Accepted ManuscriptSafety assessment of nanocomposite for food packaging applicationJen-Yi Huang, Xu Li, Weibiao ZhouPII: S0924-2244(15)00169-7DOI: 10.1016/j.tifs.2015.07.002Reference: TIFS 1681To appear in: Trends in Food Science & TechnologyReceived Date: 9 December 2014Revised Date: 1 July 2015Accepted Date: 4 July 2015Please cite this article as: Huang, J.-Y., Li, X., Zhou, W., Safety assessment of nanocomposite for foodpackaging application, Trends in Food Science & Technology (2015), doi: 10.1016/j.tifs.2015.07.002.This is a PDF file of an unedited manuscript that has been accepted for publication. As a service toour customers we are providing this early version of the manuscript. The manuscript will undergocopyediting, typesetting, and review of the resulting proof before it is published in its final form. Pleasenote that during the production process errors may be discovered which could affect the content, and alllegal disclaimers that apply to the journal pertain.MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT1Safety assessment of nanocomposite for food packaging application36 37 Huang, Jen-Yi a, Li, Xu b and Zhou, Weibiao a, b, c, *38 a Food Science and Technology Programme, c/o Department of Chemistry, National39 University of Singapore, 3 Science Drive 3, Singapore 11754340 bInstitute of Materials Research and Engineering, Agency for Science,Technology41 and Research, 3 Research Link, Singapore 11760242 c National University of Singapore (Suzhou) Research Institute, 377 Linquan Street,43 Suzhou Industrial Park, Jiangsu, 215123, Peoples Republic of China44 *Correspondingauthor;e-mail:chmzwb@nus.edu.sg,tel.:+6565163501,FAX:45 +65 6775 789546 47 Abstract48 Whilecompetitionisintenseandinnovationisvitalinthedomainof49 nanotechnology,utilisationofnanocompositesinfoodpackaginghasbecomethe50 mostdevelopedareainthefoodindustry.Migrationofnanocomponentsfrom51 nanocompositepackagingcontactingwithfoodstuffs,isoneofthemostimportant52 concernsinhumanexposuretonanomaterialsandthereforepotentialhealthrisks.53 Risk assessment of nano-packaging materials provides an unique challenge for food54 safety.Thisarticleexploresthecurrenteffortsonmigrationassessmentfor55 nanocompositesinfoodpackaging,andprovidesabetterunderstandingofthe56 existing relevant regulatory setups for protecting public health.57 58 Keywords: nanomaterial; migration; food packaging; safety assessment; regulatory59 framework60 61 62 MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT21.Introduction63 Nanotechnology is by definition the manipulation and utilisation of structures with64 atleastonedimensioninthenanometre-length(atomic,molecular,macromolecular)65 scale,whichcreatesuniquepropertiesandfunctionsfornovelapplications.66 Nanomaterials can occur naturally (e.g. in ashes, as soil particles or biomolecules), be67 produced unintentionally (e.g. in diesel exhaust) or be intentionally engineered (Tiede68 et al. 2008). Nano-food packaging is a new generation of packaging technology based69 onnanomaterials,whichhasbecomeoneofthemostdevelopedareasin70 nanotechnologyandrepresentsaradicalalternativetotheconventionalfood71 packaging.Fillersareentitiesincorporatingintotraditionalpackagingmaterials,72 mostly in low fractions, to improve the properties of the original material (Cushen et73 al.2014b).Reinforcementbyengineeredornaturalnanomaterialsasfillers,has74 appearedtobeaninterestingstrategyforimprovingthefunctionalpropertiesof75 syntheticandbiosourcedmaterials.Twomainapproachesareusedtoproduce76 nanomaterials.Inthetop-downapproach,nanometricstructuresareobtainedby77 size reduction of bulk materials, involving grinding, chemical and laser abrasion. The78 alternativebottom-upapproach,allowsnanostructurestobebuiltfromindividual79 atoms or molecules capable of self-assembling, involving a metal salt that is reduced80 with a reducing agent to reveal the metal in the nanoform.81 Polymer