Hazard identification of pyrogenic synthetic amorphous silica (NM-203) after sub-chronic oral exposure in rat: A multitarget approach
Graphical abstract
Introduction
Synthetic amorphous silica (SAS) has been used as food additive for decades under the name of “Silicon dioxide” or “E551” and in the European Union is authorized under Regulation (EC) No 1333/2008. E551 is a material comprised of nanosized (<100 nm) primary particles which can variably agglomerate and aggregate depending on the conditions of production and use (van Kesteren et al., 2015). According to the EU specifications, the forms of SAS used as food additive include fumed (pyrogenic) silica and hydrated silica (precipitated silica, silica gel and hydrous silica) depending on their manufacture process (thermal or wet) (Fruijtier-Polloth, 2012). In two studies, foodstuffs containing SAS have been shown to contain silica in the 5–200 nm size range in quantities up to 43% and 80% of the total silica content, respectively (Peters et al., 2012; Dekkers et al., 2011); another study detected 11% of the total silicon present in a coffee creamer as silica particles in the size range 1–100 nm (Heroult et al., 2014).
In the recent re-evaluation of E551, the former EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS) noted limitations in the sub-chronic, reproductive and developmental toxicological studies and, because of the limitations in the available database, it did not confirm the ‘not specified’ acceptable daily intake status of the food additive (EFSA, 2018a). The Panel also highlighted that the EU specifications are insufficient to adequately characterize the food additive E551 and that a clear characterization of the particle size distribution is required (EFSA, 2018a). Taking into account the nanoparticulate nature of the material and the potential for accumulation in tissues following daily consumption, a nanospecific risk assessment of E551 highlighted that more insight in the health risk of SAS in food is warranted (van Kesteren et al., 2015). Indeed, one important feature of the nanosized portion of SAS is the increased surface area, which affects reactivity and may result in increased ability to translocate across biological barriers (EFSA, 2018b). The absorption, distribution, metabolism and excretion parameters of materials comprising nanosized particles are influenced by the size, shape, solubility, surface charge and surface reactivity of these particles and may deviate substantially from the corresponding conventional chemicals (EFSA, 2018b). In fact, the primary particles present in E551 may partly bind to form agglomerates in the food matrix, but after in vitro gastrointestinal digestion, the gut epithelium appears to be predominantly exposed to nanosized silica (Peters et al., 2012). Notwithstanding the low gastrointestinal absorption, SAS is a biopersistent material prone to bioaccumulation upon long-term exposure (van Kesteren et al., 2015). Earlier studies gave evidence that the gastrointestinal absorption decreases with increasing dose, leading to the recommendation of studying dose-effect relationships in future investigations at lower dose levels, more representative of the current consumers' exposure (van der Zande et al., 2014).
The NANoREG project aimed to provide legislators with decision-making tools by gathering data and performing pilot risk assessment of a selected number of nanomaterials (NMs) used in consumer products, as well as to develop long-term new testing strategies adapted to a high number of NMs relevant for their environmental and health impact (http://www.nanoreg.eu/). Within this project, the aim of the present study was to apply an integrative approach for sub-chronic toxicity testing of NMs by oral administration, as recommended by EFSA (EFSA, 2011 now superseded by EFSA, 2018b), in order to provide suitable data for NMs hazard assessment. NM-203 - a reference, well-characterized pyrogenic SAS NM complying with the E551 specifications (Rasmussen et al., 2013) - was used as test material in a 90-day oral toxicity study on the basis of the OECD test guideline (TG) 408 (OECD, 1998). NM-203 was selected since it was studied and characterized in the frame of the Nanogenotox joint action, where toxicokinetics has been evaluated by intravenous and oral administrations on male and female Sprague-Dawley rats (De Jong et al., 2013); moreover, NM-203 was short-listed in the NANoREG project for its relevance in food safety (Fruijtier-Polloth, 2016).
This paper describes the overall approach of the study which involved a large network of expertise to deal with a broad range of endpoints beyond those listed in the OECD TG 408. The results presented in this paper include characterization of NM-203 dispersions, general toxicity, tissue distribution, biomarkers of liver and kidney toxicity, thyroid hormones, blood count and immunotoxicity endpoints, and histopathological analysis of target tissues, namely liver, kidney, thyroid, gastrointestinal tract, spleen, and adrenals. Other results concerning reproductive systems and genotoxicity will be described separately.
