State of the safety assessment and current use of nanomaterials in food and food production

Trends in Food Science & Technology xx (2014) 1e11

* Corresponding author.

http://dx.doi.org/10.1016/j.tifs.2014.08.0090924-2244/� 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Bouwmeester, H., et al., State of the

Trends in Food Science & Technology (2014), http://dx.doi.org/10.101

State of the

safety assessment and

current use of

nanomaterials in

food and food

production

Hans Bouwmeester*,
Puck Brandhoff,
Hans J.P. Marvin, Stefan Weigeland Ruud J.B. Peters

RIKILT e Wageningen UR, 6700AE Wageningen,

The Netherlands (e-mail: [email protected])

Nanomaterials are developed for and applied in food, food ad-

ditives, supplements and food contact materials. In an inven-

tory of internet databases 140 products in the food and food-

related sectors were identified that claim to contain nanomate-

rials. A great diversity of nanomaterials is applied, ranging

from inorganic metal and metal oxides to organic nanomateri-

als that carry bioactive ingredients. Here we present an over-

view of nanomaterial applications that are currently

available, discuss state of the art analytical chemical character-

ization and toxicological assessment methods, as well as cate-

gorization methods to support the safety evaluation of the

application of nanomaterials throughout food production.

IntroductionNanomaterials have unique functional properties that aremainly size related i.e. due to the much larger surface tomass ratio compared to the larger-sized bulk materials.Because of the unique functional properties of

safety a

6/j.tifs.2

nanomaterials they are being used by many industries,including the food and agricultural sectors, and anincreasing number of products that contain nanomaterialscan already be found on the market (Chaudhry et al.,2008; Dur�an & Marcato, 2013; Nanotechnologies; Weiss,Takhistov, & McClements, 2006). The main drivers behindthe application of nanosized ingredients and additives infood and food-related products are directed to improvingfood production processes, extending the shelf-life of freshproducts, improving the consistency, stability and texture ofproducts, and enhancing the uptake and bioavailability ofnutrients. Nanomaterials could have a substantial impacton the food sector in the future, potentially offering benefitsfor industry and the consumer, although the specific proper-ties and characteristics of nanomaterials need to be consid-ered for any potential health risks.

Companies and institutes worldwide are currently study-ing and developing nanomaterial applications to improve orreplace pesticides and antibiotics in agricultural food pro-duction, to modify the mechanical and sensorial propertiesof food, increase the nutritional value of food (Alejandro &Rubiales, 2009; Perlatti et al., 2012; Shi, Xu, Feng, &Wang, 2006; Verma, Singh, & Vikas, 2012). In fact,possible applications of nanotechnology can be identifiedin the complete food production chain. It should be notedthat some substances have always contained nanoscale par-ticles but have not been recognized as such. Fatty acid lipo-somes used in health products are another form ofconventional substances that occur in nanosized micelleform. Proteins or protein micelles in many products havesizes that bring them into the nano-range. For instancecasein, a natural protein in milk, spontaneously forms clus-ters and micelles with sizes ranging from 2 to 200 nm (deKruif & Huppertz, 2012). In this paper we are concernedwith engineered nanomaterials, mostly metal and metal ox-ide nanoparticles, and lipid-, carbohydrate- and protein-based nano-encapsulates in food, food packaging andsupplements.

While the special properties of nanoparticles have led toinnovative products, there are also concerns about theirsafety, especially because of our still limited knowledgeof human health effects of these materials. In response tothis uncertainty more up-to-date information is requiredon the state-of-the-art of applications of nanotechnologyas pesticides, food additives, food contact materials andfeed additives, i.e. the use of nanoparticles in the

ssessment and current use of nanomaterials in food and food production,

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Delta:1_surname

Delta:1_given name

Delta:1_surname

Delta:1_given name

mailto:[email protected]

http://dx.doi.org/10.1016/j.tifs.2014.08.009



2 H. Bouwmeester et al. / Trends in Food Science & Technology xx (2014) 1e11

agricultural, feed and food sector. In this review, we willbriefly discuss the currently used definitions of nanopar-ticles, and its applicability for food and the food-relatedsectors. In order to frame the need for a safety assessmentof nanoparticles in these domains we present an overviewof nanoparticles containing products that are currently onthe market. This overview of most applied nanoparticlesand product matrix combinations will direct the require-ments for experimental procedures to generate data forthe safety assessment. Recent advances in analyticalmethod development will be discussed in relation the hu-man exposure data generation as well as in relation toadvanced models for hazard assessment and characteriza-tion of nanoparticles in food and food-related products.Finally, categorization approaches are briefly discussed asa screening method for safety assessment as long asadequate toxicological and analytical methods are not inplace.

Definitions of nanoparticlesNanotechnology is defined by the International Organi-

zation for Standardization as the “application of scientificknowledge to manipulate and control matter at the nano-scale in order to make use of size- and structure-dependent properties and phenomena, as distinct from thoseassociated with individual atoms or molecules or with bulkmaterials” (ISO, 2010). The word “nano” refers to abillionth of a metre (10�9 m) and nanotechnology can beunderstood as the fabrication, characterization and manipu-lation of particles with sizes <100 nm (EFSA, 2011).

