In vivo evaluation of activities and expression of antioxidant enzymes in Wistar rats exposed for 90 days to a modified clay.

Although clays are wildly used in a range of applications, the toxicity assessment of these new materials is still scarce. In the present study, oxidative stress induced by Clay 1, a novel clay, was determined in rats after 90 d of oral exposure. The activities of antioxidant enzymes, namely, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione S-transferase (GST), were examined. In addition, genetic expressions of SOD and CAT and relative protein abundance of CAT were also determined. Data showed that most of the biomarkers assayed remained unaltered. Only CAT activity, as well as its genetic and protein expressions, appeared enhanced in the kidney. Therefore, further studies are needed to clarify the relevance and consequences of these findings to ensure the safety of this clay.

In vivo toxicity evaluation of the migration extract of an organomodified clay-poly(lactic) acid nanocomposite.

The food packaging industry is in continuous development in order to obtain more secure and stable food and beverages. The incorporation of inorganic and organic materials with plastic polymers leads to polymer composites. Among the inorganic compounds, clays such as montmorillonite (MTT) and its derivatives are of great interest due to their advantageous properties. The Technological Institute of Packaging, Transport,and Logistics (ITENE) developed a novel nanocomposite based on a poly(lactic) acid (PLA) polymer using an MMT derivative, named Clay1, as filler, to be used in the beverage industry. The improvement of the technological properties of this new material was demonstrated, but safety issues are also of concern. In the present study, a histopathological examination by optical and electron microscopy of organs from Wistar rats exposed for 90 d to a migration extract of PLA-Clay1 nanocomposite was carried out. Moreover, different clinical biochemistry, inflammation,and oxidative stress biomarkers were determined. Results showed no apparent evidence of damage, indicating that this nanocomposite has a good profile to be used in the food packaging industry, although further research is still needed.

The influence of organic modification on the cytotoxicity of clay particles to keratinocytes, hepatocytes and macrophages; an investigation towards the safe use of polymer-clay nanocomposite packaging.

Organically modified clays can be used as nanofillers in polymer-clay nanocomposites to create bio-based packaging with improved strength and barrier properties. The impact of organic modification on the physico-chemical properties and toxicity of clays has yet to be fully investigated but is essential to ensure their safe use. Two organoclays, named N116_HDTA and N116_TMSA, were prepared using a commercially available sodium bentonite clay and the organic modifiers hexadecyl trimethyl ammonium bromide (HDTA) and octadecyl trimethyl ammonium chloride (TMSA). An in vitro hazard assessment was performed using HaCaT skin cells, C3A liver cells, and J774.1 macrophage-like cells. Organic modification with HDTA and TMSA increased the hazard potential of the organoclays in all cell models, as evidenced by the higher levels of cytotoxicity measured. N116_TMSA caused the greatest loss in viability with IC50 values of 3.2, 3.6 and 6.1 µg/cm2 calculated using J774.1, HaCaT and C3A cell lines, respectively. Cytotoxic effects were dictated by the amount of free or displaced organic modifier present in the exposure suspensions. The parent bentonite clay also caused distinct cytotoxic effects in J774.1 macrophage-like cells with associated TNF-α release. Such information on the hazard profile of organoclays, can feed into risk assessments for these materials.

Effects of two organomodified clays intended to food contact materials on the genomic instability and gene expression of hepatoma cells.

Globally, food industries have made significant progress in order to increase the shelf-life of food products and have fewer economic losses. In this sense, the use of organomodified clays destined to be incorporated in polymer matrices play a novel role, leading to improved materials named nanocomposites with enhanced technological profiles. Due to the presence of these clays into the package, the safety of the consumers is a main concern. Cloisite(®)30B and Clay1 are two organomodified clays containing quaternary ammonium salts as modifiers, that can be potentially used to reinforce packaging polymers. Available toxicity data about these clays, specifically genotoxicity, is still limited and inconclusive in some aspects. Thus, the purpose of this work was to evaluate both clays ability to induce genomic instability through the cytokinesis block micronucleus cytome assay (CBMN) and for the first time, their influence in the modulation of several genes involved in genotoxicity and cell death mechanisms. Overall, no genotoxicity response was obtained in any case at the conditions tested. On the other hand, significant expression changes were observed on the genes selected. Nevertheless, further studies are highly needed to elucidate and increase the knowledge about the molecular mechanisms of clays toxicity.

