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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.

Layered silicates are largely used to develop polymer nanocomposites with improved physical properties compared to the original polymeric matrix. In this work, the silylation of sodium montmorillonite (Mt) was performed in distilled water using 3-aminopropyltriethoxysilane and vinyltrimethoxysilane, obtaining two different silane modified clay minerals named Clay3 and Clay4, respectively. Clay3 indicated a better profile with a notable increase in the interlayer space compared to Mt. For this reason, Clay3 was selected to scale up its production and to be incorporated in polypropylene (PP) matrix. The PP-Clay3 nanocomposite showed an enhancement in elasticity, a reduction of 15% in the oxygen transmission rate and important antimicrobial effects in comparison to pristine PP. Also, for the first time, cytotoxic effects in human cell lines exposed to Clay3 and Clay4 were evaluated. No cytotoxic damage was observed after Clay3 exposure but an important decrease in cell viability was produced by Clay4. The PP-Clay3 nanocomposite is an attractive alternative for the food packaging industry although further work is needed to get better results in the case of Clay4. Moreover, individual case by case evaluation of clay minerals under different conditions is required in order to obtain the most complete toxicity information.

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.

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.

The exploitation of polylactide (PLA) nanocomposites (which integrate organically modified clays (organoclays) into polymers) in packaging for the cosmetics industry could provide a biodegradable alternative to the use of conventional plastics. In this study nanocomposites with a PLA polymer matrix and clay fillers organically modified with the quaternary ammonium salts, hexadecyl trimethyl ammonium bromide (HDTA) or octadecyl trimethyl ammonium chloride (TMSA), were produced and tested for their safe use in cosmetic packaging. To address concerns over the potential release of constituents from such nanocomposites, levels of total overall migration in a range of simulants (e.g. vegetable oil, aqueous media, and cosmetic formulations) was assessed 10 days post incubation at 40 °C following EU-Plastics Regulation 10/2011 concerning materials and articles in contact with foodstuffs. Total overall migration levels calculated for all PLA nanocomposites tested (maximum of 0.88 ± 0.44 mg/dm2) were well below the total established legislative migration limit (10 mg/dm2). Toxicity of the nanocomposite migration extracts to the skin was assessed in vitro. Exposure of skin cells (HaCaT immortalized human keratinocytes) and a full thickness epidermal skin model (EpiDerm™) to migration extracts did not result in any significant loss in cell viability or skin irritation (OECD TG 439). The results therefore indicate that the levels of migration from the nanocomposite measured was low, and that the nanocomposite migration extracts stimulated minimal toxicity to the skin. Up until now, the hazard of migration extracts from polymer-organoclay nanocomposites following dermal exposure has not been investigated and thus our study addresses a gap in knowledge. These findings can inform the safe design of bio-based biodegradable nanocomposite packaging (used by the cosmetics, and other industries) in the future to promote a more sustainable and greener economy for the plastics industry.

Two organo-modified clays for food contact applications were developed to produce hydrophobically modified montmorillonite and hence to obtain better compatibility between the biopolymer and the filler (nanoclay). These nanofillers were characterised by Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction and thermogravimetric analysis (TGA) in order to study their composition, structure and thermal stability. The fillers were used to reinforce polylactic acid (PLA) bottles, which were characterised using different techniques such as mechanical and barrier properties, morphology and thermal stability. The results were compared with conventional PLA bottles. The use of the modified clay in PLA bottles was found to lead to an improvement in mechanical and barrier properties. Finally, cytotoxicity tests were carried out with the organo-modified clays using Caco-2 and HepG2 cell lines, with uptake of neutral red as a basal cytotoxicity biomarker.