline injury; moreover, it caused mitochondrial destabilization as a downstream consequence of alkaline injury. Collectively, our findings suggest that reline toxicity arises from alkaline stress caused by thermally generated byproducts, whereas freeze-drying mitigates such liabilities. With the increasing adoption of DESs in bio-relevant settings, these findings indicate that the safety of a DES not only depend on its composition, but possibly also on its preparation history. This study highlights that the preparation method of a DES as a critical yet often-overlooked determinant of DES safety, which is especially relevant when evaluating DESs for future applications.