Understanding the Presence and Impact of Silver Nanoparticles in the Food Additive E174

Abstract

E174 (silver) is an EU-approved food additive used in confectionery coatings, chocolate decorations, and liquors, regulated under quantum satis conditions. Despite its authorization, the European Food Safety Authority (EFSA) raised concerns in its 2016 evaluation regarding insufficient data on particle size distribution, undisclosed particle quantities, and the lack of relevant toxicity studies on the material used. This research addresses these gaps by investigating the nanoparticle (NP) fraction of E174 and its potential implications for human health. Nanoparticles (1–100 nm) exhibit unique physicochemical properties compared to bulk materials due to their high surface area, surface energy, and quantum effects, which may alter their toxicological behavior. Silver nanoparticles (Ag NPs) are of particular concern as their toxicity largely depends on ionic silver release, influenced by their physicochemical characteristics. This study characterizes Ag NPs in E174-containing products from the Belgian and broader European markets, assessing their potential hazard using an in vitro human gut cell model. Transmission electron microscopy (TEM) and single-particle inductively coupled plasma-mass spectrometry (spICP-MS) revealed that E174-derived Ag NPs are readily released upon water elution. The NPs exhibited spheroidal and irregular morphologies, averaging 11 ± 4 nm in food additives and 18 ± 7 nm in products. Across all samples, over 97% of the particles were nano-sized, despite the bulk of the silver being in the form of flakes. Silver NPs occurring in all twenty applications showed a crystalline phase and similar surface topology, as verified by electron diffraction and TEM. These particles were composed solely of silver, with no other elements co-localising with the Ag X-ray specific signal, as demonstrated by scanning electron microscopy (SEM)-energy dispersive X-ray spectroscopy (EDX) and scanning transmission electron microscopy (STEM)-EDX. The observations that a higher amount of released NPs was observed in the products than in the sheet-like food additives and that a higher release coincided with a rougher surface of the larger particles, as seen in certain products, suggests that the NP release can be linked to different production processes of E174. Method validation demonstrated a total measurement uncertainty of 16% (spICP-MS) and 18% (TEM), crucial for reliable risk assessment. The fate of E174-derived Ag NPs during digestion is simulated in an exploratory study, revealing that Ag NPs remain stable in saliva but may undergo dissolution under gastric conditions (pH 2.5), with proteins that may partially mitigate dissolution. Intestinal conditions suggested near-complete dissolution, although in one exceptional case, compact Ag NPs were observed in a sample including proteins, suggesting partial protection by protein coronas. Further analysis using scanning transmission electron microscopy (STEM-EDX) demonstrated intracellular Ag NP uptake by Caco-2 cells, with particles undergoing physicochemical transformations, including sulphur co-localization, suggesting NP sulfidation. Cytotoxicity emerged at concentrations ≥32.2 µg/mL, with higher doses (128.7 µg/mL) inducing mitochondrial alterations, oxidative stress, and HMOX1 gene activation. These findings highlight the need for a revised safety assessment of E174, emphasizing the importance of linking NP properties to biological effects for robust regulatory decisions.