Sara Maisanaba

In vitro toxicological evaluation of essential oils and their main compounds used in active food packaging: A review

Essential oils (EOs) and their main constituent compounds have been extensively investigated due to their application in the food industry for improving the shelf life of perishable products. Although they are still not available for use in food packaging in the market in Europe, considerable research in this field has been carried out recently. The safety of these EOs should be guaranteed before being commercialized. The aim of this work was to review the scientific publications, with a primary focus on the last 10 years, with respect to different in vitro toxicological aspects, mainly focussed on mutagenicity/genotoxicity. In general, fewer genotoxic studies have been reported on EOs in comparison to their main components, and most of them did not show mutagenic activity. However, more studies are needed in this field since the guidelines of the European Food Safety Authority have not always been followed accurately. The mutagenic/genotoxic activities of these substances have been related to metabolic activation. Therefore, in vivo tests are required to confirm the absence of genotoxic effects. Considering the great variability of the EOs and their main compounds, a case-by-case evaluation is needed to assure their safe use in food packaging.

In vitro toxicological assessment of clays for their use in food packaging applications

Montmorillonite based clays have a wide range of applications that are going to contribute to increase human exposure to these materials. One of the most promising uses of clays is the development of reinforced food contact materials that results in nanocomposites with improved barrier properties. Different organoclays have been developed introducing modifiers in the natural clay which is commercially available. However, the toxicological aspects of these materials have been scarcely studied so far. In the present study, the cytotoxic effects of a non-modified clay (Cloisite Na+) and an organoclay (Cloisite 30B) have been investigated in the hepatic cell line HepG2. Only Cloisite 30B showed cytotoxicity. In order to elucidate the toxic mechanisms underlying these effects, apoptosis, inflammation, oxidative stress and genotoxicity biomarkers were assayed. Moreover, a morphology study with light and electron microscopy was performed. Results showed genotoxic effects and glutathione decrease. The most relevant ultraestructural alterations observed were mitochondrial degeneration, dilated endomembrane systems, heterophagosomes formation, fat droplets appearance and presence of nuclear lipid inclusions. Cloisite 30B, therefore, induces toxic effects in HepG2 cells. Further research is needed to assess the risk of this clay on the human health.

Toxic effects of a modified montmorillonite clay on the human intestinal cell line Caco-2

The incorporation of the natural mineral clay montmorillonite into polymeric systems enhances their barrier properties as well as their thermal and mechanical resistance, making them suitable for a wide range of industrial applications, e.g., in the food industry. Considering humans could easily be exposed to these clays due to migration into food, toxicological and health effects of clay exposure should be studied. In the present work, the cytotoxic effects induced by two different clays (the unmodified clay Cloisite® Na+, and the organically modified Cloisite® 30B) on Caco‐2 cells were studied after 24 and 48 h of exposure. The basal cytotoxicity endpoints assessed were total protein content, neutral red uptake and a tetrazolium salt reduction. Our results showed that only Cloisite® 30B induced toxic effects. Therefore, the effects of subcytotoxic concentrations of this clay on the generation of intracellular reactive oxygen species, glutathione content and DNA damage (comet assay) were investigated. Results indicate that oxidative stress may be implicated in the toxicity induced by Closite® 30B, in regards of the increases in intracellular reactive oxygen species production and glutathione content at the highest concentration assayed, while no damage was observed in DNA. The most remarkable morphological alterations observed were dilated cisternae edge in the Golgi apparatus and nucleolar segregation, suggesting impairment in the secretory functions, which could be related to inhibition in the synthesis of proteins. Copyright

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.

Use of nanoclay platelets in food packaging materials: technical and cytotoxicity approach

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.

Evaluation of the mutagenicity and genotoxic potential of carvacrol and thymol using the Ames Salmonella test and alkaline, Endo III- and FPG-modified comet assays with the human cell line Caco-2

Currently, direct antimicrobial and antioxidant additives derived from essential oils are used in food packaging and are perceived by consumers as low-health-risk compounds. In this study, we investigated the potential mutagenicity and genotoxicity of carvacrol and thymol, major compounds in several essential oils, using the Ames Salmonella test and the alkaline, Endo III- and formamidopyrimidine glycosylase (FPG)-modified comet assays, respectively. Thymol did not show any mutagenic activity at any concentration assayed (0-250 μM), whereas carvacrol exhibited mutagenic potential, displaying greater activity in presence of the metabolic fraction (29-460 μM). The genotoxic effects were evaluated in the human colon carcinoma cell line Caco-2, and the standard comet assay revealed that neither carvacrol (0-460 μM) nor thymol (0-250 μM) had any affects at 24 and 48 h. The FPG-modified comet assay showed that the highest concentration of carvacrol (460 μM) caused DNA damage, indicating damage to the purine bases. These results should be used to identify the appropriate concentrations of carvacrol and thymol as additives in food packaging. Moreover, further studies are necessary to explore the safety and/or the toxicity mechanisms of these compounds.

New advances in active packaging incorporated with essential oils or their main components for food preservation

ABSTRACT The food industry is developing new packaging systems (active packaging) through the incorporation of essential oils or their main compounds into the films. This could confer them the antimicrobial and/or antioxidant properties already extensively described for these substances, improving the shelf life of perishable food products. However, a safe range of concentrations for their use in active food packaging should be established to reach the market proposals and avoid risks for consumers. The present work reviews the scientific literature concerning these two properties together with the toxic effects induced by these substances on human cell lines.

In vitro genotoxicity testing of carvacrol and thymol using the micronucleus and mouse lymphoma assays

Currently, antimicrobial additives derived from essential oils (Eos) extracted from plants or spices, such as Origanum vulgare, are used in food packaging. Thymol and carvacrol, the major EO compounds of O. vulgare, have demonstrated their potential use as active additives. These new applications use high concentrations, thereby increasing the concern regarding their toxicological profile and especially their genotoxic risk. The aim of this work was to investigate the potential in vitro genotoxicity of thymol (0-250 μM) and carvacrol (0-2500 μM) at equivalent doses to those used in food packaging. The micronucleus (MN) test and the mouse lymphoma (MLA) assay on L5178Y/Tk(±) mouse lymphoma cells were used. The negative results for thymol with the MN with and without the S9 fraction and also with the MLA assay reinforce the view that this compound is not genotoxic in mammalian cells. However, carvacrol presented slight genotoxic effects, but only in the MN test at the highest concentration assayed (700 μM) and in the absence of metabolic activation. The lack of genotoxic response in the MLA assay after 4 and 24h of exposure indicates a low genotoxic potential for carvacrol. Alternatively, the general negative findings observed in both assays suggest that the MN results of carvacrol are marginal data without biological relevance. These results can be useful to identify the appropriate concentrations of these substances to be used as additives in food packaging.

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.

Cytotoxic and mutagenic in vitro assessment of two organosulfur compounds derived from onion to be used in the food industry.

Edible members of the Allium family are widely used since they exhibit antioxidant and antibacterial related to the organosulphur compounds. One the most promising use of Allium species, hence, onion essential oil, could be in the packaging food industry. The present work aims to assess the safety of two organosulphur compounds present in onion essential oil; dipropyl disulphide, dipropyl sulphide and their mixture. For this purpose, cytotoxicity, reactive oxygen species and glutathione contents, and ultrastructural cellular damages were studied in the human intestinal cells, Caco-2, exposed to these organosulphur compounds. Moreover, their potential mutagenicity was also assessed. The results revealed no significant adverse effects. Additionally, reactive oxygen species scavenger activity was observed for both compounds. Therefore, they could be a good natural alternative to other synthetic antioxidant and antibacterial substances used in the food industry.