Nanotechnology presents many possibilities for the food industry, offering potential benefits like targeted nutrient delivery, improved food preservation, and enhanced sensory experiences. However, due to their unique properties at the nanoscale, nanomaterials (NMs) might necessitate additional testing requirements. To implement the information requirements, the European Food Safety Authority (EFSA) has published two guidance documents outlining the criteria for evaluating materials at the nanoscale. These guidance documents address defined “engineered nanomaterials” (More et al., 2021a) and food additives containing a fraction of smaller particles (More et al., 2021b). The EFSA guidance recommends a tiered analysis of nanospecific considerations revolving around the behavior of the NM within the gastrointestinal (GI) tract. This includes dissolution dynamics, cellular uptake, transcytosis, and potential disruption of the intestinal barrier. Additionally, genotoxicity and the accumulation potential of the NM are assessed. This tiered analysis offers a unique opportunity to integrate in vitro testing into the regulatory process. The guidance document additionally proposed a stepwise approach, focusing on in vitro assays related to cytotoxicity (cell death), oxidative stress, (pro) inflammation, and impairment of the intestinal barrier. Furthermore, in vitro dissolution testing under simulated lysosomal and GI conditions is recommended to obtain a more exhaustive picture of the complex biological interactions of ingested NMs in the GI tract. In order to develop case studies implementing the two guidance documents, the EFSA launched a pilot project using nanocellulose (NC) as an emerging material in the food sector, and additional case studies are ongoing in the remit of an EFSA-funded project called NAMS4NANO, where various NMs are characterized and tested in different in vitro models.

The gut barrier plays a crucial role as both a protective barrier that separates the internal blood from the luminal content and a selective barrier that regulates the flux of water, ions, and essential dietary nutrients. The intestinal barrier consists of a multilayer system encompassing a chemical layer containing the antibacterial proteins secreted by Paneth cells, a mucus layer secreted by goblet cells, an epithelial layer, and the cellular immune system. Enterocytes and goblet cells are the most studied cell types. Alteration of one or many of those layers leads to increased intestinal permeability, also called intestinal hyper-permeability or leaky gut syndrome, enhancing translocation of bacteria, bacterial products (such as lipopolysaccharides), and undigested nutrients from the intestinal lumen into the systemic circulation (Kinashi and Hase, 2021). Many diseases arise or are exacerbated by a leaky gut, including diarrhea, inflammatory bowel disease, celiac disease, autoimmune hepatitis, or type 1 diabetes (Aleman et al., 2023; Marasco et al., 2022; Miele et al., 2009; Anand and Mande, 2022). In addition, individuals with a leaky gut are more vulnerable to toxicity driven by chemical exposure as dietary pesticides or food chemicals can more easily enter the systemic blood and reach the target organs (Gama et al., 2022). Although it can be argued that intestinal barrier disruption is an intermediate event toward various clinical outcomes, given its central role, in this study, we advocate considering this event as an adverse outcome when assessing the toxicity of food NMs.

Although the underlying mechanisms remain evasive and evidence has not been systematically gathered or produced, a mechanistic-based approach would be highly instrumental in assessing the impact of food NMs on intestinal barrier integrity. Such a risk assessment based on mechanistic reasoning requires relevant adverse outcome pathways (AOPs). The OECD defines the AOP as a description of a logical sequence of causally linked events (key events, KEs) at different levels of biological processes, which follows exposure to a stressor and leads to an adverse outcome (AO) in humans or wildlife (Garcia-Reyero and Murphy, 2018). Hence, a KE describes a measurable and essential change in a biological system that can be quantified in experimental or clinical settings. Then, the strength of the relationship between two KEs (key event relationship, KER) is first established by biological plausibility (e.g., binding to a receptor induces a signaling cascade) and then by evaluating the current evidence supporting this causal link; this approach can also highlight gaps in the knowledge or inconsistencies between studies. Data discrepancies can arise from variations in experimental settings and physicochemical properties of NMs. The development of an AOP is a dynamic process built from hypothesized KEs toward the refinement of KEs and KERs by assembling evidence found in the literature or newly produced (Vinken, 2018). AOP development guidance was established by the OECD (OECD, 2016), and already developed AOPs describing various AOs are stored in the publicly accessible repository platform called AOP-Wiki (https://aopkb.oecd.org/). There is no explicit quality assurance for AOPs in AOP-Wiki, except for those that have undergone the OECD review process (OECD 2018), which makes them compliant AOPs compliant for potential regulatory use.