nanocomposites can potentially be used as food packaging materials, and82 can be categorised into four types based on the purpose of use (Chaudhry et al. 2008):83 (1)Improvedpackaging:polymerswithnanofillers,i.e.polymernanocomposite,84 aimingatimprovingthepackagingproperties,suchasbarrierpropertiesagainst85 oxygen, carbon dioxide, volatiles and flavour, temperature control, moisture stability86 and ultraviolet blocking properties, have received much attention due to the potential87 to increase the shelf life of fresh and processed food which are packed under modified88 MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT3atmosphere.Polymernanocompositecanimprovethequalityoffresh,frozen,and89 processedmeat,poultry,andseafoodproductsbyretardingmoistureloss,reducing90 lipid oxidation and discolouration, and enhancing product appearance.91 (2)Activepackaging:incorporationofnanofillerswithantimicrobialorantioxidant92 activities (e.g. silver, zinc oxide, magnesium oxide), which results in an inhibiting or93 retardingeffectonthetargetedacceleratingfactorsofmicrobialgrowthandfood94 spoilage. These active packaging systems can extend the products shelf life, enhance95 food quality and safety and ultimately lead to less food waste (Cushen et al. 2012).96 (3) Intelligent packaging: incorporation of nanosensors into food packaging materials97 improves detection and tracking of the food condition during storage and transport for98 safetyandbiosecuritypurposes.Itcommunicatesinformationto theconsumerbased99 on its ability to monitor, trace, or record external or internal changes in the products100 environment.101 (4) Degradable or compostable biopolymers: biopolymers are notorious for their low102 mechanicalstrength,poorgasbarrierproperties,reducedthermalstabilityandlow103 meltviscosity.Theincorporationofnanofillers,resultinginbiopolymer104 nanocompositematerials,hasrisenasasolutiontotheseproblemsistoimprovethe105 chemical and physical properties of the biopolymer. Biopolymer nanocomposites can106 be used to extend the shelf-life of the fresh products such as fruits and vegetables by107 controlling of respiratory exchange (Akbari et al. 2007).108 As commercialisation of nanotechnologies grows, the public and the governments109 becomemoreconcernedaboutthesafetyeffectsofthewidespreaduseof110 nanocompositesasfoodpackagingmaterials,suchaswhethernanomaterialscan111 migratetowardsthepackagedfoodstuffsanddrinksfrompackagingaswellasthe112 potentialhazardconsumerhealthafternanomaterialsmigrated(Huangetal.2011).113 Thereasonforthisstatusisthatpropertiesofmaterialsatnano-sizecanbe114 MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT4substantially different from conventional bulk forms as well as individual molecules,115 andtheirtoxicityisnotyetcompletelyunderstood.Duetolimitedtoxicitydata116 available, some nanomaterials are not approved in the European Union (EU) (Cushen117 et al. 2014a). In addition to the physicochemical and biological nature, toxicity can be118 dependentontheparticlesize,morphology,andsurfacebehavioursofthe119 nanomaterials(Magnusonetal.2011).Theconsumersafetyimplicationsfrom120 nanotechnologyapplicationsinfoodareintrinsicallylinkedtothelikelihoodand121 extent of exposure through its consumption. Most consumers are concerned about the122 applicationofnano-containingfoodpackagingmaterialsbeforeitssafetyisverified123 byappropriateandaccuratemigrationtestingandexposureassessment.124 Nanomaterialsthathavebeenprovedtomigrateininsignificantlevelsornotto125 migrateareapproved(EFSA2012).Therefore,quantifyinghumanexposuresto126 nanoparticlesmigratingfrompackagingmaterialsisanimportantprocedureto127 determine the risk from an increased use of nanomaterials (Cushen et al. 2014b).128 Migration tests are important for the food packaging sector, and must therefore be129 performed during the introduction process of a nano-packaging material (Cushen et al.130 2013). Approval