Evaluation of the cyto- and genotoxicity of two types of cellulose nanomaterials using human intestinal cells and in vitro digestion simulation

Emerging cellulose nanomaterials (CNMs) may have commercial impacts in multiple sectors, being their application particularly explored in the food sector. Thus, their potential adverse effects in the gastrointestinal tract should be evaluated before marketing. This work aimed to assess the safety of two CNMs (CNF-TEMPO and CMF-ENZ) through the investigation of their cytotoxicity, genotoxicity (comet and micronucleus assays), and capacity to induce reactive oxygen species in human intestinal cells, and their mutagenic effect using the Hprt gene mutation assay. Each toxicity endpoint was analysed after cells exposure to a concentration-range of each CNM or to its digested product, obtained by the application of a standardized static in vitro digestion method. The results showed an absence of cytotoxic effects in intestinal cells, up to the highest concentration tested (200 µg/mL or 25 µg/mL, for non-digested and digested CNMs, respectively). Of note, the cytotoxicity of the digestion control limited the top concentration of digested samples (25 µg/mL) for subsequent assays. Application of a battery of in vitro assays showed that CNF-TEMPO and CMF-ENZ do not induce gene mutations or aneugenic/clastogenic effects. However, due to the observed DNA damage induction, a genotoxic potential cannot be excluded, even though in vitro digestion seems to attenuate the effect. The lowest digested CNF-TEMPO concentration induced chromosomal damage in Caco-2 cells, leading to an equivocal outcome. Ongoing research on epigenotoxic effects of these CNMs samples may strengthen the lines of evidence on their safety when ingested, paving the way for their innovative application in the food industry.