Initially developed to support new approach methodology (NAM)-based chemical risk assessment, the framework was recognized as instrumental in prioritizing and developing testing strategies for NMs (Halappanavar et al., 2019). Most AOPs developed for chemicals should also be applicable to NMs, considering certain adaptations at the molecular initiating event (MIE) and early KE levels (Clerbaux et al., 2022). For example, AOP 144, leading to liver fibrosis, was proven to be nano-relevant as endocytic lysosomal uptake is described as a nano-relevant MIE connected to lysosomal disruption and mitochondrial dysfunction, leading to cell death in the liver (Gerloff and Landesmann, 2024; Murugadoss et al., 2021; Gerloff et al., 2017). Simple in vitro models for testing the potency of NMs were associated with the MIE and early lysosomal and mitochondrial KEs in this AOP (Murugadoss et al., 2021). These early KEs lead to cell death.

Building on “tissue injury,” Halappanavar et al. proposed a methodology for identifying NM-relevant KEs based on the plausibility, measurability, and regulatory importance of the KEs (Halappanavar et al., 2019). The authors identified inflammation (increased pro-inflammatory mediators and leukocyte recruitment/activation), oxidative stress, and cell death as the main upstream KEs to tissue injury. Reported endpoints and associated assays were identified, allowing for a quantifiable measurement of these KEs. Finally, in line with the increasing recognition of the central role of the leak gut, ‘intestinal barrier disruption’ was recently added to AOP-Wiki in the COVID-19 context (Clerbaux et al., 2022). However, although the ingestion of food NMs increases the possibility of an adverse outcome on the intestinal barrier, there are currently no AOPs supporting the toxicity of NMs on the intestinal barrier following their cellular uptake in the intestine.

In this study, we developed an AOP proposing a toxicological pathway toward intestinal barrier disruption, following the intestinal uptake of NMs via lysosomal and mitochondrial dysfunction. This AOP was developed by leveraging information from existing AOPs published in AOP-Wiki and based on biological plausibility. Following the establishment of the AOP, we extracted current evidence from the scientific literature for a targeted selection of NMs highly relevant to the food sector, including ZnO, CuO, FeO, SiO2, and Ag NMs and nanocellulose. They have gained increasing interest due to their potential applications in different areas (biocides or pesticides, cosmetics, nutrient feed and food additives, food supplements, and food contact materials) and have also been extensively investigated in nanotoxicology studies. However, conflicting data on NM oral exposure in humans, uncertainties regarding their fate in the human body, the pristine NM physicochemical changes potentially occurring during the digestive process, and the low human relevance of animal models (e.g., pH in the stomach) still in use highlight the need for further investigation. This also emphasizes the importance of developing integrated approaches to testing and assessment (IATAs) and AOPs, as recommended by the EFSA Roadmap (Escher et al., 2022) With this purpose, our in-depth literature review allowed us to highlight current knowledge gaps and data inconsistencies guiding future research.

In the prototypical stressor section of AOP-Wiki, the keywords “nanoparticles,” “NPs,” “nanosized particles,” “nanomaterials,” and “nanotubes” were used to filter AOPs in AOP-Wiki, which report a nanosized material as a stressor. For each AOP, the AOP number, MIE, AO, and status information have been extracted.

