## 5 Conclusions  
Our review of recent publications revealed divided opinions regarding the potentially harmful effects of E171/TiO\({}_{2}\) NPs based on both in vitro and in vivo studies. Food products and drinks can differ substantially in terms of their composition and structure, which has a direct bearing on their behavior inside the gastrointestinal tract. At the same time, given the diversity and agglomeration capacity of NPs, they can show different properties in terms of absorbability and activity within food matrices. E171 has especially sparked concerns about its potential adverse effects on human health, primarily because of the presence of a nanoparticle fraction (fraction under 100 nm). In the present study, we assessed the possible toxicity and pro-inflammatory effects of three different E171 samples (containing NP fractions) and one TiO\({}_{2}\) NP sample in concentrations found physiologically in the body (60-600 \(\upmu\)g mL\({}^{-1}\)) on Caco-2 colon cancer cells. The findings showed that all four TiO\({}_{2}\) samples had no significant effect on Caco-2 cell viability after 24 h of exposure but as many as three substances out of the four tested caused a reduction in cell proliferation after 72 h (XTT assay). Additionally, a decline in the metabolic activity of Caco-2 cells (ATP test) was detected at concentrations of 300 and 600 \(\upmu\)g mL\({}^{-1}\) for all tested substances. While exposure for 24 h led to minimal LDH release, prolonged exposure (72 h) resulted in an increased release of LDH, indicating potential toxicity. Furthermore, all TiO\({}_{2}\) samples prompted elevated secretion of two key pro-inflammatory cytokines, IL-1\(\upbeta\) and TNF-\(\upalpha\), in a manner that was not dose-dependent. The discrepancies in the obtained results can be caused by the variation in the share of individual sizes of TiO\({}_{2}\) particles within each of the four products tested. Therefore, it is difficult to formulate general observations concerning the effect of E171 on human cells because of the different compositions of these industrial products. For scientific purposes, the separation of E171 NPs into fractions of various dimensions within 1-100 nm (e.g., using microfiltration) is reasonable. In this way, a better understanding of what different sizes and shapes of TiO\({}_{2}\) NPs exist in E171 will be gained. However, this outcome will have limited usability as E171 will still be applied industrially as a mixture of various fractions of NPs within 1-100 nm and above 100 nm. Except for the properties of particular nanoparticles, the actual effects are also impacted by the conditions within the GIT itself. To recapitulate, the current scientific knowledge in this context remains incomplete, and further efforts should be made to continue research in this important area, given the ever-more widespread use of nanoparticles. The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app14209387/s1](https://www.mdpi.com/article/10.3390/app14209387/s1)]([https://www.mdpi.com/article/10.3390/app14209387/s1](https://www.mdpi.com/article/10.3390/app14209387/s1)), **Figure S1:** Percentage distribution of individual molecule fractions in the tested samples (S1-S4); **Figure S2:** Figures A and B present the analysis of the 4-parameter logistic (4 PL) model applied to the data obtained from the ATP and LDH assays, respectively. The fitted curves demonstrate the dose-response relationship for each tested substance (S1-S4), the dashed vertical line represents the EC50 value, indicating the concentration at which half of the maximum effect is observed; **Figure S3:** Figures A and B present the analysis using the 4-parameter logistic (4 PL) model for the data obtained from the pro-inflammatory cytokine assays, specifically IL-1\(\beta\) and TNF-\(\alpha\), respectively.