According to the framework on risk assessment of nanoparticles in applications related to food and feed falling within the remit of the European Food Safety Authority (EFSA) - i.e. nutrients and nutrient sources, novel foods, food contact materials, food additives, food flavourings, feed additives, and pesticides - nanoscale specificities are integrated in the risk assessment process as nanoscale-based hypotheses. These hypotheses encompass potential human exposure to particles (‘Is toxicokinetics driven by particle uptake and distribution?’) and particle-related hazards (‘Has the material particles within a biologically relevant nanoscale size? Does this size influence cellular uptake? Is there bioaccumulation of nanoparticles or other biological interactions related to the nanoscale?) [Schoonjans et al., 2023]. The Guidance on Nano - Risk Assessment of the EFSA Scientific Committee points out New Approach Methodologies (NAMs) as the first choice to generate information for addressing these hypotheses and improve mechanistic understanding of processes at the nanoscale [EFSA Scientific Committee, 2021]. Integrated Approaches to Testing and Assessment (IATAs) are suggested to be used for the integration of human, animal and NAMs-derived evidence.
Recently, the production and use of cellulose at the nanoscale has attracted increasing interest. Nanocellulose (NC) is an emerging material in the food sector with several application areas, including prospective use as a novel food or as food additive. Three main classes of NC exist, i.e. NC fibres produced by bacterial species (bacterial NC, BNC) and other NCs obtained by technological modification of cellulose from plants or other origins, leading to cellulose nanofibres (nanofibrillated cellulose, NFC) or nanocrystals (cellulose nanocrystals, CNC). The biological sources and processing conditions affect the size, morphology, and several other physicochemical parameters of NC. Although all NC materials typically have a high aspect ratio, CNC usually consists in rod-shaped crystals, whereas NFC consists in fibrils composed of fibres with a length up to 2-3 μm; nanofibers are even longer in BNC and organized in networks. For all the NC types, the diameter can be very small (as low as 5-10 nm).
The potential hazards of ingested NC are insufficiently characterised and NC nanoscale features require a nano-specific assessment. A recent review of current knowledge on (possible) adverse health effects of NC upon oral exposure indicated that toxicity data, especially from in vivo studies, are limited and outcomes are not unambiguous [Brand et al., 2022]. The hazard assessment is further complicated by the diversity in morphologies and surface modifications, the lack of standard reference materials, the limited knowledge about intestinal fate and absorption, the analytical difficulties in detecting NC in biological matrices, the dispersion issues, and the possible presence of impurities and interferences within biological assays.
The use of human relevant NAMs appears as the best option to generate the needed information for the hazard assessment of NC oral exposure for several reasons. First, laboratory animals do not appear to be appropriate models because the digestive physiology, microbiome and rate of fibre degradation differ from humans. Second, the variability in the physicochemical properties of NC makes NAMs ideal for efficient testing. Third, the needed studies are technically easier to implement using in vitro methods than in vivo studies. Unfortunately, the available evidence from in vitro studies is contrasting [Brand et al., 2022; Stoudmann et al., 2020; Ventura et al., 2020]. Besides, in the vast majority of the cases, data are unsuitable for the hazard identification of NC oral exposure owing to methodological shortcomings, i.e. (i) insufficient physicochemical characterisation of the pristine materials; (ii) absence of suitable dispersion protocols; (iii) use of extremely high concentrations (well above 100 μg/mL); (iv) no confirmation of cellular exposure.
In the EFSA-funded project NANOCELLUP, a NAM-based IATA for addressing data gaps in the assessment of potential hazards associated to NC oral exposure was designed [Vincentini et al., 2023]. This IATA focused on three main pillars, i.e. (i) assessment of the uptake and potential crossing of the intestinal barrier by NC, (ii) assessment of local effects, including inflammation and genotoxicity, on the gastrointestinal epithelia, and (iii) assessment of any digestion or degradation of NC by the human microbiome. Eight NC samples belonging to the three NC types, plus a comparator in the micro-range, were selected as study materials and submitted to a thorough physicochemical characterisation.
A battery of in vitro tests was used to provide insight into NC hazard and mode of action according to a tiered approach, which lead to selection of three materials belonging to the three main NC types for in depth-testing. Uptake by the intestinal epithelium and any potential crossing was studied using a triculture model based on co-culturing three different human-derived cell lines (Caco-2 cells, HT29-MTX cells, and Raji B lymphocytes), which was applied for the first time to the study of nanofibres. This triculture model incorporates microfold (M) cells and mucus secreting cells, both of key importance in nanoparticle uptake, which enhances its physiological relevance compared to Caco-2 cell monocultures.