for the application of a new food packaging material depends on its131 migration assessment. However, limited empirical data are currentlyavailable on the132 potentialmigrationofnanomaterialsfrommanufacturedproductsandtheir133 subsequenteffectonfoodstuff,althoughnumerousnanotechnology-derivedfood134 packaging materials are commercially available in several countries (Song et al. 2011).135 Once the test results are available, potential human exposure assessments are a logical136 progressionbasedonmigrationdata,especiallyinthecaseofmigrationintoreal137 foodstuffs(Cushenetal.2014a).Thoroughexposureassessmentofnanotechnology138 applicationinthefoodpackagingcouldenhanceconsumersknowledgeaboutthe139 risks of migration. A sound foundation should be provided on which products can be140 MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT5commercialisedwithconfidence,orwithdrawntopreventconsumersfrompotential141 hazards.142 Therestofthisarticlepresentsacomprehensivereviewofthelatestmigration143 assessmentsofnanomaterialsfromfoodpackaging.Itstartswithanintroductionof144 variousnanomaterialsrecentlyimplementedintofoodpackaging.Furthermore,an145 overviewofthedifferentanalyticaltechniquesavailableforidentification,146 characterisationandquantificationofnanomaterialsinfoodisprovided.Itthen147 highlightssomerecentresultsofmigrationtestsfornanocompositepackaging.The148 lastpartofthisarticleistosummarisethecurrentregulatoryframeworksinvarious149 judiciaries for new nano-sized components in both food matrix and contact materials150 focusingonthesafetyaspects.Sincerelativelylittleworkhasbeenreportedtodate,151 thisreviewisbeneficialtodevelopappropriatemigrationstudyprotocolsfor152 addressing nanomaterials used in food packaging.153 154 2.Nanomatarials in food packaging155 Because of the unique physical and chemical properties of nanomaterials, various156 inorganicnanomaterialshavebeenrecognisedaspossibleadditivestopolymersto157 enhancetheirperformance.Bybottom-upapproach,nanomaterialscanprovide158 multifunctionalactivity.Reinforcingnanofillersareusefulforvariousapplications,159 includingclayandsilicatenanoplatelets,silicananoparticles,carbonnanotubesand160 graphene(Duncan2011).Thereisabundantevidencetobuildthebenefitsof161 inorganicnanofilleronfoodpackagingmaterials,amongwhichthereareenhanced162 maintenanceofflavour,colourandtexture,improvedstabilityduringstorageand163 transport,decreasedspoilageandbetterappearance(Sorrentinoetal.2007).Metal164 andmetaloxidenanomaterialshavereceivedmuchattentioninantimicrobialactive165 food packaging. Their successfulapplication depends upon controlled synthesis. The166 MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT6solution-phase techniques allow high consistency among products (de Azeredo 2013).167 Metaloxideshavealsobeenincorporatedintocommercialisedproductsdisplaying168 lightactivatedmicrobeinactivation.Packagingmaterialscontainingmetal169 nanoparticleshavebeencommerciallyavailableoutsidetheEUformanyyears,e.g.170 nano-Agembeddedbabybottles(Cushenetal.2012),nano-ZnObasedfilmsfor171 wrapping foodstuffs (Top Nano Technology, Taipei, Taiwan).172 This section summarises the development of the new nano-sized components that173 havebeenusedandproposedintheresearchliteratureandscientificreportsas174 applicabletofoodpackagingstrategies.Table1isasummaryofthelatest175 implementationofengineerednanomaterialsintofoodpackagingsystems,andthe176 findings on their actual performance.177 178 2.1.Clay179 Polymer incorporated with nanoclay is one of the first polymer nanocomposites to180 appear on the market as a novel material for food packaging. Nanoclays are the widest181 commercialapplicationofnanoparticlesandconstitutealmost70%ofthemarket182 volume (Silvestre et al. 2011). This is not only due to the availability and low cost of183 claybutalsotheirsignificantenhancements,relativelysimpleprocessability,high184 