1. Chemistry

Engineered nanomaterials (NMs) are intentionally manufactured materials, with one or more external dimensions of less than 100 nm [1-3]. One distinguishes two large groups, nanostructured materials and nanoparticles. Nanostructured materials are solids composed of structural elements, such as crystallites. The mobility of nano-sized elements in these materials is constrained by their firm inclusion in the larger structure. Nanoparticles, on the other hand, are individual particles in the nanometer range, which may be applied to surfaces or integrated into other bulk materials, for example polymers. They can be freely released into the environment and pose a potential risk to human health [2]. While NMs are often named according to their primary material, like nanosilver, they may widely differ in size, surface structure and attached functional groups [2].These characteristics influence function, exposure as well as toxicity.

2. Applications and market data

NMs may be manufactured via a ‘top-down’ or a ‘bottom-up’ approach. In the ‘top-down’ approach larger particles are broken down into nano-sized material. Processes include milling and homogenization. In the ‘bottom-up’ approach more complex molecular nanostructures may be assembled from smaller compounds and manufacturing processes include self-assembly, crystallization, layer-by-layer deposition, solvent extraction/evaporation and biomass reactions [3].

Due to the large variety of their properties, NMs have found a correspondingly wide array of applications in consumer products. They enhance material functionalities such as improved durability, flexibility, temperature and flame resistance, barrier properties, optical and recycling properties [1-3]. They have been applied in medicine, cosmetics, agriculture and food processing, including food packaging. Within the food packaging sector, NMs have been primarily used as antimicrobials and to improve barrier function. Both applications aim to extend the shelf life of packaged food products. Barrier function improvements are obtained by including nanoparticles in a polymer matrix, which slows down the diffusion of gases into the food [3, 4]. NMs may also be used to create intelligent packaging, which can alert the consumer to the presence of microbes, fungi, chemical contaminants or gases indicating spoilage [3]. NMs have been incorporated into polymeric packaging materials such as polyamides (PA), nylons, polyolefins, ethylene-vinylacetate copolymer, polystyrene (PS), epoxy resins, polyurethane, polyvinyl chloride (PVC) and polyethylene terephthalate (PET) [3, 5]. Metal and metal oxide NMs (silver, gold, zinc oxide, silica, titanium dioxide, alumina and iron oxides), carbon based NMs and nano-sized polymers are most commonly used. Nanoparticles differ widely in size and other characteristics. Thus, the migration potential of NMs from food contact materials into food cannot be easily predicted. Even within one batch of nanoparticles, size may differ significantly, and the primary components of the nanoparticles may still remain in the mixture as impurities [6].

Table 1. A selection of the most important nanomaterials used in Food Contact Materials (FCMs) including their application and regulation

Nanomaterial Application Used in Authorized
Titanium nitride Improvement of thermal properties [7], antimicrobial and deodorant agent [8],UV filter [9] PET, fridges [8] EC 10/2011 (ND,  PET)
Carbon black Additive [10] Rubber, Silicones, printing inks EC 10/2011 (2,5% w/w in polymer, 10-300 nm); U.S. FCS
Silicon dioxide Anti-slip agent Printing inks, paper & board, rubbers, silicones EC 10/2011 (1-100 nm)
Aluminum Filler in polymers, scratch- and abrasion-resistance in coatings [10], improvement of barrier properties, UV-filter [8] Not authorized in the EU, listed as GRAS by the U.S. FDA
Silver Antimicrobial [11], anti-biotic, antistatic agent Reusable food containers[8] Not authorized in the EU, no conclusive information available for the U.S.
Nanoclay (bentonite) Improvement of barrier properties[12] PE, PET, PP, PS, TPO and nylon [8, 13] Not authorized in the EU, listed as GRAS by the U.S. FDA
Zinc oxide UV filter, antimicrobial and fungistatic agent [14], deodorant [8] Plastic glasses, plastic films Not authorized in the EU, listed as GRAS by the U.S. FDA

