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.

In vitro toxicity evaluation of new silane-modified clays and the migration extract from a derived polymer-clay nanocomposite intended to food packaging applications.

The clay montmorillonite (Mt) is among the nanofillers more frequently used for food packaging applications. The organomodification of clays with different modifiers, such as silanes, is an important step in the preparation of improved polymer/clay materials known as nanocomposites. However, the toxicological data about these nanofillers is still scarce. In the present study, an in vitro toxicological evaluation in Caco-2 cells of two silane-modified clays based on Mt, Clay3 and Clay4 (0-250µg/ml), was performed. The cytotoxicity, cell death, genotoxicity and oxidative stress produced by both organoclays were evaluated after 24 and 48h of exposure. Moreover, the migration extracts obtained from nanocomposites of polypropylene (PP) + Clay3 and only PP were also investigated. Only Clay4 induced cytotoxicity, showing a reduction of cell viability to 63% of the control, as well as oxidative stress in a concentration-dependent manner. Regarding the PP-Clay3 migration extract, no cytotoxic effects were observed after exposure to the tested concentrations (0-100%). Moreover, significant differences in the presence of Ca, Mg and Si compared to the PP extract were obtained, although migration levels were in accordance with the food contact materials regulation. These findings indicate that a case-by-case toxicological assessment of organoclays should be performed.

Induction of micronuclei and alteration of gene expression by an organomodified clay in HepG2 cells.

Clay2 is an organomodified montmorillonite developed by the Technological Institute of Packaging, Transport and Logistic (ITENE) in order to improve polymeric materials used in food packaging. There is not much known on Clay2 toxic potential, particularly at DNA level, therefore it is mandatory to assess its toxicity prior to its commercialization. In the present study the human hepatoma cell line (HepG2) was exposed to non-cytotoxic concentrations of Clay2 and the genomic stability was studied with the Cytokinesis block micronucleus cytome assay, by determining the formation of micronuclei (MN), nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs). Moreover, the expression of various genes involved in the mechanisms of its action using the real-time quantitative PCR was studied. The results obtained provide the evidence that Clay2 is potentially genotoxic as it increased the frequency of micronuclei. In addition it deregulated genes involved in the metabolism, immediate-early response/signaling, DNA damage and oxidative stress showing new valuable information on the cellular response to Clay2. Nonetheless, further studies are highly needed to elucidate the molecular mechanisms of clays toxicity.