stabilityandbenignity(Sorrentinoetal.2007).Themostcommonclaynanoparticle185 utilisedinthesenanocompositesismontmorillonite(MMT),whichisatypeof186 comprehensively available natural clay mineral originated from rocks or volcanic ash.187 MMTclaysbelongtothestructuralfamilyofthe2:1layeredphyllosilicatesor188 smectites,consistingofalayerofedge-sharedaluminumormagnesiumhydroxide189 octahedral locating between two silicon oxide tetrahedral layers. Their dimensions, 1190 nmthickand100to500nminlateralextension,resultinplateletswithhighaspect191 ratio (i.e. the largest to the smallest dimension) of 100 to 500. Clay structure is formed192 MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT7byhundredsofnegativelychargedsilicatesheetlayersperiodicallystackedinto193 particles or tactoids of 8 to 10 mm in diameter (Rodriguez et al. 2013). The imbalance194 of the surface negative charges is due to isomorphous substitution of Si4+ for Al3+ or195 Al3+ for Mg2+ within the silicate layers and it is compensated by exchangeable cations196 (typically Na+ and Ca2+) occupying the space of the interstitial layers (Sorrentino et al.197 2007). The formation of the layer into stacks is separated by a regular van der Waals198 gap, which is called the interlayer or gallery (~1.26 nm) and allows for the multi-layer199 polymer assemblies to be constructed at appropriate conditions.200 Ananocompositecanbeexfoliatedorintercalated,whichdependsonthe201 exfoliationdegreeofnanoparticlesinpolymericmatrices.Ifthenanoparticlesare202 fullydispersedbetweenthepolymericmatrices,thenanocompositeisexfoliated(de203 Abreu et al. 2010). Nanoclay has a natural nano-scaled layer structure, which however,204 comeinplateletclusterswithlittlesurfaceexposed.Therefore,theyshouldbe205 exfoliatedasindividualplateletsandhomogeneouslydispersedwithinthepolymer206 matrix in order to take full advantage of the potentially high surface area, in excess of207 750 m2 g1.208 When dispersed into polymers, nanoclays inherently resist the permeation of gases209 or other substances, and provide substantial improvements in barrier properties of the210 nanocomposite.Theenhancedbarrierperformanceofclay/polymernanocomposites211 arewidelyexplainedonthebasisofamazestructurecreatedbyimpermeableclay212 plates,forcingthepenetratingmoleculestotravelalongertortuouspathtodiffuse213 throughthefilm.Theincreaseinpathlengthisafunctionoftheaspectratioofthe214 clay filler, their volume fraction, degree of dispersion and orientation in the composite.215 The packaging materials incorporating exfoliated nanoparticlesexhibit higher barrier216 performancethanthatwithintercalatednanoparticles(deAbreuetal.2010).217 Nanoclays embedded in food packaging films decrease gas transmission rate in order218 MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT8tokeepthefreshnessofoxygen-sensitivefoodsandprolongtheirshelflife.Afew219 Breweries have been reported to be already using the technology in their beer bottles.220 Thestoragetimeofbeerincreasesfrom11to30weekswhennanoclayis221 incorporated in the bottles (Silvestre et al. 2011).222 Besidesthesubstantialimprovementsingasbarrierpropertiesofpolymer223 composites,nanoparticlescanbeutilisedascarrierofantibacterialagentsand224 additives.Somestudieshavereportedtheincreasedtortuosityeffectofdispersed225 nanoclaysonstabilisingadditivesandefficientlyprolongingretentionorcontrolling226 transfer of antibacterial agents by polymer matrices (Chakraborti et al. 2012; Girdthep227 etal.2014).Acontrolledreleasefrompackagingfilmtothefoodsurfacehasmany228 benefitscomparedtosprayinganddipping.Thiscontrolcanbeespeciallyimportant229 for long-term storage of foods or for imparting specific desirable characteristics, such230 asflavour,toafoodsystem.Asaconsequenceofthis,suchsystemsarevery231 promising in the active packaging field (Sorrentino et al. 2007).232 233 2.2.Silver234 Nanosized particle of silver has emerged as a promising substance in a wide