The AOP was first built based on biological plausibility and by leveraging existing KEs in another NM-relevant AOP. We started with the apical outcome, “intestinal barrier disruption,” which we postulated was due to “cell death/injury” of the different intestinal cell types, identified as a main KE upstream of tissue injury in nano-relevant AOPs (Halappanavar et al., 2019), and this rationale is based on the fact that the intestinal barrier is composed of different layers of specific cell types, whose death or injury impairs the barrier function. In this study, we focused on enterocytes and goblet cells. The identification of the intermediate cell-specific KEs between “cell death/injury” and “intestinal barrier disruption” was based on biological plausibility and further refined, following a literature search. On the other side of the pathway, the MIEs and initial KEs defined in the nano-relevant AOP for the liver, considering NM-induced ‘mitochondrial dysfunction’ leading to “cell death/injury,” were extrapolated via a tissue analogy-based approach as upstream KEs (Figure 1).

To verify the relevance of the proposed AOP for the food sector, we focused the literature research on NMs used as food additives and novel foods. Zinc oxide (ZnO) NMs are micronutrient supplements and food fortification ingredients that can release Zn ions intracellularly after being absorbed by enterocytes (Youn and Choi, 2022). Silicon dioxide (SiO₂) NMs (E551) are food additives that exhibit size-dependent cytotoxicity in in vitro studies (Dong et al., 2020). Iron oxide (FeO) NMs (E17) serve as food additives and flavorings (Ansari et al., 2019); their impact on the intestinal barrier is yet to be explored. Nanosilver (Ag NM, E174) is an antimicrobial material used as a colorant or food contact material that most properly exerts its toxicity through silver ion release, but conclusive studies are lacking (Bredeck et al., 2021). Copper oxide (CuO) NMs are used as nutrients, food supplements, and pesticides and share potential ion-driven toxicity mechanisms with ZnO and Ag NMs (European Food Safety Authority EFSA, 2009).

The literature search was performed both automatically using AOP helpFinder and manually in PubMed based on selected keywords, followed by a selection procedure of the relevant studies based on predefined exclusion criteria.

To date 15 AOPs are present in the AOP-Wiki that report nanosized material(s) as a stressor with only AOP173 endorsed by OECD (Table 1), refers to the correlation between the NM cellular uptake by endocytic lysosomal absorption and the consequent mitochondrial dysfunction leading to liver fibrosis (Gerloff and Landesmann, 2024). Other AOPs illustrate the NM-mediated lung toxicity (shown as gray in Table 1). AOP 237, 303, 302, and 319 are under development as part of different OECD projects. Finally, there are several AOPs proposed that are not currently part of the OECD process. Not pertaining to the lungs, AOP 209 connects silica nanoparticle-induced disruption of cholesterol to hepatotoxicity, while AOP 207, AOP 208, and AOP 210 suggest pathways leading to infertility.

In addition, outside AOP-Wiki, at least five AOPs identifying NMs as stressors and leading to lung outcomes are described in the literature in humans. Two AOPs starting from an increased substance interaction and leading to lung emphysema and lung fibrosis, respectively, have been proposed (Halappanavar et al., 2020). Literature reports suggesting potential hazards of TiO₂ served for the development of one putative AOP, leading to lung cancer (Nymark et al., 2021; Braakhuis et al., 2021). An AOP for graphene-family nanomaterial-induced lung damage was developed (Ding et al., 2023), as well as an AOP describing Ag NM toxicity toward the respiratory tract (Nicholas et al., 2021). Regarding intestinal outcomes, a plausible AOP was proposed following the ingestion of TiO₂ nanoparticles, which could eventually lead to colorectal cancer (Rolo et al., 2022). This AOP is not included in AOP-Wiki. An AOP-oriented study also assessed cytotoxicity, oxidative stress, genotoxicity, perturbation of the cell cycle, and apoptosis in human intestinal cells, following Ag NM exposure, based on KEs present in AOPs reporting Ag NMs as a stressor; however, no intestinal outcomes were reported (Kose et al., 2023).