While nanoparticles, or nanomaterials consisting of suchparticles, are generally accepted as those with a particlesize below 100 nm, this size limit is fairly arbitrary. Theregulatory approaches addressing nanoparticles differ perregion. In the USA, the FDA did not issue strict definitionsof nanoparticles and considers food manufacturing pro-cesses that involve nanotechnology in the same manner asany other food manufacturing technology (FDA, 2011).The FDA recognizes that nanoparticles in food may havenew properties and that additional or different testingmethods may be necessary to assess the safety of thefood substance. The variation in biological activity thatmay result from engineering food substances in the nano-metre range may raise questions about the applicability oftraditional safety tests for these materials. The FDA clearlystates that, as with any studies to support the safety of foodsubstances, studies to establish the safety of food sub-stances manufactured using nanotechnology should havebeen appropriately validated for these materials (FDA,2012). The same holds true for Europe, where in additiona proposal for a nanomaterial definition has been issued.The European Commission adopted the Recommendationon the definition of nanomaterials 2011/696/EU on 18October 2011 (EC, 2011). According to this recommenda-tion, a “nanomaterial” means:

Please cite this article in press as: Bouwmeester, H., et al., State of the safety a

Trends in Food Science & Technology (2014), http://dx.doi.org/10.1016/j.tifs.2

- A natural, incidental or manufactured material contain-ing particles, in an unbound state or as an aggregate oras an agglomerate and where, for 50% or more of theparticles in the number size distribution, one or moreexternal dimensions are in the size range1 nme100 nm.

- In specific cases and where warranted by concerns forthe environment, health, safety or competitiveness thenumber size distribution threshold of 50% may be re-placed by a threshold between 1 and 50%.

- By derogation from the above, fullerenes, grapheneflakes and single wall carbon nanotubes with one ormore external dimensions below 1 nm should beconsidered as nanomaterials.

It is expected that this definition will be used primarilyto identify materials for which special provisions mightapply (e.g. for risk assessment or ingredient labelling).However, there are various scientific and/or technical chal-lenges related to the measurement of materials in the imple-mentation of the recommended nanomaterial definition.

Apart from technical issues about the EU definition of ananomaterial there is some discussion whether all particlessmaller than 100 nm should be considered in this recom-mendation (Bleeker et al., 2013). Legal labelling obliga-tions and information requirements may require furtherspecification to separate natural, incidental and manufac-tured nanoparticles (natural nanoparticles being materialsthat are already present in many food and feed productslike for instance casein proteins in milk products); differen-tiate between soluble and non-soluble nanoparticles (mate-rials in which nano-specific risks are limited because theydissolve quickly in media changing into non-nanoparticles), and organic and inorganic nanoparticles(soft nanoparticles being materials such as micelles, emul-sions and liposomes for which nano-related risks are negli-gible, for example in case they fall apart in thegastrointestinal tract). Furthermore, new directions maybe required for nanoparticles with a history of use. Theseare materials which are already on the market for a longtime, whereas the authorization of these materials may bebased on a dossier that is not suitable to investigate nano-related properties and/or effects. This may be the case fora number of current food additives as synthetic amorphoussilica (SAS, E551) and titanium dioxide (TiO2, E171), andmaterials that have a history of use in their colloidal form,such as silver.

Applications of nanoparticles in food and food-relatedproducts

Nanoparticles are used in food and agriculture in severalapplication domains. In the primary production (agricul-ture) nano-formulated pesticides, fertilizers and other agro-chemicals are being developed. The application ofnanoparticles has also enabled the development of innova-tive packaging materials that can improve the safety and


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3H. Bouwmeester et al. / Trends in Food Science & Technology xx (2014) 1e11

shelf life of products. And lastly, nanoparticles are devel-oped to prevent microbial spoilage of packaged food, toimprove colours, flavours, taste and texture and increasethe bioavailability of vitamins and minerals.

Generally, nanoparticles can be divided into three cate-gories: inorganic nanoparticles, combined organic/inor-ganic (surface modified) nanoparticles and organicnanoparticles as shown in Fig. 1. Inorganic nanoparticlesconsists of metal, but more often metal oxides, that areused for that antimicrobial properties. Surface modifiednanoparticles are nanoparticles that add certain types offunctionality to the matrix, such as antimicrobial activityor a preservative action through absorption of oxygen andgenerally consists of surface modified clay. In recent yearsall these materials have attracted great interest.

In order to understand how many products containingnanoparticles are already available to consumers, a compre-hensive database on types and uses of such nanoparticleswould be useful. The European Union commissioned toproduce a list of nanoparticles on the EU market and thatis focused on applications already on the market althoughit cannot be excluded that some of the information relatesto products in the development stage (S.W.P. Wijnhovenet al., 2010). In addition, it is not certain that all these ma-terials are nanoparticles, some information relates to thebulk form of the substance, some to the nanoform. Mate-rials included are metal and metal oxides as silver, gold,synthetic amorphous silica, titanium dioxide, zinc oxide,aluminium oxides and hydroxides, cerium dioxide and cal-cium carbonate, but also carbon based materials such as thewell-known fullerenes, carbon nanotubes (CNT) and e fi-bres, carbon black and graphene, and finally nanopolymers,nanoclays and nanocomposites.