range235 ofapplications,includingfoodpackaging.StableAgnanoparticlescanbeexsitu236 synthesised via the regular borohydride reduction of Ag+ ions and then dispersed into237 a polymerisable formulation. It is however difficult due to the limitation of controlling238 thenanoparticlemonodispersity.Ontheotherhand,nanoparticlescanbeinsitu239 producedinapolymerisablemediumfromprecursorswithbetterdispersionability.240 ThereductionofAgsaltsuchasAgNO3producescolloidalAgwithparticle241 diametersofseveralnanometers.TheAgnanoparticlesproducedbyphysical242 reductionpresentregularshapeandbetterdispersion,thechemicalreduction,onthe243 other hand, exhibits agglomerated particles (de Azeredo 2013). Properties of nano-Ag,244 MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT9such as a low redox potential, could increase the capacity of smaller Ag nanoparticles245 toreleaseAg+comparedwiththeequivalentbulkmaterial.Agnanoparticlesalso246 have a novelty, which can endure high temperatures and has low volatility (Cushen et247 al. 2013).248 Nano-Agisthemostfrequentlyusedmaterialaslaundrydetergents,disinfectant249 sprays,andkitchenutensilsto inhibitthegrowthofmicroorganismsonsurfacesand250 insolutionsduetoitsbroad-spectruminhibitoryactivities.Itiseffectiveagainst251 Gram-positiveandGram-negativebacteria,andhasasignificantantimicrobial252 performanceagainstmultidrug-resistantmicroorganisms(Birlaetal.2009;Lietal.253 2011).WhenAgnanoparticlesareincorporatedandimmobilisedinapolymerfilm,254 theparticlesdisperseevenlyandkeeptheirnano-sizeintothefilm.Such255 Ag-incorporatedfilmmayshowantibacterialactivityforprolongingfoodstuffshelf256 life,whichhasalreadybeenfoundinseveralcommercialfoodcontactmaterials.257 Antibacterial effect in food packaging applications can thus be performed without Ag258 nanoparticlemigration,withpolymeractingascarriersofnano-Agreservoirs259 (Llorens et al. 2012b).260 The small dimension, quanta and large external area effect favour interaction with261 microbialcells,whichgivenano-Agmoreeffectiveantibacterialactivitythanthe262 normal-sizedAgparticles.Itwasfoundtobe10100timesmoreefficientthan263 traditionalAgNO3.Thistranslatedintothat,tobeaseffectiveasAgNO3,only264 0.050.5mgmL1ofAgnanoparticleswasneeded.Laraetal.(2010)reportedthat265 Agnanoparticleswereabletorestrainthemicroorganismgrowthfromtheinitial266 contactwithpathogens,andshowedtheirantibacterialeffectonbacteria.Besides,267 directdamagetocellmembranes,therearebasicallysomecommonproposed268 mechanismsoftheantimicrobialactivityofAgnanoparticles.Metallicnano-Ag269 interacts with dissolved O2 and H+, inducing its antimicrobial effect. Lok et al. (2007)270 MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT10confirmedthattheoxidisedsurfaceofAgatomsoftheAgnanoparticlesisan271 importantparttoperformantimicrobialactivities.Kimetal.(2007)havesuggested272 theproductionofreactiveoxygenspeciesbyAgnanoparticlesandAgionsasa273 mechanismfortheirtoxicity.Inaddition,metallicAgnanoparticlescanactas274 efficientvehiclestoinsertwithinthecellmembraneduetotheirsmallsizeand275 subsequently feasible release Ag ions to the interior of cells in a short time, i.e. Trojan276 Horsemechanism(Limbachetal.2007),inhibitingDNAreplicationandATP277 production. Thus, their antibacterial activity is affected by the availability of ionic Ag278 for bacterial contact (Magaa et al. 2008). The toxicity of Ag nanoparticles is closely279 related to particle size. Regardless of the mechanism, best antimicrobial performance280 wasobtainedfrom110nmofnon-aggregated,welldispersednanoparticleswith281 larger surface areas for Ag+ release, which have higher efficiencies of protein binding,282 andpenetrateporesinmicrobialmembranesmoreeasily.Moreover,aggregation283 degree,surfacecharge,solubilityandsurfacecoatingofAgnanoparticlealsoaffect284 the antibacterial activities (Duncan 2011).285 Besidestheantimicrobialactivity,nano-Agcouldcatalysetheabsorptionand286 