The analytical challenge of identifying NC crystals and fibres was addressed by fluorescence detection using two alternative staining methods, which were compared in terms of selectivity and sensitivity. Uptake and crossing were assessed by using Confocal Laser Scanning Microscopy (CLSM) and a quantitative approach was developed for screening internalisation whereby uptake of NC crystals (CNC) or fibres (NFC, BNC) was measured as percentage of NC-containing cells on the total number of counted cells at a given magnification. This methodology allowed a quantitative estimation of the proportion of cells involved in the uptake process. Cell uptake of the three selected materials was demonstrated, and such uptake was greater in a triculture model as compared to Caco-2 monolayers. Uptake was the greatest in repeated exposure conditions, in which intestinal barrier crossing was demonstrated for CNC. Pro-inflammatory responses accompanied by massive NC uptake in macrophages and barrier function impairment were also observed, whereas no indications for genotoxicity were obtained. Finally, no formation of smaller particles following potential colonic fermentation of NC was observed using an in vitro model of human colon (ARCOL) [Vincentini et al., 2023].
For the integration of these results in regulatory hazard assessment of NC after oral exposure, prospective use of NC as novel food or as food additive were considered, as these are the most likely applications leading to direct exposure of consumers. It was considered that NC is a non-digestible carbonaceous nanomaterial for which, based on the results obtained, a potential for biopersistence and bioaccumulation cannot be excluded upon chronic exposure. In addition, notwithstanding marked differences in several physicochemical parameters were observed among the different NC materials, no clear boundaries could be identified in terms of cell uptake and adverse effects, albeit CNC, composed of smaller particles, appeared as the material type of higher concern. The differences in toxic responses noted within each type of materials highlighted that complex relationships exist between physicochemical and (adverse) biological effects. The variability in toxic responses within each class of NC material observed might limit read across, unless physicochemical determinants of such variability are identified and understood. All these elements were recommended to be considered in future studies on NC hazards as well as in the assessment of the risks associated to NC oral exposure [Vincentini et al., 2023].
In NANOCELLUP, as an essential requirement, all experimental studies were performed to ensure relevant and reliable results in the perspective of their use for regulatory risk assessment. A detailed physicochemical characterisation was performed and material-specific dispersion protocols ensuring maximal deagglomeration were developed. A specific SOP detailing the protocol for NC dispersion was developed to ensure that a similar level of dispersion was achieved through the full dose/concentration range, with a maximum concentration applicable in in vitro studies of 30 µg/mL. For in vitro testing, in absence of OECD validated methods, scientifically ‘valid’ methods covering the different endpoints were selected, taking into account the recommendations from international bodies, e.g. OECD [OECD, 2108a; OECD, 2018b], from the literature (e.g. Drasler et al., 2017), the European Union Reference Laboratory for alternatives to animal testing (EURL ECVAM) and the EFSA SC Guidance on Nano - Risk Assessment [EFSA Scientific Committee, 2021]. In the design of the tests as well as in the reporting, a number of requirements were complied with, including: (i) detailed cell characterisation and description of cell culture methods, (ii) exposure and post-exposure times defined and justified with respect to the individual tested parameters, (iii) check for the absence of interference, (iv) quality controls including negative and positive controls and assay reagent controls, and (v) replication of key studies in different laboratories.
The successful outcome of this project paved the way to a wider international research effort funded by EFSA, namely NAMS4NANO (‘Integration of New Approach Methodologies results in chemical risk assessments: Case studies addressing nanoscale considerations’ - GP/EFSA/MESE/2022/01). This is a multiannual project including several individual projects funded as separate lots. The overall aim of this ongoing action is to promote the use of NAMs for nanospecific risk assessment, covering both nanomaterials and conventional materials (i.e. not engineered at the nanoscale) containing a fraction of small particles.
1. Brand W, van Kesteren PCE, Swart E, Oomen AG. 2022. Overview of potential adverse health effects of oral exposure to nanocellulose. Nanotoxicology 16:217.
2. Drasler B, Sayre Ph, Steinhäuser KG, Petri-Fink A, Rothen-Rutishauser B. 2017. In vitro approaches to assess the hazard of nanomaterials. NanoImpact 8, 99.