Since 2006 there is a voluntary database, the WoodrowWilson Inventory, that is accessible for consumers (PENdatabase). This inventory lists a large number of chemicallyand structurally diverse nanoparticles used in product appli-cations in medicine, communication, foodstuffs and foodpackaging, dietary supplements, over-the-counter drugs,and many other consumer products ranging from cosmeticsand toothpaste to paints and clothing. The inventory alsoshows some statistical analysis which shows that the

Fig. 1. Three categories of nanomaterials for food and food-related ap-plications: Inorganic nanomaterials (metal, metalloids, fullerenes andcarbon nanotubes): Combined organic/inorganic nanomaterials (sur-face modified clay, metal or metalloids): Organic nanomaterials

(nano delivery systems, capsules, polymers and emulsions).



number of consumer products containing nanoparticleshas risen steadily over the past 5 years, with the additionof about 250 new products per year to a current total of1317 products. The increase in products containing nano-particles appears to be a linear increase and not an expo-nential explosion as sometimes mentioned in the morepopular literature. It does however show a 500% increasein the number of listed products since the start of the Wood-row Wilson Inventory in 2006.

There is also a set of European inventories of productsavailable to consumers with a claim of containing nanopar-ticles. The ANEC/BEUC inventory lists products that claimto contain nanoparticles and that are available to Europeanconsumers (ANEC & BEUC, 2010). The inventory can bedownloaded from the site and lists 475 products, only 2 ofwhich are related to food. The BUND (Friends of the EarthGermany) product database focuses on consumer productsin Germany that claim to contain nanoparticles (BUNDdatabase). The BUND database contains about 200 prod-ucts of different product groups. For food related nanopar-ticles 32 products are listed. The Nanotech-data is adatabase of nanotechnologies for Luxembourg and areasin Germany and Belgium (Nanotech-data, 2013). Nanopro-ducts.de is a freely accessible database that deals with themarketing of products containing nanoparticles and/orproducts produced with nanotechnology on the internet.The database contains more than 450 nanotechnology prod-ucts in the area of comprising process engineering, ana-lytics, materials and commercial products (Nanoproducts,2013). Finally, the Nanodatabase is an on-line databasepublished by the Danish Consumer Council and the DanishEcological Council listing more than 1200 consumer prod-ucts available in the EU and purported to contain nanopar-ticles (Nanodatabase, 2014). Interestingly, the databaseprovides a traffic light indicator of potential risk to humanhealth and the environment. Products are assessed accord-ing to system published in 2011 (EPA, 2011), which usesknowledge of the physical structure of the material (nanoand bulk form), and any information on exposure andhazard.

From these databases, literature and internet references,a list of currently applied food-related products that claimto contain nanoparticles has been compiled. Whencompiling the list it was noted that there is little or no con-trol whether claims made by producers, suppliers or otherswho placed the information in the database, are actuallycorrect, e.g. some of these products may not contain nano-particles after all. The current listing consists of a table of140 food related products that can be found in the supple-mental material (Table S1). The table contains the productnames, the name of the producer/supplier, the product cate-gory (agriculture, food additive, food packaging or foodsupplement), the type of nanoparticle (e.g. organic micelle,silica, silver) and a reference where this information can befound on the internet. The statistics of this list of nanoprod-ucts is summarized in Fig. 2.


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Fig. 2. Schematic presentation of the applications of nanomaterialsfound in commercially available food related products.


Only a few (6) nano-products were identified in the agri-culture sector. This seems in agreement with the literaturewhich mentioned that many nano-products for the agricul-tural sector are in development, but only a few really existpresently (Perlatti et al., 2012). Of the six agriculture nano-products five are pesticides which are used in the form ofemulsions, and one is a nanoclay intended as a binder formycotoxins. The list of food-related nano-productscompiled in this study is only a snapshot and it is expectedthat it will change over time since new products will enterthe market. On the other hand, some products currently onthe market or referenced in the literature may disappear intime or be no longer available on the internet.

The vast majority of the available nano-products arefood supplements although the difference with food addi-tives is not always clear. Most of these food supplementsare organic micelles or liposomes used as nano-encapsulates or nano delivery systems for vitamins or other“health-promoting” substances. The sizes of these micellesand liposomes are typically in the range of 10e100 nm and100e300 nm, respectively (R. Peters et al., 2011). Thenano-encapsulates generally consist of a core containingthe (bio)active ingredients surrounded by a wall or barrierand have their roots in the pharmaceutical industry, wheresynthetic polymeric nano-encapsulates are often employed(Antunes, Fierro, Guerrante, Mendes, & Alencar, 2013;Puri et al., 2009). In case of the application in food and sup-plements only food-grade polymers based on lipids, pro-teins, and/or polysaccharides are suitable (Graveland-Bikker & de Kruif, 2006; Luykx, Peters, van Ruth, &Bouwmeester, 2008; Mozafari et al., 2006). Of the threetypes of organic nanoparticles, lipid-based nanoparticlesare among the most applied organic nanoparticles sincethey can be produced using natural ingredients on an indus-trial scale and have the ability to encapsulate compoundswith different solubilities. Protein-based nanoparticles areoften prepared using a ‘bottom up’ approach, where



structures are built from molecules capable of self-assembly, e.g. micelle like structures. For example, glob-ular proteins such as whey proteins from milk have the abil-ity to form particles with sizes of 40 nm while 95% of thecaseins are naturally self-assembled micelles with a50e500 nm diameter. Up to now, chitosan, a naturallybiodegradable and biocompatible polysaccharide, andstarch appear to be the most used polysaccharides for nano-particles in pharmaceutical and biomedical applications(Luykx et al., 2008).