decomposition of ethylene emitted from fruit metabolism, which has been postulated287 as anethylene blocker.Fruit and vegetable ripeningcaused by thegaswas therefore288 retarded with the extension of product shelf life.289 290 2.3.Zinc oxide291 ZnOnanoparticlesiscommerciallyproducedbyphysicalvapoursynthesisor292 mechanochemicalprocessing(Casey2006).Theproductioncanbeachievedby293 chemical reactions using different precursors as well, and synthesis methods have also294 beenused,suchashydrothermalsynthesis,thermaldecompositionandprecipitation295 (Espitia et al. 2012). Engineered nanoparticles of ZnO is hoped to enable its use as a296 MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT11 moreaffordableandsaferfoodpackagingsolutionnotonlybecauseoftherecent297 discoveryofitsbiocidalactivity(Emamifaretal.2010;Emamifaretal.2011),but298 alsowhiteappearance,UVblockingpropertiesandcheapnesscomparedwithAg299 nanoparticles.IncorporatingZnOnanoparticlesinpolymermaterialsisbeneficialto300 improve their packaging properties such as barrier properties, mechanical strength and301 stability (Espitia et al. 2012).302 In order to express the antibacterial activities, ZnO nanoparticles must contact or303 penetrate into microbial cells. Depending on the ZnO concentration, their antibacterial304 activitiesmaybebactericidalorbacteriostatic.Moreover,Premanathanetal.(2011)305 reportedthatZnOnanoparticlespresentamoresignificanteffectivenesson306 Gram-positivethanGramnegativemicroorganism.Yamamoto(2001)demonstrated307 that ZnO shows antimicrobial effect stimulated by visible light which increases when308 particlesizedecreases.TheincorporationofZnOnanoparticlesinthepolymerofa309 food packaging material allows interactions between the packaging and the food, and310 thus has a dynamic effect on its preservation (Espitia et al. 2012).311 312 2.4.Titanium dioxide313 Nano-sizedTiO2particlesaresynthesisedbyvariousmethods,inwhichsol-gel314 processingisthemostcommonone.TiO2isawidelystudiedoxidenanoparticlefor315 itsUVblockingpropertyasitisanefficientshort-wavelengthlightabsorberwith316 highphotostability.TiO2nanoparticle-incorporatedfoodpackagingfilmscouldhave317 theextraadvantageofpreventingfoodcomponentfromtheoxidationbyUV318 irradiation while retaining good transparency (Duncan 2011). TiO2 nanoparticles also319 exhibitphotocatalyticactivities,whichareusefulasselfcleaningandantimicrobial320 agents under UVA or black-light illuminations. In food processing, the biocide effect321 ofTiO2nanocompositeswithpolymermaterials,suchasEthylenevinylalcohol322 MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT12(EVOH)(Cerradaetal.2008)andchitosan(Diaz-Visurragaetal.2010),hasbeen323 tested.Xingetal.(2012)reportedthatTiO2showsamorepronouncedantibacterial324 effectonGram-positivethanGram-negativemicroorganism.Inaddition,TiO2325 nanoparticles can be used to produce oxygen scavenger films (Xiao-e et al. 2004).326 327 2.5.Copper/Copper oxide328 NanoparticlesofelementaryCucanbegeneratedbythermalorsonochemical329 reductionmethodsfromcopperhydrazinecarboxilatecomplexesinaqueousmedia.330 NanoparticlesofmetallicCuareeasilyoxidisedduetothesmallredoxpotentialof331 Cu0/Cu2+(Llorensetal.2012a).CombiningCufeatureswiththesize-dependent332 enhancementofnanostructures,finelydispersedbioactiveCunanoparticlesare333 expected to exert an improved disinfecting effect due to their size. It provides higher334 mobilityofreleasedCuions,allowingthemtointeractcloselywithbacterial335 membranes(Conteetal.2013).CuOismainlyproducedbyreductionwith336 borohydride,whichcanbeeasilyoxidisedtoshowantibacterialeffectin337 ammonia-contained media (Kotelnikova et al. 2007).338 339 2.6.Carbon nanotube340 Carbonnanotubesarecylinderswithnanoscalediameterswhichmayconsistof341 one-atom thick single-wall nanotube, or a number of concentric tubes which is called342 multi-wallnanotube.Carbonnanotubeshaveextraordinarilyhighaspectratiosand343 