On February 19, 2014 the peer-reviewed journal Food Additives & Contaminants: Part A published the article “Use of nanoclay platelets in food packaging materials: technical and cytotoxicity approach” investigating physical properties of nanoclay reinforced polyactic acid (PLA) plastic bottles and assessing the toxicity of nanoclay platelets in vitro. The bioplastic PLA is based on renewable sources and is often deemed the most promising biodegradable and compostable plastic material. It is used in food contact materials (FCM) like bottles and trays due to its favorable mechanical, thermal and chemical properties. The addition of modified nanoclay platelets enhances properties like strength, heat resistance, gas and water permeability, and flammability. The authors of the study, Jorda-Beneyto from the Packaging, Transport and Logistics Research Institute in Valencia, Spain, and colleagues from two further European institutes, compared physical properties of native and two types of nanoclay reinforced PLA bottles. The toxicological assessment was performed using a neutral red (NR) uptake assay in the two human cell lines Caco-2 (colon cells) and HepG2 (liver cell). The NR assay measures cell viability through the cells’ potential to take up color pigments. A reduced color uptake indicates fatal damages to the cell’s membrane, and hence permits measuring the toxicological endpoint of “cytotoxicity”.

The nanoclay platelets are so-called organo-modified layered silicates (OMLS), where the cations in the clay layer are exchanged for organic cations like alkylammoniums or alkylphosphoniums, making them more hydrophobic and more strongly absorbed in the polymer matrix. The two organo-modified clays were added at 4% w/w: CLAY1 contained hexadecyltrimethyl-ammonium bromide (HDTA; CAS 200-311-3) and CLAY2 contained an HDTA and acetylcholine (ACO; CAS 200-128-9) chloride mixture.

The CLAY1-PLA samples showed best elastic properties in the compression test. The thermal treatment test revealed that only native PLA bottles showed structural deformation at 60 °C, whereas the reinforced bottles kept their shape. While increased clay concentration decreases transparency, it also reduces water transmission rates (WVTR) and oxygen permeability (PO2). A reduction of up to 40 % was achieved when CLAY1 was used compared to conventional PLA bottles. Jorda-Beneyto and colleagues argue that CLAY1-PLA displays better properties when compared to CLAY2-PLA due to different and more suitable polarity of CLAY1 leading to a better interaction with the PLA polymer matrix. Structural analysis by transmission electron microscopy (TEM) revealed the presence of three coexisting structural size classes: < 20 nm, < 100 nm and > 100 nm. The researchers state that further improvements will yield better transparency properties by increasing the percentage of smaller particles.

For the cytotoxic test, both cell lines were exposed to the two types of nanoclay platelets in two time settings of 24 h and 48 h, and at various concentrations. CLAY1 exposed Caco-2 cells did not display any alteration in the NR uptake assay after both exposure times. HepG2 cells exposed to the same type showed significant reductions for the highest concentration of 8 μg ml–1 already after 24 h of exposure compared to the control group. The reduction did however not allow for the calculation of an EC50value. CLAY2 exposed Caco-2 cells showed a significant concentration dependent reduction for concentrations of 40 μg ml–1 and 20 μg ml–1 for the 24 h and 48 h setting, respectively. The EC50 levels obtained were 51.8 μg ml–1 for 24 h and 27.8 μg ml–1 for the 48 h setting. HepG2 cells exposed to CLAY2 showed a significant decrease in number starting from 62.5 μg ml–1 for both time settings. The corresponding EC50 values were 109.1 μg ml–1 (24 h) and 60.3 μg ml–1 (48 h). Jorda-Beneyto and colleagues argue that CLAY2 probably displays higher cytotoxicity due to a different composition of the above described organo-modifiers. Comparing EC50 values of CLAY2 exposure for the two cell lines indicates that the Caco-2 cell line is more sensitive than HepG2, with EC50 values twofold lower. Both cell lines also have different origins and display different metabolic characteristics facing toxic injuries. The scientists conclude that further studies have to clarify the potential risk of nanoclay platelets in FCMs for human health, in particular the risk through oral exposure. Of interest is also how the toxicity is influenced by exchanging the organic cations for other organic cations, in order to estimate the toxicities arising from the nanoclay platelets themselves.


Jorda-Beneyto et al. (February 19, 2014). “Use of nanoclay platelets in food packaging materials: technical and cytotoxicity approach”. Food Additives & Contaminants: Part A.