Nano-silica and -silver can also be found in many sup-plements, nano-silica as a carrier delivering minerals andnano-silver for its history as an antibacterial agent. Silicondioxide, or silica, has been used in food processing andfood applications for many years in a form which is calledsynthetic amorphous silica (SAS). It is registered in the EUas E551 and consists of primary particles, aggregates andagglomerates. While the primary particles and part of theaggregates in SAS seem to consist of particles with sizes<100 nm (R. Peters et al., 2012), SAS is regarded as ananostructured material (Bosch, Maier, & Morfeld, 2012).A similar situation exists for titanium dioxide, anotherapproved food additive with number E171. While theaverage particle size is 200e300 nm, up to 36% of thebulk material may contain particles with sizes <100 nm(Weir, Westerhoff, Fabricius, Hristovski, & Von Goetz,2012). Many other metals in the form of nano-sized parti-cles are available as food- or health supplements. Theseinclude nano-selenium (Xu, Yang, An, & Hu, 2007),nano-calcium (Hannig & Hannig, 2010), nano-iron(Sekhon, 2010), and colloidal suspensions of metal parti-cles, e.g. copper, gold, platinum, silver, molybdenum,palladium, titanium, and zinc (Park, Li, & Kricka, 2006).

Nanoparticles used in food packaging consist mostly ofclay or silver. Clay is used to manufacture impermeablelayers in packaging materials, especially for plastic bottles,to offer mechanical strength or form a barrier against gases,volatile components (such as flavours) or moisture. Thenano-clay mineral is mainly montmorillonite; natural clayobtained from volcanic ash/rocks (also termed bentonite).The clay is surface modified to form an organo-clay whichhas a natural nano-scaled layer structure and is organicallymodified to bind to polymer matrices (de Paiva, Morales, &Valenzuela D�ıaz, 2008). Surface modified nanoparticles canalso be used to add certain types of functionalities to thematrix, such as antimicrobial activity or a preservative ac-tion through absorption of oxygen. Other materials inciden-tally used in food packaging are silica, titanium dioxide andstarch. While silver is by volume not the most used mate-rial, it is the fastest growing nanomaterial application infood, food supplements and food packaging as an antimi-crobial. This is also the case for refrigerators and food stor-age boxes. In this study about 40 applications of silver infood and food-related products were found, most of themin food supplements. While no current applications of silverin food were identified, patents have described the use of


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silver to prepare antibacterial wheat flour and as an additivein animal feed (Park et al., 2006). More recently silver hasbeen studied as an alternative for the antibiotics used in thepoultry production (Pineda et al., 2012).

Hazard assessment of engineered nanoparticles infood

Nanoparticles are used in the food and agriculture sec-tors because of their unique properties. This is mainlybecause of their small size and consequently their muchlarger surface to mass ratio as compared to larger sized ma-terials. As a consequence, nanoparticles exhibit differentphysico-chemical properties and biological effectscompared to larger sized materials (Oberd€orster,Oberd€orster, & Oberd€orster, 2005). Because of this, discus-sions were initiated on how to assess the safety of nanopar-ticles in general and within food and feed productsspecifically. This resulted in various (draft) opinions issuedby FDA and EFSA on the adequateness of the risk assess-ment paradigm and guidelines for industry regarding thesafety evaluation on nanoparticles (EFSA, 2011; FDA,2011, 2012).

Up to now only for a few nanoparticles a safety assess-ment is available in the scientific literature (S. Dekkerset al., 2013; S. Dekkers et al., 2011; S. W. P. Wijnhovenet al., 2009). These safety assessments are surrounded byuncertainties, mainly due to the lack of reliable character-ization data of the nanomaterial in the product, and inade-quate material characterization in the toxicological studiesperformed. The clear need to develop, implement and vali-date routine analytical methods for the characterization ofnanoparticles in food has been recognized before. In addi-tion experimental (tiered) approaches for the hazard assess-ment of nanoparticles are developed. In the sections belowboth developments are summarized.

Detection and characterization of engineerednanoparticles in food

There is a vast array of analytical techniques to charac-terize pristine nanoparticles, as powder or in clean aqueousliquids (Oberd€orster et al., 2005; Powers et al., 2006; Tiedeet al., 2008), however, food, biological samples and agricul-tural samples are heterogeneous mixtures and thereforerequire separation or pre-treatment to isolate the target an-alyte from the interfering matrix components. Due to itshigh reactivity nanoparticles can change in compositionand size as a response to changes in their environment. Re-sults obtained after sample preparation do not, or onlypartially, reflect the original situation in the original sam-ple. Ideally, sample preparation is kept minimal (Szakalet al., 2014). Roughly three categories of detection andcharacterization techniques can be observed: imaging, sep-aration and spectroscopic techniques.