Abbreviations
FCS = Food Contact Substance Notifications, GRAS = Generally Recognized As Safe, ND=not detectable in food PE= polyethylene, PET= polyethylene terephthalate, PP= polypropylene, PS= polystyrene, TPO= thermoplastic polyolefin

The nano-enabled food and beverage packaging market has grown significantly over the past 20 years. It is difficult to estimate the exact volume of the nano-enabled food and beverage packaging market. The table below is therefore only a selection of available estimates. Principia Markets has estimated the nano-enabled food and beverage packaging market volume more cautiously, with GBP 1 billion in 2010.

Table 2. Volume of nano-enabled food and beverage packaging market

Year Market Volume
(in billion USD)
2002 0.15
2004 0.86
2008 4.13 [13]
2009 4.21 [13]
2014 Prognosis >7 [13]

According to Innovative Research and Products (iRAP), active technology has the largest market share with USD 4.35 $billion market volume in 2014 [13]. The majority of the market is in the US, while Europe accounts for around 30% of the global market share [13].

3. Analytical techniques to detect NMs

NMs may be characterized using a large variety of methods, depending on which properties of the NM shall be characterized. Electron microscopy, X-ray microscopy, scanning electron microscopy, field-flow fractionation, chromatography, light scattering, Raman spectroscopy and mass spectrometry have all been used to analyze NMs [1]. A complete list of methods for characterization of different properties may be found in the Organisation for Economic Co-operation and Development (OECD) report ENV/JM/MONO(2012)40 of 2012. It is often necessary to supplement various identification techniques with one another to describe nanoparticles accurately. However, a major obstacle to the generation of comparable and reproducible results remains the lack of analytical standards. In 2012, the OECD indicated that OECD certified analytical standards existed only for gold, titanium dioxide, single wall carbon nanotubules and polystyrene (ENV/JM/MONO(2012)40) [6]. The issue is further complicated by a lack of consistent methodologies for measuring and identifying their specific properties including mass, shape and surface properties of NMs and the large variability between different batches of industrially produced NMs [6]. As it remains difficult to accurately quantify and characterize nanoparticles in an economically feasible manner, predictive modeling of transport, fate and subsequent toxicity of NMs in the environment is impossible [6].

 4. Exposure

The potential environmental exposure to engineered NMs increases with the number of applications in which NMs are used. The maximum internal exposure is limited by the external exposure and elimination kinetics in the body [9]. Individuals may be exposed to similar or similarly acting NMs through different consumer products including food packaging, cosmetics, food additives and detergents, and via different routes of exposure. Assessment of NM exposure during production, use and disposal is complicated by a lack of good measurement techniques. Traditional surveillance measurements do not measure those characteristics of NMs that were also shown to be linked to a biological response, such as surface-to-mass ratio, mechanical strength, durability, conductivity, reactivity, solubility, and the ability to adsorb and carry other chemicals [15]. In 2011, Lorenz and colleagues published a study estimating the exposure of German consumers to engineered nanoparticles [9]. The researchers found exposure to depend most heavily on a product’s market share, product labeling and the consumers’ awareness of engineered NMs. This means that consumers can actively reduce exposure. Lorenz et al. did not include food contact materials (FCMs) in their analysis.

Exposure to NMs may occur via inhalation, ingestion and dermal contact. Exposure via ingestion is the most significant source with regard to FCMs [3]. Exposure to NMs contained in packaging may either occur through migration from the packaging into the food or through degradation of FCMs containing NMs in the environment. The health risks posed by engineered NMs from FCMs  is determined by the toxicity of the NM, the rate of migration and the consumption rate of the particular food [3]. Migration studies have shown that migration increases with storage duration and content of the NM in the FCM [5, 16]. Temperature is generally thought to increase migration [16, 17], but not all studies have been able to confirm this finding [5]. A model developed by Simon et al. requires information about the packaging matrix and the nanoparticles to predict migration [18]. Though not included in the study, characteristics of the food contained in the packaging material may also be of importance.

