**8. Potential Toxicity, Limitations, and Regulatory Aspects of Nanoemulsions**

Nanoemulsions, due to the nanometric size of the droplets, may partially remain intact during digestion, representing potential safety risks related to the compounds used for their production (such as surfactants). They can be of concern in metabolic or hormonal dysregulation due to their rapid absorption compared to conventional emulsions, their ability to increase the bioavailability of bioactive agents to a toxic level, and the possibility of increased absorption by epithelial cells which can cause changes in the functionality of the gastrointestinal tract [97]. However, as they have a high surface area, nanoemulsions can also be quickly digested by enzymes from the gastrointestinal tract, reducing the possible toxic effect that can occur due to their accumulation in organ cells [98].

In vitro studies were performed using cell cultures, usually models of normal cells such as fibroblasts, to investigate potential toxicity of nanoemulsions. Kaur et al. [99] reported that nanoemulsions based on tocopheryl polyethylene glycol succinate (TPGS), lemon oil, Tween-80, and water did not show toxicity in Hep G2 cells. In another study, Marchese et al. [100] observed that bergamot essential oil nanoemulsions showed cytotoxic activity against Caco 2 cells at high concentrations. A limitation of these studies is the fact that authors have not previously exposed the nanoemulsions in simulated conditions of the gastrointestinal tract before contact with the cells.

Knowledge about the potential toxicity of nanoemulsions in vivo is still limited and should be investigated [97]. The effect of nanoemulsions based on antimicrobial compounds, such as essential oils, on the gastrointestinal tract is also poorly reported in the literature. This effect must be carefully studied, as antimicrobial compounds can influence the intestinal microbiota or epithelial cells of the gastrointestinal tract.

In a recent study, Hort et al. [101] evaluated the toxicity of Miglyol and egg lecithin nanoemulsions using an in vivo model (male Wistar rats). The nanoemulsions were orally administered to rats for 21 days at lipid concentrations of 200, 400, or 800 mg/kg of body weight. The results of biochemical, hematological, oxidative stress, and genotoxicity parameters showed that nanoemulsions could be considered safe for oral administration, but high doses by the parenteral route could cause toxic effects.

The few studies suggest that nanoemulsions formulated with GRAS ingredients do not exhibit strong cytotoxic effects. The nanometer size of the droplets suggests that they are rapidly transformed into monoglycerides and free fatty acids in the small intestine, which are normal digestion products and should not have toxic effects [57].

As for regulatory aspects, essential oils and other antimicrobial agents are mainly regulated by the European Food Safety Authority (EFSA) in Europe and the Food and Drug Administration (FDA) in the United States [102]. However, for nanoemulsions there is no international authority that makes this regulation. The FDA addresses the regulation of nanotechnology products as guidance for industries. The European Council and Parliament have regulated food nanotechnology as new food products or food ingredients [103].