Classical light scattering techniques as dynamic lightscattering (DLS) or the more recent nano tracking analysis(NTA) provide a size distribution in terms of hydrodynamic



diameter (Filipe, Hawe, & Jiskoot, 2010; Finsy, 1994).Electron microscopy (EM) techniques are widely used todetermine size, shape and other properties from nanopar-ticles in food. Coupled with energy dispersive X-ray spec-troscopy (EDX) EM is used to determine elementalcompositions of nanoparticles while the EM is used todetermine size, shape and size distribution (Dudkiewiczet al., 2011; Luykx et al., 2008; R. Peters et al., 2011).Recently, by using TEM/EDX in combination with DLSthe size, shape and elemental characteristics of silver nano-particles in pears were determined (Zhang, Kong,Vardhanabhuti, Mustapha, & Lin, 2012). A drawback ofimaging techniques is that they are often not quantitative.

The most widely used separation techniques for nano-particles in food and agricultural samples are hydrody-namic chromatography (HDC) and field flow fractionation(FFF), often in combination with detectors such as multipleangle light scattering (MALS) and inductive coupledplasma mass spectrometry (ICP-MS) (Hassell€ov,Readman, Ranville, & Tiede, 2008; Kammer, Legros,Hofmann, Larsen, & Loeschner, 2011; R. Peters et al.,2011). HDC is a chromatographic technique that made arevival the last years since it provides reliable size separa-tion that is largely independent from the matrix. The sepa-ration mechanism is based on different samplings of theflow velocity profile due to differences in the effectivediameter (Striegel & Brewer, 2012). Larger particles aretransported slightly more rapidly along the column whichis filled with non-porous polystyrene microspheres. Withasymmetric-flow field flow fractionation (AF4) high-resolution separation is achieved within a very thin flowagainst which a perpendicular force field is applied. Thesmaller particles are transported much more rapidly alongthe channel than the larger particles (Gray et al., 2012).

The detection limits of DLS and EM techniques are typi-cally in the mg/L range while detection limits of combina-tions of HDC or AF4 with MALS and ICP-MS are in themg/L and mg/L range respectively. As a consequence sam-ple preparation and analyte concentration steps are oftenrequired prior to instrumental analysis. An alternativeapproach that has been developed in recent years is singleparticle ICP-MS (sp-ICPMS) in which nanoparticles canbe detected with limited sample preparation (Degueldre,Favarger, & Wold, 2006; Laborda, Jim�enez-Lamana,Bolea, & Castillo, 2011). In sp-ICPMS individual nanopar-ticles are atomized and ionized in the ICP plasma and theresulting plume of ions is detected by the MS. Detectionlimits are generally in the ng/L range. A great advantageof sp-ICPMS is that it determines a number-based size dis-tribution as required by the European Commission recom-mendation for the definition of nanoparticles (EC, 2011).Recently, a fully validated method was published for thequantification and characterization of silver nanoparticlesin chicken meat (R. J. B. Peters et al., 2014).

While there exist many techniques to detect, quantify,and measure the properties of inorganic nanoparticles,


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this is not the case for organic nanoparticles composed ofpolymers, lipids, proteins and polysaccharides. Imagingtechniques are limited and EM is generally only useful ifstaining techniques are applied. Separation techniques asHDC and AF4 can be used but care has to be taken notdisrupt the micelle-like structures. While ultraviolet detec-tion (UV) can be used for quantification, matrix assistedlaser desorption ionization (MALDI) in combination withMS techniques can be used for chemical characterization(Helsper, Peters, Brouwer, & Weigel, 2013; R. Peterset al., 2011).

From the above it is clear that there is no single analyt-ical method that can be used to detect all types and sizes ofnanoparticles in all matrices. In addition, the EU definitionof nanoparticles poses many analytical challenges. Inparticular, the requirement of measuring the constituentparticles inside aggregates, the difficulty to convert experi-mentally measured signals into accurate number-based sizedistributions, and to detect and count particles at the lowersize range of the definition, i.e. smaller than 10 nm. Analyt-ical methods able to identify materials as nanoparticles ac-cording to the recommended nanomaterial definition are indevelopment. However, most current methods have a detec-tion limit higher than 1 nm or a lower sensitivity for smallerparticles. As a consequence, they can only be used for apositive test to prove that a material is a nanomaterial,but not for a negative test to prove that a material is not ananomaterial. Presently, none of the currently availablemethods is able to determine for all kinds of potential nano-particles whether they fulfil the definition or not. Thereforea range of measurement methods is required to investigatewhether nanoparticles fulfil the regulatory definition(Linsinger et al., 2012). Internationally validated andharmonized analytical detection methods are importantfor the mutual acceptance of data and dossier requirementfor the regulatory safety assessment. For the exposureassessment and the characterization of nanoparticles usedin toxicological testing it is important that the dynamicwindow (sensitivity), robustness and repeatability of theused analytical methods is adequately reported.