PLA coated paper containing active inorganic nanoparticles: Material characterization and fate of nanoparticles in the paper recycling process.

For paper and paperboard packaging, recyclability plays an important role in conserving the resources and reducing the environmental impacts. Therefore, when it comes to the nano-enabled paper packaging material, the recyclability issue should be properly addressed. This study represents our first report on the fate of nanomaterials in paper recycling process. The packaging material of concern is a PLA (Polylactic Acid) coated paper incorporating zinc oxide nanoparticles in the coating layer. The material was characterised and assessed in a lab-scale paper recycling line. The recyclability test was based on a method adapted from ATICELCA MC501-13, which enabled to recover over 99% of the solids material. The mass balance result indicates that 86-91% zinc oxide nanoparticles ended up in the rejected material stream, mostly embedded within the polymer coating; whereas 7-16% nanoparticles ended up in the accepted material stream. Besides, the tensile strength of the recycled handsheets suggests that the nano-enabled coating had no negative impacts on the recovered fibre quality.

Cytotoxicity and mutagenicity assessment of organomodified clays potentially used in food packaging.

Modern food packaging has made great advances as result of global trends and consumer preferences, which are oriented to obtain improved food quality and safety. In this regard, clay minerals, and mainly Montmorillonite (Mt) are attracting considerable interest in food packaging because of the improvements developed in mechanical and barrier properties. Hence, the present work aim to assess the toxicity of four Montmorillonite-based clay minerals, an unmodified clay, Cloisite®Na+ (CNa+), and three modified Mt clays: Cloisite®30B (C30B), a commercial clay, and Clay1 and Clay2, two novel modified organoclays developed by the Packaging, Transport, & Logistics Research Institute (ITENE). First, the cytotoxic effects were studied in the Human Umbilical Vein Endothelial Cells (HUVEC). In addition, the potential mutagenicity of the clays was evaluated by the Ames test. Clay1 did not induce any cytotoxic effects in HUVEC, although it exhibited potential mutagenicity in TA98 Salmonella typhimurium strain. In contrast, Clay2 produced cytotoxicity in endothelial cells but no mutagenicity was recorded. However, CNa+ was not cytotoxic neither mutagenic. And finally, C30B showed positive results in both assays. Therefore, results showed that clay minerals have a different toxicity profile and a case by case toxicity evaluation is required.

Cytotoxicity and mutagenicity studies on migration extracts from nanocomposites with potential use in food packaging.

Clays are used in the food packaging industry to obtain nanocomposites. The use of these new materials is a concern, because they could reach consumers by oral exposure through possible migration, and potential toxic effects could be derived. In the present study, several in vitro basal cytotoxicity and mutagenicity tests on migration extracts obtained from a nanocomposite material with poly (lactic) acid (PLA) and two modified clays, Clay1 and Clay2, are shown. Migration extracts in distilled water showed values of 0.1 ± 0.2mg/dm(2) in all samples. Also, the content of characteristic metals of the clays structure (Al, Ca, Mg, Fe, Si) was studied and no statistical differences were observed. For the cytotoxicity assays, the human intestinal Caco-2 and human liver HepG2 cells were selected. Cells were exposed to concentrations between 2.5% and 100% extracts determining three different biomarkers of cellular viability. No significant differences were observed in the cytotoxicity assays. Finally, mutagenicity was evaluated by the Ames test and resulted in the absence of mutagenic response at all the concentrations assayed. Taking in account all above mentioned, these new materials show a good profile for their use in food packaging although further research is still needed.