5. Toxicity

NMs behave differently from bulk materials because of surface and quantum effects. Due to these two effects NMs are often significantly more reactive than bulk materials [2]. Toxicity can generally not be correlated with nanoparticle mass dose, which is why exact characterization, including surface area, of NMs is very important. Dose can often be better correlated with an effect when surface area per volume is measured instead of mass per volume. [2]. However, the difficulty of accurately measuring surface area per volume has led scientists to continue measuring dose in mass per volume. Other determinants of NM toxicity are chemical composition, size, aggregation, crystallinity, surface functionalization, and change in particle characteristics over time and water chemistry [2, 19].

Inhalation as exposure route for NMs has been most extensively studied as nanoparticles are abundant in the air and can reach the lower respiratory tract [20]. The inhalation of nanoparticles has been linked to epithelial cell proliferation, fibrosis, emphysema and the appearance of tumors [2]. With regards to ingestion, the Organisation for Economic Co-operation and Development (OECD) suggests in its report ENV/JM/MONO(2012)40 to use in vitro models simulating the gastrointestinal tract’s environment in order to predict whether the stomach milieu will disperse or agglomerate the nanoparticles in question. For repeated dose toxicity testing, drinking water has been used as a medium, which is in accordance with the oral repeated dose toxicity OECD test guidelines (TG 407408). Chronic studies are best performed by feeding the nanoparticles to test animals with their diet, but care should be taken not to confound effects with contaminants that may be carried by the NMs, according to OECD report ENV/JM/MONO(2012)40.

A few studies have considered the effects of nanoparticles if exposure takes place via ingestion. NMs in the gastrointestinal tract have been linked to ulcerative colitis, colon cancer and Crohn’s disease. However, clinical trials testing whether a reduction in NMs in the diet can reduce the symptoms of Crohn’s and inflammatory bowel disease (IBD) have produced contradictory results [1]. Further, it is not possible to extrapolate any of these findings across different NMs and different batches of the same material. In June 2013, Bergin and colleagues published an article in theInternational Journal of Biomedical Nanoscience and Nanotechnology reporting that the ingestion of nanoparticles at levels typically present in the environment is unlikely to have any acute adverse effect. However, they also state that current literature is inadequate to assess the potential of NMs to accumulate in tissues [21]. The lack of conclusive research into potential long term effects and the increased presence of different NMs in the environment precludes any definite risk assessment.

At the cellular level, nanoparticles may cause damage via mechanisms of inflammation. Larger nanoparticles can enter cells through phagocytosis, whereas nanoparticles smaller than 0.7 nm are thought to enter cells through ion channels and pores [22]. Fluorescent or radioactively labeled nanoparticles have been used to investigate the distribution in the body after exposure [20]. Nanoparticles may accumulate in organelles such as mitochondria. They may also translocate into the circulatory and lymphatic system, body tissues and organs [23-26]. Further, it has been shown that nanoparticles smaller than 70 nm may enter cell nuclei [1]. Positively charged nanoparticles may also cross the blood-brain barrier, a mechanism that has been extensively researched to deliver drugs to the brain [27]. In vitro tests have shown that nanoparticles can create reactive oxidative species, which damage cells by peroxidizing lipids, altering proteins, disrupting DNA, interfering with signaling functions, and modulating gene transcription. The incurred cell damage activates an inflammatory response in the body. Further results are the release of antioxidants and possibly DNA damage [28]. Excess levels of inflammation are thought to cause disease [2]. NMs that enter the circulatory system (< 30 nm) are linked to arteriosclerosis, blood clots, arrhythmia, heart disease and cardiac arrest [29].

The section below elucidates the toxicity of nanosilver in an exemplary manner to depict difficulties arising from the toxicological evaluation of NMs.