Toxicological assessment of nanoparticles in foodThe (regulatory) safety assessment of nanoparticles still

relies mainly on in vivo data generated according to OECDguidelines. For these studies the need of additional nano-specific requirements are frequently debated. In additionto the long lists of required parameters for an adequate ma-terial characterization, requirements for nano-specific bio-logical endpoints have been discussed (Bouwmeesteret al., 2011). Chronic (or sub-chronic) oral studies arebest performed by administering the nanoparticles directlyvia the diet as this is most realistic compared to human oralexposure. Alternatively oral gavage might be used to reli-ably administer the materials to the animals. It is very likelythat the nanoparticles interact with the feed matrix orgavage solutions. Clearly during transit through the



digestive tract the physico-chemical properties of the nano-particles are changing. This has been shown for silver andsilica nanoparticles using an in vitro digestion model(Mwilu et al., 2013; R. Peters et al., 2012; Walczaket al., 2013).

Given the enormous diversity in nanoparticles applied infood and feed it is scientifically and ethically not defend-able to only rely on animal experiments in future safety as-sessments. The incorporation of alternative testingapproaches in a tiered toxicological safety assessment hasbeen suggested before (Cockburn et al., 2012; Szakalet al., 2014). In the first tier of assessment physiochemicalproperties of the pristine nanomaterial and its stability insuspension are assessed. As mentioned for this a broadrange of analytical procedures is available. The secondtier of assessment of nanoparticles following oral exposureis proposed to consist of an assessment of solubility andagglomeration using in vitro digestion models. Severalin vitro digestion models with increasing complexity areavailable, ranging from static to dynamic models thathave been used for conventional chemicals (Lefebvreet al., 2014). Gastric and combined gastric and intestinalmodels have been used to study the fate of silver and silicananoparticles, both in the presence and absence of a foodmatrix (Mwilu et al., 2013; R. Peters et al., 2012;Walczak et al., 2013). For example for synthetic amorphoussilica (SAS) we showed that, in the mouth stage of thedigestion, nano-sized silica particles with a size range of5e50 and 50e500 nm were present in food products con-taining E551 or added SAS. However, during the successivegastric digestion stage, this nano-sized silica was no longerpresent. Most likely due to the low gastric pH combinedwith high electrolyte concentrations in the gastric digestionstage large silica agglomerates are formed. This can be ex-plained theoretically by the extended DLVO theory. Impor-tantly, in the subsequent intestinal digestion stage, thenano-sized silica particles reappeared again. These findingssuggest that, upon consumption of foods containing SAS,the gut epithelium is most likely exposed to nano-sized sil-ica (R. Peters et al., 2012). This behaviour has also beenobserved for other nanomaterial like silver (Walczaket al., 2013), where dissolution might also be an issue(Mwilu et al., 2013).

If persistence of the nanoparticles in the gut content hasbeen identified, uptake or bioavailability of the nanopar-ticles needs to be assessed, this would be tier four. Classi-cally monolayers of gut epithelial cells in two chambersystems are used for this. In recent years more complexmodels involving amongst others mucus secreting cellshave been developed that more adequately simulate thecomplex human anatomy of the gut epithelium (Lefebvreet al., 2014). Several studies indicated in vitro intestinaltranslocation potential of nanoparticles (Mahler et al.,2012; des Rieux et al., 2007). However, in these studiesnanoparticles were not exposed to the harsh conditions ofthe human digestive tract that are known to change the


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physicochemical properties of nanoparticles. Physicochem-ical properties of nanoparticles play a crucial role in the in-teractions between nanoparticles and cells. Smallernanoparticles generally translocate more efficiently throughcell layers than larger particles (Mahler et al., 2012; Ohet al., 2011). Also surface charge of nanoparticles affectsthe mucus entrapment and passage of the gut epithelium(Hussain, Jaitley, & Florence, 2001; des Rieux et al.,2007). The combination of size, charge and surface chem-istry affects the formation and composition of a proteincorona around nanoparticles. The composition of the pro-tein corona has been shown to also influence biological in-teractions (Lundqvist, 2013; Meder et al., 2012). Thusrelevant in vitro gut epithelium translocation studies shoulduse nanoparticles that have passed the digestive tract, as itcan be expected to affect the protein corona of the nanopar-ticles. Coupling of in vitro digestion and in vitro transloca-tion models has been performed for the in vitrobioavailability assessment of metals (Oomen, Tolls, Sips,& Groten, 2003) and is now explored for nanoparticlesusing.

At the moment there is no combination of in vitro ap-proaches that can truly replace chronic oral in vivo studies.While rapidly more knowledge is obtained on the applica-bility and robustness of the in vitro methods using nanopar-ticles, none of the methods have been validated yet. Thus ina last tier of safety assessment of nanoparticles dedicatedin vivo studies might still be required. The number availableoral studies evaluating the uptake, distribution and potentialeffects of nanoparticles is increasing, however only fewstudies can be used to assess the contribution of nanopar-ticle related properties on the observed toxico-kinetic ortoxico-dynamic effects. For nanoparticles that are proneto dissolution like zinc oxide and silver it is important tobe able to distinguish between potential ionic or nanopar-ticle effects. Two 28 day oral exposure studies using silverin nanoparticle and ionic from showed highly comparableorgan distribution and biological effects of both forms ofsilver (Loeschner et al., 2011), and indicated up to twomonth retention of silver in some tissues (M. van derZande et al., 2012). Both studies heavily relied on a com-bination of analytical techniques like the afore mentionedSP-ICP-MS and TEM-EDX.

