A key aspect of REACH regulation is the progressive substitution of the most dangerous chemicals with suitable alternatives. At the same time, in order to reduce the number of tests with animals, REACH regulation strongly encourages the use of alternative approaches, such as in vitro methods at cellular and sub-cellular level. If there is a therapeutic response then the major advantage of DESs would be in varying their constituents and molar ratios to improve their pharmacological properties for desired therapeutic applications. In this work, the cytotoxicity of selected ammonium-based DES towards five human cancer cell lines and one normal cell line was investigated. The DESs are based on ChCl combined with four HBDs, namely glycerine, ethylene glycol, triethylene glycol and urea. MCF-7 cells were Talazoparib pretreated with the solvents for 24 h and stained with DHE dye to determine the influence of DES exposure on ROS production. The fluorescent intensities of DHE oxidization by ROS were measured using a fluorescence microplate reader and flow cytometer. As shown in Fig. 6, exposure to the four DESs causes an increase in the ROS level of the treated MCF-7 cells. These changes are more significant at the concentrations close to the IC50 of the solvents. The highest oxidative stress was exerted by 13DES, in which the fluorescent intensity of the cells treated with 25 ��g/mL was two times higher than that of the control cells. This increment in ROS can be ascribed to the burden of DESs on the antioxidant enzyme superoxide dismutase. SOD is responsible for clearing off the free radicals generated by the ROS. Increased ROS production by DESs can overwhelm the SOD functions by inducing apoptosis in targeted cells. This hypothesis is to some extent supported by Dai et al., who reported that mixtures of many abundant primary metabolites from all kinds of organisms can form NADESs when mixed in adequate ratios. Various materials were found to be soluble in NADES, such as some non-water soluble bioactive natural products, gluten, starch, DNA, proteins and polysaccharides. The high solubilizing capacity is related to their BAY-60-7550 supramolecular structure and broad polarity range. The existence of NADES in plants and their properties indicate that NADES might be involved in the biosynthesis and storage of various non-water soluble metabolites in cells and imply the role of NADES in protecting organisms from extreme conditions. Overall, the relatively high cytotoxicity and low selectivity index of the solvents demonstrated considerable risks associated with their applications. The results also indicated a significant role for the HBD on the cytotoxic activity of the DES. However, further investigations may contribute to clarify such effects of HBD on the activity of DES. Brain irradiation is a first-line treatment for both primary and metastatic intracranial malignancies. Irradiation-induced cognitive impairments, including dementia, occur in 50�C90% of patients with brain tumors who survive >6 months postirradiation. Moreover, the incidence and severity of these impairments increase over time.