5a. Case study of nanosilver

Nanosilver is a NM with a core consisting of metallic silver. It may have different compositions, surface areas and functional groups that influence its particular behavior in the environment and organism. Nanosilver is used as an antimicrobial in a variety of consumer applications including food containers and refrigerators [2]. In Europe, it is not authorized for the application in food grade materials. In the U.S. nanosilver can reasonably be assumed to be used in food contact materials (FCMs), even if the registrations do not indicate conclusively whether they concern nano- or bulkmaterials.

Huang et al. (2011) demonstrated that nanosilver migrates in the nanoparticular form from polyethylene (PE) composite packaging into food [17]. Studies by Huang et al. (2011) and Song et al. (2011) found temperature to increase the migration of nanosilver [16, 17]. In the first study migration from PE plastic bags increased over 15 days irrespective of the food simulant, whereas in the latter study migration reached its steady state after six hours [16, 17]. Huang and colleagues reported the spherical particle around 300 nm, Song and coauthors did not report the size of their nanoparticles. Another research group observed particles sized 10-20 nm to aggregate in complexes of about 50 nm [30].

Nanosilver has been observed to induce oxidative stress, genotoxicity and apoptosis in a variety ofin vitro studies [26, 31-34]. During tests carried out for the nanoGEM project, exposure to nanosilver resulted in positive responses in the micronucleus test. However, no genotoxicity was found with the Ames test (Schnekenburger, nanoGEM closing conference, June 13, 2013). In a study by Marambio-Jones and colleagues, exposure to silver ions was found to result in increased membrane permeability, loss of the proton motive force and disruption of DNA replication in bacteria [19]. Silver ions seem to affect eukaryotic cells similarly by creating oxidative species [35]. In toxicity testing under anaerobic conditions, adverse effects were not found to be particle-specific but rather due to the toxicity of the silver ion, Ag+ [36]. It is assumed that the toxicity of nanosilver arises from the toxicity of Ag+ released from the nanosilver or present as impurities [37]. Yet, the presence of ionic silver together with silver nanoparticles has been found to be more toxic than ionic silver present on its own [37].

Few studies have investigated the effects of silver nanoparticles in in vivo rodent models. In a subacute inhalation mouse model, exposure to 3.3 mg/m3 (over 10 days) of 5 nm (in diameter)  silver nanoparticles induced minimal inflammation [38], likely due to the accumulation of NMs in macrophages and the inability of macrophages to effectively destroy these particles. A similar study by Ji et al. (2007) did not report any significant change in weight or hematology at exposure levels around the American Conference of Governmental Industrial Hygienists (ACGIH) silver dust limit of 100 µg/m3 [39]. Zhang et al. (2013) investigated the effect of injections of 45 mg/kg/day of 7 nm (in diameter) silver nanoparticles on Sprague-Dawley rats [40]. Exposure to Ag-NPs over three days was found to result in significant weight loss, reduced activity, and even death [40].

Two studies investigated the effects of ingested nanosilver particles. The in vivo study by Park et al. (2010) found ingested nanosilver particles smaller than 73 nm to be distributed to organs including brain, lung, liver, kidney and testis, whereas large sized particles (323 nm) could not be detected in these tissues (1 mg/14 days) [41]. Inflammatory markers were increased in animals exposed to the smaller nanoparticles. After 28 days, the authors observed adverse effects on liver and kidney in the group treated with the highest dose of 1 mg Ag-NPs (42 nm diameter)/kg. Inflammatory makers were increased in a dose dependent manner [41]. Similarly, van der Zande et al. reported in 2012 that nanosilver was present in all examined organs after 28 days oral exposure, irrespective of the nanosilver coating [42]. After 8 weeks post-dosing, nanosilver was cleared from all organs other than the brain and testis. In contrast to Park and colleagues, van der Zande et al. did not observe hepato- or immunotoxicity irrespective of the significantly higher exposure levels administered (90 and 9 mg/kg). These results point out that traditional dose-response toxicology cannot adequately predict NM toxicity, unless it includes other relevant NP characteristics such as surface size, coating/functional groups and size.

