Progetto NANOCELLUP

Four EU research institutions, with additional support from the EC-JRC, collaborate under the coordination of ISS to this project funded by the European Food Safety Authority (EFSA) (Grant Agreement number GP/EFSA/SCER/2020/04).

National Institute of Health (ISS, Italy)

• Francesco Cubadda (Principal Investigator)
• Olimpia Vincentini (Deputy)
• Francesca De Battistis
• Gabriele Moracci
• Serena Cecchetti
• Andrea Raggi
• Francesca Ferraris
• Francesca Iacoponi

French Agency for Food, Environmental and Occupational Health & Safety (ANSES, France)

• Valerie Fessard
• Kevin Hogeveen
• Ludovic Le Hégarat
• Anne-Louise Blier

Sciensano (Belgio)

• Jan Mast
• Eveline Verleysen
• Lisa Siciliani

National Research Institute for Agriculture, Food and the Environment (INRAE)-University of Clermont Auvergne (UCA, Francia)

• Stephanie Blanquet-Diot
• Lucie Etienne-Mesmin
• Sylvain Denis
• Morgane Brun

European Commission Joint Research Centre (EC-JRC, Italia)

• Susanne Bremer-Hoffmann
• Francesco Sirio Fumagalli
• Alessia Bogni
• Deborah Stanco

Nanotechnologies are a vast research and application area with impact in all production sectors included, ever more, the food sector. Nanotechnologies are introducing dramatic changes by enabling management of characteristics such as materials’ constituting particle size and morphology for the improvement or development of new process and product properties. Although making materials smaller can generate novel and useful properties, concerns have been raised on potential risks related to the interactions of nanoparticles at the molecular or cellular levels, which may ultimately harm human health and the environment. Reducing the size of particulate materials to the nanoscale can impart certain changes in properties and biokinetics behaviour, which may also lead to altered toxicological effects compared with the corresponding conventional (non-nanomaterial) substance. However, approaching the safety assessment of products of nanotechnology is a challenge, since new concepts and tools are needed.

Nanocellulose (NC) is an emerging material in the food sector and comes in three main forms, i.e. bacterial NC (BNC), the nanofibrillated one (NFC) and the nanocrystals (CNC). Prospective application areas are food contact materials, food additives and Novel Foods. The biological sources and processing conditions has been shown to affect several physicochemical parameters of NC (e.g. size, aspect ratio, morphology, polydispersity, surface charge, surface chemistry and crystallinity index). The potential hazards of ingested NC are insufficiently characterized and NC nanoscale features require a nano-specific assessment, which in addition should cover the variability in NC physicochemical parameters.

According to the EFSA Guidance in risk assessment of nanomaterials (2021), a nano-specific assessment is needed with focus on:

• local adverse effect in the gut
• uptake in/crossing of the intestinal barrier
• possible degradation of NC by the human microbiome, potentially delivering smaller fibres

New Approach Methodologies (NAMs) are the best option to generate the needed information since:

• laboratory animals are not appropriate models because the digestive physiology, microbiome and rate of fibre degradation differ from humans
• the variability in the physicochemical properties of NC make NAMs ideal for efficient testing
• the needed studies are technically easier to implement using in vitro methods than in vivo studies

The project is based on the use of NAMs for addressing the current data gaps on hazards associated to NC ingestion. The duration of the project is 28 months (March 2021-July 2023).

A NAM-based IATA (Integrated Approach to Testing and Assessment) for addressing data gaps in the assessment of potential hazards associated to NC oral exposure was considered. The IATA focused on three main pillars, i.e.

• assessment of the uptake and potential crossing of the intestinal barrier by NC
• assessment of local effects including inflammation and genotoxicity, on the gastrointestinal epithelia
• 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 characterization. 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. Cell uptake of these materials was demonstrated and such uptake was greater in a triculture model, which better simulates the barrier properties of the human intestinal epithelium, 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, indicative for potential immunotoxicological effects, and barrier function impairment were observed, whereas no indications for genotoxicity were obtained. Finally, no formation of smaller particles following colonic fermentation of NC was observed. 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 was considered.