theirelasticmoduluscanbeashighas1TPa(LauandHui2002).Theycouldnot344 onlybeusedinfoodpackagingtoenhancethemechanicalpropertiesofpolymeric345 matrices, but exert powerful antimicrobial effects, possibly because the long and thin346 carbon nanotubes puncture the microbial cells, causing irreversible damages (Kang et347 al. 2007).348 MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT133.Techniques for nanomaterial analysis349 Identification,characterisationandquantificationofnanomaterialsinfood350 matrices are very important for research into the risks of nanoparticles to consumers.351 Risk of a given material cannot only be quantified without an accurate description of352 it.Thefunctionalitiesofthenanomaterialscanchangeindifferentfoodmatrices,353 dependingoncompoundsandthermodynamicconditions.Thisisusuallytruefor354 metal nanoparticles because they need reactive surfaces to achieve theiractivity, and355 thuswouldinteractheavilywithfoodcomponents(Llorensetal.2012b).More356 detailedanalyticaltechniquesareneededtodetectmigrationofcomponentsof357 packaging material into foods due to the complexity among nanomaterials.358 Whenmeasuringnanomaterialsindifferentmatrices,theanalyticaltechniques359 shouldbesensitiveenoughtodetectlowconcentrations,assmallparticlesnormally360 represent only a small part of the total mass. They are not only necessary to generate361 data on concentration and composition but the physical and chemical properties of the362 engineered nanomaterials within the sample. Nevertheless, the actual determination of363 theamountofnanomaterialspresentinthefoodasconsumedisnotalwaysfeasible364 because of the lack of methods for the detection of engineered nanomaterials in food365 matrices.Insuchcomplexmatrices,nosingletechniquecanprovideallthe366 information; extra fractionation procedures and combined methodologies for detection367 areneeded.Forexample,testingofnanoparticlemigrationintofattyfoodsis368 particularlytediousbecauseitisdifficulttoisolateanddeterminelipophilic369 componentspresentatlowconcentrationsinthefatmatrix(deAbreuetal.2010).370 Thus,analyticalmethodologiesmustbeestablishedandstandardisetodetectthe371 presence of nanomaterials in foods or food simulants.372 Inthissection,aselectionofdifferentapproachesthathavebeenshowntobe373 applicabletotheanalysisofnanomaterialsisdiscussed.Drawingexamplesfromthe374 MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT14literature,Table2summarisestheapplicationoftheseanalyticaltechniquesto375 different media, together with the migration studies performed (refer to Section 4).376 377 3.1.Microscopy techniques378 The ideal methodologies to detect and visualise nanoparticle for various properties,379 suchassize,shape,structure,dispersion,andcoagulationstatearemainlyelectronic380 microscope (EM) techniques and related facilities. EM techniques, based on the use of381 accelerated electrons as a source of illumination, have a much higher resolution. The382 mostsalienttoolsaretransmissionelectronmicroscopy(TEM),scanningelectron383 microscopy (SEM) and atomic force microscopy (AFM). Depending on the technique,384 resolutionsdownto0.11nmcanbeachieved.Nonetheless,sinceaverysmall385 proportionofsamplesareanalysed,togetarepresentativedata,hundredsand386 thousands of particles have to be counted, which is exacting, tiresome, and inefficient387 (Liuetal.2012).Moreover,EMisusuallydestructive,indicatingthatthesame388 sample cannot be further analysed by another method for verification.389 In TEM,electrons are transmitted through a specimen to obtainan image, which390 provides2Dinformationaboutnanoparticles:theirshape,morphology,size391 distribution,uniformityandaggregationdegreewitharesolutionat0.1nm(high392 resolutionTEM)(Llorensetal.2012b).Thehigh-resolutionTEMcanevenclearly393 demonstratetheatomlayersofcrystallinesamples.Nevertheless,noliquidsamples394 can be placed in the TEM chamber and proper sample