## 2 Toxicity, Bioactivity and Biodegradability  
An essential characteristic of DESs, and especially NADEs, is their low toxicity. These solvents can be applied directly in food and pharmaceutical formulations if made with primary metabolites of cells, which have recently been called NADESs [7]. According to [PERSON] et al. [8], no cytotoxic effect is detected in the components of NADESs. Proof of their low toxicity is that many of them even lack antimicrobial activity, as demonstrated by the work of [PERSON] et al. [9] in which the toxicity of NADESs was verified against two Gram positive bacteria (_S. aureus_ and _L. monocytogenes_) and two Gram negative (_E. coli_ and _S. enteritidis_) and no inhibition of microbial growth was detected. However, this is highly dependent on the compounds forming NADESs. In the study by [PERSON] et al. [10], the NADESs used were mono- and diterpene-based and showed that all the combinations had some toxicity in Caco-2 cells but could also inhibit the proliferation of HT-29 cells. Additionally, all terpenoid-based NADESs were able to inhibit the growth of _S. aureus_ and _E. coli_. Likewise, in the study by [PERSON] et al. [11], there were specific DESs, especially those containing organic acids, which demonstrated an antimicrobial effect against Gram negative and Gram positive bacteria due to their low pH. Figure 1: Some examples of natural eutectic solvents (NADESs) and NADESs components. Vial 1, sucrose; vial 2, fructose; vial 3, glucose; vial 4, malic acid; vial 5, sucrose:fructose:glucose (1:1, molar ratio); vial 6, sucrose:malic acid (1:1, molar ratio. Reprinted from [PERSON]; [PERSON] [PERSON]; [PERSON]; [PERSON]; [PERSON]; [PERSON]; [PERSON]; [PERSON] Are Natural Deep Eutectic Solvents the Missing Link in Understanding Cellular Metabolism and Physiology? Plant Physiol., 2011, 156 (4), 1701–1705 with permission from Oxford University Press [5]. However, the application of NADESs in foods must be controlled and deeply studied. [PERSON] et al. [12] investigated the toxicity of a NADES composed by betaine:glycerol in rats. The rats were randomized into two groups of six animals each and fed by gavage for 14 days at 9:00 and 17:00 h either with 3 mL of water or 3 mL of NaDES extract (containing 28.01 mg of phenolic compound and 5.89 mg of caffeine dissolved in betaine:glycerol). The selected NADES provided the higher extraction rate. They observed specific adverse effects of oral administration of a high dose of betaine:glycerol. The absorption of the mixture induced mortality in two rats out of six. Additionally, dietary restriction, excessive water consumption, weight loss, adipose tissue loss, hepatomegaly, plasmatic oxidative stress, and increased blood lipid levels were observed. All this occurred because the extract was rich in chlorogenic acids, which have proved beneficial effects. The authors proposed a decomposition into its original constituents in the stomach or while being absorbed, the overall effect results from the individual positive and/or negative effects of each constituent (glycerol, betaine, phenolic compounds). In this case, glycerol is associated with an increase of liver glycogen [13]. Therefore, careful selection of NADES components and their doses is necessary. One important aspect of DESs to take into account is their own bioactivity. For example, protecting the extracted compounds, thus maintaining the initial biological activity. The study by [PERSON] et al. [14] shows how blackberry extracts extracted with ChCl:glucose can behave as a protective agent for anthocyanins. The eutectic solvent behaves as an antioxidant for compounds that are sensitive to the environment (oxygen, light, temperature, and pH). The proposed form of protection is based on the hydrogen bond donor (HBD) interaction and the hydrogen bond acceptor (HBA) that form the NADES which can behave as a substitute in the anion or cation exchange when the compounds are exposed to extreme conditions.