Thus, although food NMs enter the human body through the oral route, increasing the possibility of adverse effects on the gut barrier, there are currently no AOPs developed supporting the toxicity on the intestinal barrier function, following cellular uptake by intestinal cells of ingested NPs. A structured approach to assess these effects can be instrumental. In this study, we aimed to develop an AOP specifically focused on the impact of NMs on the intestinal barrier. This AOP construction relied on biological plausibility to link NM cellular uptake with the disruption of the intestinal barrier. We then aimed to evaluate this framework by extracting evidence from published research. Finally, our analysis aimed to identify any knowledge gaps or inconsistencies in the evidence supporting this AOP.

NM-relevant MIEs identified in AOPs in other tissues might be linked to the intestinal tissue. AOP 144 is at the second highest stage of AOP development. The MIE, endocytic lysosomal uptake, leads to lysosomal disruption, which induces mitochondrial dysfunction and leads to cell injury/death. This is of interest because a recently proposed AOP network linked NM-induced mitochondrial dysfunction to existing AOs in the lung, liver, and cardiovascular and nervous systems (Murugadoss et al., 2023). The authors identified that NM-induced mitochondrial toxicity is crucial for many tissues, but interestingly, they did not mention the intestinal epithelium.

Hence, we propose an AOP that is biologically plausible (Figure 2).

The abovementioned table highlights evidence gaps, particularly for KER1775 and KER993, where lysosomal disruption was almost never evaluated (Table 8). Furthermore, our quantitative understanding is currently limited. In addition, data were not consistent among studies that used various stressors, biological systems, and E-related assays.

Endocytosis is of particular interest in nanotoxicology due to its responsibility for the Trojan horse effect, in which NMs facilitate the internalization of contaminants or serve as precursors, releasing breakdown products that would otherwise exhibit limited cellular permeability. This holds true for several metal oxide NMs. Upon internalization, these NMs can undergo degradation, leading to the uncontrolled release of metal ions and bypassing the regulatory mechanisms associated with metal transporters. In our study, we could reveal that relevant NMs for the food sector can be taken up by intestinal cells in vitro.

In our study, three of the analyzed NMs (ZnO, CuO, and Ag NMs) are known to release ions after accumulating in the acidic environment of lysosomal vesicles. The ionic forms Zn²⁺, Fe²⁺, Ag⁺, or Cu²⁺ can disrupt the stability of the lysosomal membrane through interactions with the phospholipid bilayer, altering its structure or suppressing enzyme activities involved in maintaining the lysosomal integrity (Wang et al., 2018). The resulting leakage of digestive enzymes can further damage the cellular components and trigger cell death. In addition, an overload of positive ions in lysosomes also triggers anionic and water influx, ultimately leading to a disruption of the lysosome. In addition, the NM itself can exhibit cationic characteristics depending on the surface of the nanoparticles and the specific environments. Such characteristics are described for FeO and SiO₂ NMs that can lead to an influx of anions and water (proton sponge effect). Lysosomes can swell and lead to the disruption of the lysosomal membrane (Wu et al., 2017; Lee and Hong, 2020), leading to lysosomal dysfunction. However, none of the investigated studies measured the disruption of lysosomal structures in enterocytes or goblet cells, preventing us from establishing a direct link between endocytosis and lysosomal disruption, as described in KER1775 for other NMs.