From a holistic point of view in vivo studies using nano-particles could be used in a classical setup, without support-ing information on agglomeration or dissolution behaviouror without data on the nanoparticle tissue concentrations.However, in trying to understand the mechanism ofobserved effects, or to extrapolate observed effects to hu-man most likely additional information is needed. Thus,when performing oral studies with nanoparticles solubility,and changes in agglomeration state of nanoparticles needsto be assessed. Ideally this is assessed in gut content andtissues directly from exposed animals. In practice this isnot always possible due to low concentration of nanopar-ticles or interference of the matrix with the analytical



instrumentation. Therefore alternative, in vitro digestionapproaches as described above are needed to support thein vivo experiments. This becomes clear from the followingstudies. Compared with TiO2 nanoparticles, ZnO nanopar-ticles demonstrated higher absorption and more extensiveorgan distribution when administered orally. The higher ab-sorption of ZnO than TiO2 nanoparticles might be due tothe higher dissolution rate of ZnO in acidic gastric fluid(Cho et al., 2013), which was not assessed in this study.In a recent 28 and 90 day oral study using synthetic amor-phous silica (SAS) an in vitro digestion model was used toexplain the rheological and dissolution behaviour of SASand this consequences for observed limited silicon uptakein tissues (Meike van der Zande et al., 2014). It was shownthat SAS partially dissolves during passage of the gastroin-testinal tract and that increasing concentrations of SASinduced the formation of stronger gel-like properties ofthe gut content (Meike van der Zande et al., 2014). Oral up-take of organic nanoparticles is studied in relation to nano-medicine and targeted release of bioactive compounds,these studies are not discussed here.

In conclusion, the toxicological assessment of nanopar-ticles in food still relies heavily on oral in vivo studies.The scientific quality of in vivo studies with nanoparticlesincreases if analytical methods are used to characterizenanoparticle related properties in the feed, gut contentand tissues if possible. As the analytical characterizationin tissues is challenging, studying the nanoparticles inin vitro models like the in vitro digestion model can behelpful to study dissolution and agglomeration behaviour.

Categorization approaches for risk assessment ofnanoparticles

In spite of the studies that are being undertaken therestill is not a good understanding of the health effects thatmight arise from exposure to nanoparticles. Knowledgegaps exist in key areas that are essential for predicting po-tential health risks such as routes of exposure, the mecha-nism of uptake of nanoparticles into the body, theparameters that are driving nanoparticles distribution oncethese are inside the body (i.e. toxicokinetics) and theways in which nanoparticles interact with the body’s bio-logical systems (i.e. toxicodynamics). Although more andmore evidence has emerged in the scientific literature thatsome nanoparticles might have negative effects, the rangeof nanoparticles for which comprehensive hazard data areavailable is small. Given the wide diversity of nanoparticlesand observations that different nanoforms with the samechemical composition can have different toxicologicalproperties, it is likely that new approaches that do notrely on conventional toxicity testing will need to be foundto assess potential hazards of nanoparticles.

A number of concepts and approaches have been devel-oped to estimate the risks of nanoparticles. Examples ofthese include the Control Banding Nanotool (Paik, Zalk,& Swuste, 2008; Zalk, Paik, & Swuste, 2009), the


014.08.009


precautionary matrix for synthetic nanoparticles (H€ocket al., 2013) and the Stoffenmanager Nano (Van Duuren-Stuurman et al., 2012). These methods are categorizationmethods to determine an overall risk level for workingwith pure nanoparticles in the laboratory or the workplaceand do not estimate risks for consumers or the environment.Alternative methods use multiple criteria decision analysis(H€ock et al., 2013), which were originally developed fortraditional chemicals but which are now being revised fornanoparticles (Davis, 2007; Hristozov, Gottardo, et al.,2014; Hristozov, Zabeo, et al., 2014; Linkov, Satterstrom,Steevens, Ferguson, & Pleus, 2007; Tervonen et al.,2009). In such a model nanoparticles are clustered invarious risk classes using not only criteria for the physico-chemical properties of nanoparticles but also the nanopar-ticles’ bioavailability, bioaccumulation and toxicpotential. In addition value of information analysis hasbeen proposed to further direct potential additional studies,to beforehand estimate the added potential added value ofthis information (Keisler, Collier, Chu, Sinatra, & Linkov,2014), and to link the outcome of research with decision-making by producers and regulators (Linkov, Bates,Canis, Seager, & Keisler, 2011).

