Hazard identification of pyrogenic synthetic amorphous silica (NM-203) after sub-chronic oral exposure in rat: A multitarget approach

https://doi.org/10.1016/j.fct.2020.111168Get rights and content

Highlights

  • Food additive E551 is a nanomaterial consisting in synthetic amorphous silica.

  • Health risks associated to E551 are not fully assessed yet.

  • 90-day oral toxicity study with pyrogenic synthetic amorphous silica is performed.

  • Rats are treated with realistic dose levels: 2, 5, 10, 20, 50 mg/kg bw per day.

  • NOAEL 5 mg/kg bw per day in male rats, LOAEL 2 mg/kg bw per day in female rats.

Abstract

Food additive E551 consists of synthetic amorphous silica (SAS), comprising agglomerates and aggregates of primary particles in the nanorange (<100 nm), which potential nanospecific risks for humans associated to dietary exposure are not yet completely assessed. In NANoREG project, aim of the study was to identify potential hazards of pyrogenic SAS nanomaterial NM-203 by a 90-day oral toxicity study (OECD test guideline 408). Adult Sprague-Dawley rats of both sexes were orally treated with 0, 2, 5, 10, 20 and 50 mg SAS/kg bw per day; dose levels were selected to be as close as possible to E551 dietary exposure. Several endpoints were investigated, the whole integrative study is presented here along with the results of dispersion characterization, tissue distribution, general toxicity, blood/serum biomarkers, histopathological and immunotoxicity endpoints. No mortality, general toxicity and limited deposition in target tissues were observed. NM-203 affected liver and spleen in both sexes. Proposed NOAEL 5 mg/kg bw per day in male rats for enlarged sinusoids in liver. In female rats, TSH and creatinine levels were affected, proposed LOAEL 2 mg/kg bw per day. Overall, these data provide new insight for a comprehensive risk assessment of SAS exposure by the oral route.

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).

References (41)

  • F. Babick et al.

    Characterization of pyrogenic powders with conventional particle sizing technique: II. Experimental Data

    Part. Part. Syst. Char.

    (2012)
  • L. Chen et al.

    The toxicity of silica nanoparticles to the immune system

    Nanomedicine

    (2018)
  • H.-. Chiu et al.

    Using analytical centrifugation to characterize the dispersibility and particle size distributions of organic/inorganic composite coatings

    J. Polym. Res.

    (2011)
  • W.H. De Jong et al.

    NANOGENOTOX, Deliverable 7: Identification of Target Organs and Biodistribution Including ADME Parameters, Final Report

    (2013)
  • P.J. De Temmerman et al.

    Quantitative characterization of agglomerates and aggregates of pyrogenic and precipitated amorphous silica nanomaterials by transmission electron microscopy

    J. Nanobiotechnol.

    (2012)
  • S. Dekkers et al.

    Presence and risks of nanosilica in food products

    Nanotoxicology

    (2011)
  • European Food Safety Authority. Re-evaluation of silicon dioxide (E 551) as a food additive

    EFSA J.

    (2018)
  • European Food Safety Authority.Guidance on risk assessment of the application of nanoscience and nanotechnologies in the food and feed chain: Part 1, human and animal health

    EFSA J.

    (2018)
  • European Food Safety Authority. Guidance on the risk assessment of the application of nanoscience and nanotechnologies in the food and feed chai

    EFSA J.

    (2011)
  • C. Fruijtier-Polloth

    The safety of nanostructured synthetic amorphous silica (SAS) as a food additive (E 551)

    Arch. Toxicol.

    (2016)
  • Cited by (16)

    • Adverse effects and underlying mechanism of amorphous silica nanoparticles in liver

      2023, Chemosphere
      Citation Excerpt :

      Adult SD rats were given 13–45 nm amorphous SiNPs via gavage at 2, 5, 10, 20 and 50 mg/kg bw per day. It was observed that dose-related hepatic lobular lymphatic infiltration, enlarged sinoid sinuses, sinoid hyperemia, focal intraclobular necrosis and hepatic cell vacuolation/steatosis (Tassinari et al., 2020). C57BL/6 or C57BL/6S mice exposed to 30 mg/L amorphous SiNPs (20 nm) in water for 18 months developed liver inflammation and accumulation of amyloid protein around liver blood vessels (Boudard et al., 2019).

    • Pyrogenic synthetic amorphous silica (NM-203): Genotoxicity in rats following sub-chronic oral exposure

      2022, Mutation Research - Genetic Toxicology and Environmental Mutagenesis
      Citation 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.

    • Subacute silica nanoparticle exposure induced oxidative stress and inflammation in rat hippocampus combined with disruption of cholinergic system and behavioral functions

      2021, NanoImpact
      Citation 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.

    • Effects of sub-chronic oral exposure to pyrogenic synthetic amorphous silica (NM-203) in male and female Sprague-Dawley rats: focus on reproductive systems

      2021, Reproductive Toxicology
      Citation 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].

    • The Authors Reply

      2020, Kidney International Reports
    View all citing articles on Scopus
    View full text