Dora Rolo

Titanium dioxide nanoparticles (TiO₂-NPs) are widely used, and humans are exposed through food (E171), cosmetics (e.g., toothpaste), and pharmaceuticals. The oral and gastrointestinal (GIT) tract are the first contact sites, but it may be systemically distributed. However, a robust adverse outcome pathway (AOP) has not been developed upon GIT exposure to TiO₂-NPs. The aim of this review was to provide an integrative analysis of the published data on cellular and molecular mechanisms triggered after the ingestion of TiO₂-NPs, proposing plausible AOPs that may drive policy decisions. A systematic review according to Prisma Methodology was performed in three databases of peer-reviewed literature: Pubmed, Scopus, and Web of Science. A total of 787 records were identified, screened in title/abstract, being 185 used for data extraction. The main endpoints identified were oxidative stress, cytotoxicity/apoptosis/cell death, inflammation, cellular and systemic uptake, genotoxicity, and carcinogenicity. From the results, AOPs were proposed where colorectal cancer, liver injury, reproductive toxicity, cardiac and kidney damage, as well as hematological effects stand out as possible adverse outcomes. The recent transgenerational studies also point to concerns with regard to population effects. Overall, the findings further support a limitation of the use of TiO₂-NPs in food, announced by the European Food Safety Authority (EFSA).

Nanomaterials (NMs) have important and useful applications in chemical industry, electronics, pharmaceuticals, food and others. Their rapid proliferation presents a dilemma to regulators regarding hazard identification and increased concerns for public health. The Adverse Outcome Pathways (AOPs) are innovative central elements of a toxicological knowledge framework, developed for supporting chemical risk assessment based on mechanistic reasoning. AOPs describe a sequence of causally linked events at different levels of biological organisation, triggered by exposure to a stressor (like chemicals or NMs) leading to an adverse health effect in humans or wildlife. The integrative analysis of the cellular and molecular mechanisms of nanotoxicity towards the identification of connected adverse outcomes drives a sequential line – an AOP landscape definition. Each defined AOP is available for crossing data, linking known and unknown landscapes, reducing the reliance on animal studies, associated costs and ethical issues. NMs have unique properties, with specific associated toxicological challenges, which may represent unknown AOP landscapes. In this chapter, an overview of AOPs as important novel strategic tools in nanotoxicology is presented, highlighting the current applications in hazard identification and human health risk assessment. Keywords Nanomaterials Adverse outcome Risk assessment Nanotoxicology

Several metallic nanomaterials (NMs), such as titanium dioxide nanomaterials (TiO₂), present beneficial properties with a broad range of innovative applications. The human population is exposed to TiO₂, particularly by ingestion, due to its increasing use as a food additive and inclusion in dietary supplements and food packaging materials. Whether this oral exposure may lead to adverse local or systemic outcomes has been the subject of research, but studies have generated contradictory results, reflecting differences in the physicochemical properties of the TiO₂ studied, effects of the surrounding matrix, and modifications during digestion. This work aimed to investigate the toxic effects of three different TiO₂ NMs (NM-103, NM-103 and NM-105) on the gastrointestinal tract cells, Caco-2 and HT29-MTX-E12, after the use of the standardized static INFOGEST 2.0 in vitro digestion method to mimic human digestion of TiO₂, contributing to hazard assessment. The results show that, for one of the digested TiO₂ NMs studied (NM-105), a more pronounced toxicity occurs after exposure of HT29-MTX-E12 intestinal cells, as compared to undigested NM, concomitantly with subtle changes in characteristics of the NM. Thus, the inclusion of the digestion simulation in the safety evaluation of ingested NMs through in vitro bioassays can better integrate the modifications that NMs suffer in the organism. It is expected that such an approach will reduce uncertainties in the hazard assessment of ingested NMs for human health.