More recently, the NanoRiskCat tool was developed as asystematic approach to assess the hazard and exposure po-tentials for a nanomaterial-based product in a regulatoryperspective (EPA, 2011). The procedure results in fivecolour-coded ranks, three for exposure and two for hazard.The first three dots indicate the potential exposure for pro-fessional end-users, for consumers and for the environment,while the other two dots indicate the potential hazard forhumans and the environment. To evaluate exposure poten-tial for a specific nanomaterial in a nanoproduct a substan-tial amount of information is needed. As this informationrarely available for nanoparticles four potential exposurecategories (high, medium, low, unknown) are used, basedon the location of the nanomaterial in the product or appli-cation. The potential human health hazard is evaluated us-ing a number of criteria including the aspect ratio of thenanomaterial, adverse effects of the bulk form of the nano-material, acute toxicity of the nanomaterial, indications thatthe nanomaterial causes genotoxic, mutagenic, carcino-genic, respiratory, cardiovascular, neurotoxic or reproduc-tive effects in humans or (laboratory) animals or hasorgan specific accumulation been documented. For eachof these endpoints the user has to review the literatureand interpret data from scientific results to guide the tool.For a number of these criteria cut-off values are given forto aid the interpretation of literature results. Combiningall information finally produces the outcome of usingNanoRiskCat which can primarily be used to understandand categorize what is known about the hazard and expo-sure potential of using a given nanomaterial in a givenapplication. A significant strength of NanoRiskCat is thatit can be used even in cases where lack of data hampersthe traditional risk assessment procedures. A significant



weakness of NanoRiskCat is that many of the cut-off valuesused primarily in the environmental hazard evaluation isbased on dose-by-mass which is probably not valid for allnanoparticles. Furthermore, the process by which thecolour code is assigned to human hazards associated withthe nanoform of a given material is based primarily on sci-entific expert judgement.

The above analysis are strongly depending on activeexpert involvement. Inclusion of the newest data in theanalysis is a continuous challenge. A system that poten-tially can overcome these constraints is a decision supportsystem (DSS) that mimics human reasoning and is able toutilize scientific knowledge directly as it becomes availablefrom multiple sources (Marvin et al., 2013). The workingprinciple of a DSS is based on the architecture of the Se-mantic Web (Domingue, Fensel, & Hendler, 2011). In thesemantic web, descriptors of an item (i.e. ontologies) allowintelligent computer applications to support the work of sci-entists in nanoscience and to facilitate the integration withrelated knowledge fields (Hastings et al., 2011). Ontologiesare open in structure. It is simple to add new concepts andrelations to them or even import a complete ontology struc-ture. As a consequence, integration and extension of theDSS with existing and future systems alike is guaranteed.In a DSS ontologies are combined with logical reasoning(i.e. knowledge rules). These rules form the engine todraw conclusions. The knowledge rules can be very diversein character ranging from general rules about nanoparticlesto rules that determine how incomplete data from a source,such as a study, should be weighed and used. Especially, thelatter is important, since many studies suffer from incom-pleteness of data. The formal use of knowledge rules anda structured process of logical reasoning also allows forbacktracking of the reasoning process. This again allowsto provide DSS users with traceability functions whichgive insight in the logical reasoning steps and associatedfactual, and conceptual knowledge (Marvin et al., 2013).A DSS thus helps to identify those nanoparticles and appli-cations that should get priority in the safety assessment.

ConclusionsWe reviewed the currently used and reasonably foreseen

applications of nanoparticles in the agri/feed/food sector.Many of these nanoparticles and applications are at bestin the development stage. Especially in the agriculturalsector only a few materials have reached the marketplaceso far. Most applications of nanoparticles or nanotech-nology are found in food packaging and especially in sup-plements like vitamin and nutrient preparations. The reviewalso shows that organic nanoparticles like nanocarriers andnano delivery systems are by far the most applied nanopar-ticles, now and likely also in the future. Engineered nano-particles in food are presently restricted to materialswhich have a history of use like silica and titanium dioxide.Other important inorganic nanoparticles are silver, silica,clay and titanium dioxide.


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Analytical techniques to characterize pristine nanopar-ticles are well established. However, food, biological sam-ples and agricultural samples are heterogeneous mixturesand therefore require separation or pre-treatment to isolatethe target analyte from the interfering matrix components.Due to its high reactivity nanoparticles can change incomposition and size as a response to changes in their envi-ronment. Only few analytical approaches have been devel-oped so far that can detect nanoparticles in food andbiological samples. Much development is needed on issueslike sample preparation and the increase the analyticalsensitivity in terms of both concentration and smaller sizesof the nanoparticles.

Toxicological assessment of nanoparticles in food reliesmainly on in vivo data. Given the enormous diversity intypes of nanomaterial and applications these cannot beevaluated using animal studies: in vitro alternatives arerequired. Yet, no combination of in vitro approaches cantruly replace chronic oral in vivo studies. In vitro ap-proaches that combine in vitro models for gastrointestinaldigestion and epithelial translocation are under develop-ment. For the time being these in vitro approaches can beused to support data obtained from in vivo experiments.Finally, categorization approaches can be used as ascreening tool to flag nanomaterial use of concern, to setdefault guidance for when regulatory measures are to beimplemented, or to prioritize research efforts or developsafe-by-design nanomaterial products.

AcknowledgementThis research was funded by the Dutch Ministry of Eco-

nomic Affairs.

Supplementary data

Supplementary data related to this article can be found athttp://dx.doi.org/10.1016/j.tifs.2014.08.009.

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http://ec.europa.eu/environment/chemicals/nanotech/pdf/study_inventory.pdf

http://ec.europa.eu/environment/chemicals/nanotech/pdf/study_inventory.pdf
































Documents

State of the safety assessment and current use of nanomaterials in food and food production