In order to better understand the potential links between nanoparticles and disease, as well as their specific mechanisms of toxicity, more knowledge about the physicochemical properties of manufactured NMs is needed. Better detection methods of NMs and a better understanding of their fate and transport, and dispersion are vital to predict exposure and release scenarios effectively. Further research into uptake, bioavailability and mode-of action in the body is also warranted. Finally, more research into potential cumulative impacts of NMs would be important to ensure safety [1, 6, 15].

6. Regulation

There is no specific regulation for NMs anywhere in the world [43]. In some places specific NMs have been authorized for FCMs, other legislations assume bulk and NMs to be equal in toxicity.

6a. US

In the U.S., the use of NMs in food packaging is regulated by the Food and Drug Administration (FDA). According to the Central Federal Registry manufacturers must obtain pre-market approval for indirect food additives, which include NMs, either through the Food Additive Petition (FAP) system dating back to 1958, or the newer Food Contact Notification (FCN) system from the 1990s. Under the FCN system, the FDA has to respond to a submitted notification within 120 days; otherwise the substance may be marketed without further approval. The approval under the FCN is not published in the Code of Federal Regulations (CFR) but published online on the webpage of the FDA. Approval is not applicable to other manufacturers using the same substance.

Pre-market authorization is not required for substances Generally Recognized As Safe (GRAS). If a company publishes a scientific risk assessment of a substance in a peer-reviewed scientific journal, it may market a substance without previously consulting with the FDA. This is also the case for substances that have been previously sanctioned, i.e. grandfathered into the regulation, because they were marketed before 1958, or fall under the threshold of regulation (TOR) rule. The FDA is yet to publish clarification on whether the approval of bulk food additives is also applicable to their nanoscale versions. This question is of particular importance for simple indirect additives, by which the description of the authorized macroscale version may also apply to the NM [44]. Traditionally, the FDA has taken the position that materials that are chemically identical to authorized indirect additives and comply with the limitations specified in that authorization may be used without further notice. It is not clear how many NMs are used for food contact materials under this position, though some materials including silver, carbon black, aluminum are known to be used in FCMs [12].

6b. Europe

In Europe, NMs in food packaging are generally regulated under the framework regulation EC 1935/2004, which states that their use in food packaging may not pose a danger to human health (Article 3). In accordance with Article 23 of regulation EC 10/2011, nanoparticles have to undergo case-by-case evaluation before being placed on the market (also see Novel Food Regulation EC 258/97). NMs have to be authorized even if the equivalent bulk material is already authorized (EEC 89/109). If a non-authorized substance is used, a migration limit of 0.01 mg/kg shall be observed through use of a functional barrier (Article 14, EC 450/2009) [5]. For plastic food packaging, currently only three NMs have been authorized, namely carbon black, titanium nitride and silicon dioxide. Titanium nitride may not be detectable in food, carbon black cannot be used at levels higher than 2.5% w/ w packaging. For silicon dioxide no specific migration limit was set (Annex I, EC 10/2011).

In 2011, the European Food Safety Authority (EFSA) published a guidance document “on the risk assessment of the application of nanoscience and nanotechnologies in the food and feed chain” (EFSA 2011) in which it indicates which physicochemical information is required from the manufacturer. It requests in vitro genotoxicity, absorption, distribution, metabolism and excretion (ADME) tests and a repeated-dose 90-day oral toxicity study. A substance may be exempted from these requirements if data indicates that no migration occurs, or complete degradation or dissolution can be verified [45]. EFSA does not specify how these requirements can be carried out in a consistent and economical manner, considering the difficulties in accurately measuring and characterizing nanomaterials. Denmark and France are currently establishing a registry of products containing NMs (Decree 2012-232 and Danish Draft Order).

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