Our hypothesis proposes that lysosomal disruption triggers the release of lysosomal proteases, consequently inducing mitochondrial dysfunction, as described in KER993. Mitochondrial dysfunction encompasses a broad spectrum of alterations in both the mitochondrial structure and function. NM-derived ions interfere with redox cycling in mitochondria, leading to elevated ROS production. In addition, physical interactions between the entire NM and mitochondria and reactive groups on the NP surface can trigger ROS generation. This aspect is confirmed by several studies related to Ag NMs (Braeuning et al., 2018; Domenech et al., 2021; Kämpfer et al., 2021; Jia et al., 2020; Rufino et al., 2021; Juling et al., 2017; Böhmert et al., 2012; Botha et al., 2019; Gioria et al., 2018; Oberemm et al., 2016; Chen et al., 2016). As the concentration of NMs/ions increases within a cell (e.g., through accumulation after chronic or high exposure), the likelihood of interactions with cellular components, particularly mitochondria, also increases. Moreover, the production of ROS can initiate a cascade of additional cellular events with an impact on the intestinal barrier including inflammatory responses (Youn and Choi, 2022; Horie and Tabei, 2021). Xu and colleagues described increased ROS production, pro-inflammatory cytokines, and cytotoxicity after treatment with smaller SiO₂ nanoparticles, affecting signal transduction pathways (Xu et al., 2022). Pathways such as RhoA/ROCK, leading to the disassembly of TJ, inflammatory signaling cascades, and cytoskeleton, can compromise cellular processes crucial for a healthy barrier (Xu et al., 2022; Jia et al., 2020; Chen et al., 2016; González-Mariscal et al., 2008). Such impairments would not automatically lead to cell death but can be of relevance for the intestinal barrier (Ude et al., 2019).

The causal relationship between mitochondrial dysfunction and cell death/injury is well-captured within KER363 in the OECD-endorsed AOP48 and AOP144. ROS plays a critical role in cell death, particularly at higher concentrations. A lower level of ROS production can act as a signaling molecule, even enhancing cellular stress resistance. However, when ROS production overwhelms the cell’s antioxidant defenses, a state of oxidative stress occurs, promoting cell death. Physicochemical properties, the susceptibility of different cell types, and exposure times, are other parameters that can influence different cytotoxicity results. Increased lipid vacuoles and changes in morphologic ultrastructure, like villi and organelles dimensions, have also been observed in NM-treated cells (Li et al., 2023). Moreover, altered expressions of cytoskeleton- and lipid-metabolism-related proteins have also been found in in vivo studies (Reichardt et al., 2009; Reichardt et al., 2012). Passive lipid uptake could be progressively normalized through a decrease in the intestinal adsorptive area because of changes in the microvilli length, width, and density of enterocytes. All these changes can contribute to membrane instability, lipid accumulation, oxidative stress, and inflammation, which ultimately lead to cell death machinery activation and intestinal epithelium homeostasis dysregulation (Fang et al., 2021; Anto and Blesso, 2022). The balance between proliferation and apoptosis of intestinal cells is dependent on the microenvironment and stress factors; the defect in balance is strongly connected with several intestinal diseases and GI injuries (Nunes et al., 2014; Subramanian et al., 2020).

The intestinal barrier is a multilayer system. Alteration of one or more layers of the intestinal barrier leads to increased intestinal permeability and decreased barrier function (Horowitz et al., 2023). Some studies have reported the role of TJs in regulating epithelial cell proliferation. For instance, overexpression of claudin-2 in human colon cells increased proliferation in vitro and accelerated tumor growth in vivo (Buhrmann et al., 2015; Abu-Farsakh et al., 2017). Moreover, it is known that pro-inflammatory cytokines such as TNF and IL1β or LPS can induce barrier loss by internalization of occludin (Nighot et al., 2017; Kaminsky et al., 2021). The administration of recombinant IL-13 in mice increases claudin-2 expression and augments intestinal paracellular cation permeability (Raju et al., 2020). The causal link between damaged/dying enterocytes and goblet cells leads to increased paracellular permeability due to disruption of the epithelial monolayer and regulation of mucus secretion and thickness; this process, well described in the literature (Horowitz et al., 2023; Paone and Cani, 2020), is now captured within KER3197 in AOP-Wiki.

Other pathophysiologic events leading to barrier disruption can occur in the gut after NM ingestion. For instance, some food additives have been shown to induce microbiota composition alterations. These microbiota alterations have been associated with a reduced mucus layer thickness and an increased gut penetrability linked with intestinal inflammation and metabolic alterations of the intestinal barrier (Paone and Cani, 2020; Chassaing et al., 2017; Viennois and Chassaing, 2018).