## 4 Discussion  
Specific characteristic properties of nanoparticles such as their size, shape, type, surface charge, or solubility determine their toxicity [17; 18], which we also observed in our studies. One of the factors affecting potential cytotoxicity is obviously the content of different particle fractions in the tested samples (Figure S1). In vivo and in vitro studies show that TiO\({}_{2}\) particles have lower toxicity compared with TiO\({}_{2}\) NPs [19]. [PERSON] et al. [20] demonstrated that the cytotoxicity of TiO\({}_{2}\) nanoparticles increased as particle size decreased. Among the three sizes of TiO\({}_{2}\) NPs (10-30 nm, 50-100 nm, 110-300 nm), the NPs in the 10-100 nm range caused cell rupture and leakage. It was shown that the smallest size of TiO\({}_{2}\) NPs caused the greatest leakage, and as particle size increased, LDH leakage gradually decreased. Because of their small size, nanoparticles are more easily absorbed by cells, which may lead to the formation of reactive oxygen species (ROS), promotion of inflammatory response, changes to cellular signal pathways, and, consequently, cytotoxicity, epigenetic changes, genotoxicity, and immunotoxicity [21; 22]. Smaller particles, with a diameter of 5-10 nm, are taken up and can trigger an inflammatory response through endocytosis via the epidermal growth factor receptor (EGFR) [23]. Also, the surface area-to-volume ratio significantly affects the behavior of nanoparticles (NPs). As the size of NPs decreases, the number of surface atoms increases exponentially. Consequently, in smaller NPs, a greater proportion of atoms are on the surface, which alters surface properties like electronic structure, energy levels, and reactivity compared with those in the interior [21]. This can result in heightened chemical reactivity and variations in absorption and excretion rates within biological systems [24; 25]. Current in vitro and in vivo research validates the toxicity of TiO\({}_{2}\) NPs, highlighting their impact on cellular cycles, nuclear membrane contraction, and apoptosis [6; 26]. The time of exposure as well as the dosage of E171/TiO\({}_{2}\) NPs can also impact toxicity, as evidenced in our own as well as other authors' studies. [PERSON] et al. [1] demonstrated that both chemical forms (TiO\({}_{2}\), TiO\({}_{2}\) NPs (60 nm)) significantly reduced the vitality of tumor cells, depending on the dosage. In particular, E171 had a pronounced inhibitory effect on the growth of the Caco-2 cell line (E171 IC50: 1.88 mg L\({}^{-1}\) Caco-2; TiO\({}_{2}\) NPs 60 nm IC\({}_{50}\): 14.3 mg L\({}^{-1}\) for Caco-2). Low-level genotoxicity was observed in Caco-2 cells, particularly after exposure to TiO\({}_{2}\) 60 nm, which indicated a slightly higher level of cytotoxicity and genotoxicity of E171. Also, [PERSON] et al. [27] concluded that E171 showed toxic effects on Caco-2 cells after a 24-th exposure to 1000 ug mL\({}^{-1}\). In an in vitro study conducted by [PERSON] et al. [28] on a model of the human intestine, it was demonstrated that exposure to TiO\({}_{2}\) NPs resulted in atrophy of the intestinal villi. The authors reported that the addition of TiO\({}_{2}\) NPs concentrated at 350 ug cm\({}^{-3}\) led to the loss of approximately 42% of intestinal microvilli. Other authors have also reported results similar to ours regarding the exposure time to TiO\({}_{2}\) NPs and its impact on toxicity levels. We demonstrated that extending the exposure time to 72 h resulted in a significant decrease in Caco-2 cell proliferation. Similar conclusions were drawn by [PERSON] et al. [29], who demonstrated that a 72-h exposure to TiO\({}_{2}\) NPs significantly reduced the vitality of Caco-2 cells in a manner that was both concentration- and dose-dependent. The authors studied five TiO\({}_{2}\) forms differing in terms of size and structure in human colorectal adenocarcinoma cells (Caco-2) and macrophages derived from THP-1 monocytes.