3. EFSA Scientific Committee, More S, Bampidis V, Benford D, Bragard C, Halldorsson T, Hernandez-Jerez A, Hougaard Bennekou S, Koutsoumanis K, Lambre C, Machera K, Naegeli H, Nielsen S, Schlatter J, Schrenk D, Silano V, Turck D, Younes M, Castenmiller J, Chaudhry Q, Cubadda F, Franz R, Gott D, Mast J, Mortensen A, Oomen AG, Weigel S, Barthelemy E, Rincon A, Tarazona J, Schoonjans R. 2021. Guidance on risk assessment of nanomaterials to be applied in the food and feed chain: human and animal health. EFSA J 19(8):6768, 111 pp.
4. OECD. 2018a. Guidance Document on Good In Vitro Method Practices (GIVIMP), OECD Series on Testing and Assessment, No. 286, Organisation for Economic Co-operation and Development.
5. OECD. 2018b. Series on the safety of manufactured nanomaterials No. 85. In: Evaluation of in Vitro Methods for Human Hazard Assessment Applied in the OECD Testing Programme for the Safety of Manufactured Nanomaterials. Organisation for Economic Co-operation and Development (OECD), ENV/JM/MONO (2018)4.
6. Schoonjans R, Castenmiller J, Chaudhry Q, Cubadda F, Daskaleros T, Franz R, Gott D, Mast J, Mortensen A, Oomen AG, Rauscher H, Weigel S, Astuto MC, Cattaneo I, Barthelemy E, Rincon A, Tarazona J. 2023. Regulatory safety assessment of nanoparticles for the food chain in Europe. Trends Food Sci Technol. 134:98.
7. Stoudmann N, Schmutz M, Hirsch C, Nowack B, Som C. 2020. Human hazard potential of nanocellulose: quantitative insights from the literature. Nanotoxicology 14:1241.
8. Ventura C, Pinto F, Lourenco AF, Ferreira PJT, Louro H, Joao Silva M. 2020. On the toxicity of cellulose nanocrystals and nanofibrils in animal and cellular models. Cellulose 27:5509.
9. Vincentini O, Blier AL, Bogni A, Brun M, Cecchetti S, De Battistis F, Denis S, Etienne-Mesmin L, Ferraris F, Sirio Fumagalli F, Hogeveen K, Iacoponi F, Moracci G, Raggi A, Siciliani L, Stanco D, Verleysen E, Fessard V, Mast J, Blanquet-Diot S, Bremer-Hoffmann S, Cubadda F. 2023. EFSA Project on the use of New Approach Methodologies (NAMs) for the hazard assessment of nanofibres. Lot 1, nanocellulose oral exposure: gastrointestinal digestion, nanofibres uptake and local effects. EFSA Supporting publication 20(9):EN-8258. 49 pp.
Francesco Cubadda has an education in Chemistry and Toxicology. Senior Scientist at the Istituto Superiore di Sanità, the Italian National Institute of Health, he leads a group with a long track record in research on analytical determination, toxicology, risk assessment of nanomaterials. Current interest lies in the use of NAMs in nanotoxicology, risk assessment, with the coordination of international projects such as NANOCELLUP and NAMs4NANO (Lot 3). He also serves as Head of the Italian delegation to the OECD's Working Party on Manufactured Nanomaterials (WPMN). He has a long standing expertise in food safety risk assessment, in both the chemical and the nutritional domain, and he served in several working groups of the European Food Safety Authority. He is a current member of the EFSA Cross cutting Working Group on Nanotechnology and national scientific expert in the EFSA Network for Risk Assessment of Nanotechnologies in food and feed since 2011.
Maria Chiara Astuto has an education in Toxicology (University of Milan, Italy). Scientific officer at the EFSA’s Methodology and Scientific Support Unit, she is involved in several scientific activities of EFSA’s Scientific Committee in the area of chemical risk assessment. Current activities are the coordination of EFSA’s Scientific Committee Working Group on Nanotechnologies and EFSA’s Network with EU Member States on Nanotechnologies in Food and Feed, as well as various experimental projects aimed at promoting the implementation of New Approach Methodologies in regulatory risk assessments.
Olimpia Vincentini has an education in Biology (Sapienza University, Rome) and a PhD in Experimental and Clinical Pathology (Alma Mater Studiorum University, Bologna). Senior scientist at the Istituto Superiore di Sanità - the Italian National Institute of Health - her interests focus on validation of New Approach Methodologies (NAMs) for risk assessment of nanomaterials, including advanced intestinal barrier models for assessing nanoparticles uptake and translocation. She’s currently contributing to the development of an OECD guidance document on an in vitro approach to determine the gastrointestinal fate of nanomaterials. She also serves as expert for the national committee on scientific evaluation of preclinical studies for phase 1 clinical trials.
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