Section snippets
Characterization of NM-203
The pyrogenic SAS material NM-203, one of the NANoREG project core materials, was obtained from the NM repository of the European Commission Joint Research Centre, Institute for Health and Consumer Protection (JRC-IHCP, Ispra, Italy). This material has been comprehensively characterized (Rasmussen et al., 2013) and the main physicochemical parameters are summarized in Table 1.
Characterization of SAS NM dispersion
The characterization work focused on the comparison of the dispersion quality obtained with a protocol developed for the
Characterization of SAS NM dispersion
Size descriptors of NM-203 at 2.56 mg/ml in 0.05% w/v BSA (Jensen et al., 2011) and at 5 mg/ml in water obtained by AF4-MALS-UV are summarized in Table 2. Fractionation gave similar results in terms of averaged size and showed in both cases two major populations of secondary particles (agglomerates/aggregates). Intra and inter-day variability were assessed and resulted <10%. Analytical results for the reference material ERM FD-100 colloidal silica in water were in good agreement with the
Discussion and conclusions
The aim of this study was to identify potential hazards of a pyrogenic silicon dioxide used as food additive (E551). The dose levels, in the range of 2–50 mg/kg bw per day, have been chosen as close as possible to the range of expected human exposure and were substantially lower than those investigated in other toxicological studies with SAS, including sub-chronic, prenatal and two-generation oral studies (van der Zande et al., 2014; Hofmann et al., 2015; Wolterbeek et al., 2015). This choice
Author contributions section
Roberta Tassinari: Conceptualization, Methodology, Investigation, Data curation, Writing - Original Draft, Writing - Review & Editing, Visualization.
Gabriella Di Felice: Validation, Data curation, Writing - Original Draft, Writing - Review & Editing.
Cinzia Butteroni: Investigation, Data curation, Writing - Original Draft, Writing - Review & Editing.
Bianca Barletta: Investigation, Data curation, Writing - Original Draft, Writing - Review & Editing.
Silvia Corinti: Investigation, Data curation,
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
The study was financially supported by the European Union FP7 Project NANoREG (grant 310584).
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2022, Mutation Research - Genetic Toxicology and Environmental MutagenesisCitation Excerpt :The primary particle size was 13–45 nm with over 78% < 100 nm and specific surface area = 203 m2/g. As described in Tassinari et al. [26], particle suspensions were prepared at a concentration of 5 mg/ml in MilliQ water by probe sonication on ice for 20 min at 40% amplitude, using a Sonopuls ultrasonic Homogenizer HD3200 series (Bandelin electronic GmbH, Berlin, Germany) equipped with a SH 213 G booster horn and a sterile KE 76 tapered tip. This dispersion was used as such for administration of 50 mg/kg bw or diluted for the administration of the lower doses.
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2021, NanoImpactCitation Excerpt :Similar to our findings, several studies reported that administration of SiNPs of initial particle size ranging between 1 and 15 nm is relatively toxic and able to alter cognitive and memory function in rats (You et al., 2018) and in adult zebrafish (Li et al., 2020). In line with these findings, SiNPs were shown to be able to penetrate through the blood-brain barrier (BBB) and to accumulate in the brain after intravenous injection (Shim et al., 2014; Aureli et al., 2020; Tassinari et al., 2020) or intraperitoneal administration in mice (Kim et al., 2006). In our study, the Si concentrations in the brain of both treated groups (25 and 100 mg/kg) were increased by 3.3 and 7.7-fold respectively, compared to controls, confirming its brain accumulation.
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2021, Reproductive ToxicologyCitation Excerpt :Moreover, EFSA stated that, although SiO2 appears to be poorly absorbed, silicon-containing material was found in tissues [11]. Biodistribution data recorded in Tassinari et al. (2020) [10] showed that the very limited increase in the silicon content present in peripheral tissues was due to the low gastrointestinal absorption of SAS (in the range <0.01 %-0.2 %) but accompanied by a steady state levels as balance between uptake and elimination. Moreover, single oral administration of SiO2 spherical NPs (15 nm and 89 nm) at higher dose levels (500 and 1000 mg/kg bw) in rats did not increase Si levels in reproductive organs [36].
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2020, Kidney International Reports