pretreatment is thus necessary395 (Mavrocordatosetal.2007).Forsolutions,thedehydrationcouldcauseunwanted396 particleaggregationandchangesinsurfaceproperties.Chemicalfixationanda397 straining step are often required for biological tissues and other complicated matrices398 to keep their original state and also enhance contrast (Liu et al. 2012).399 InSEM,afocusedbeamofelectronsinteractswithatomsinthesamplesurface,400 MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT15andsecondaryelectrons,producingback-scatteringelectronsandcharacteristic401 signalscanbedetectedforimaging.SEMhasalowerresolutionthanTEM,but402 providessamplessurfacetopographyandcomposition.SEMdevicewithdetectors403 abletoconductathighvacuum,orlowvacuum(~2Torr)isusefulforanalysing404 biologicalspecimens(Llorensetal.2012b).Thesamplesurfaceneedstobe405 conducting,itthushastobecoatedwithalayerofconductivematerial,butthiscan406 cause the information loss. Imaging of nanoparticles in their natural state is critical for407 nanomaterialresearch,noneofthesepretreatmentscanhowevercompletelyavoid408 artifactsresultingfromsampledryingorpreparation.Theseartifactscanbeavoided409 by using environmental SEM (ESEM), which allows specimens to be imaged in their410 originalstatewithoutmodificationorpretreatmentundervariablehumidity,upto411 75% (Doucet et al. 2005).412 AFM provides 3D surface information about solid or liquid samples in tapping or413 contactmodes.Anoscillatingcantileverisscanningoverthesamplesurfaceand414 electrostaticforcesaremeasuredbetweenthetipandthesurface.Thus,AFMis415 capableofrevealingtheshapeofthenanoparticlesandtheroughnessprofileswith416 high resolution, approximately 0.5 nm, with tip dimension as the limiting factor. The417 main strength of an AFM is that it images structures down to 0.11 nm under wet or418 moist conditions. Nevertheless, AFM for food related samples is limited in its ability419 toobtainqualitativeorquantitativeinformationofthesamplecomposition(Tiedeet420 al. 2008).421 422 3.2.Spectroscopic technique423 Spectroscopic techniques are used for nanomaterial characterisation and analysis,424 such as determinations of the size and agglomeration in dispersions or solution from425 1 nm to 10 m.426 MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT16Thequalitativedeterminationofcrystallinenanomaterialscanberealisedby427 applying X-ray diffraction (XRD) techniques. XRD is used to reveal data about the428 elementalcompositionorcrystallographicstructureofnaturalandengineered429 materials based on intensity of a scattered X-raybeam on the specimen. X-rays are430 usedtoproducethediffractionpatternbecausetheirwavelengthistypicallythe431 same order of a few angstroms as the interatomic distances in crystalline solids. This432 techniqueisnon-destructiveandcomplexsamplepretreatmentisnotneeded,433 resultinginitscomprehensiveapplicationinmaterialcharacterisation.XRDisa434 commonly used technique to characterise the MMT exfoliation in polymer materials435 forpackagingusages(Koo,2006).SmallangleX-rayscattering(SAXS)isan436 analyticalmethodderivedfromXRDtechnique.Ithasbeenappliedtoanalysethe437 structureoftheself-assemblednanoparticlessuchasnanotubes,andisusefulfor438 solid and liquid materials.439 Sincetheopticalpropertiesofnanomaterialsaredifferentfromthoseof440 macroscalemetalcounterparts,UV-Visspectroscopyispossibletobeusedfor441 nanoparticlecharacterisation.Duetoitslowcostandeasyoperation,UV-Vis442 spectroscopy is applied as a supporting method to detect the presence of nanoparticles443 andtocharacterisetheirquality.Typically,anincreaseinthenanoparticle444 concentrationleadstoadecreasingUV-Vistransmittance.Thisisbecausethatthe445 largersurfaceareaofnanoparticlesleadstoanincreasedUV-Visabsorption446 efficiency.ThehighestwavelengthofUV-Visabsorptionspectrumisafunctionof447 theaverageparticlesize,andtheinformationaboutparticledispersioncanbegiven448 by its full width at half-maximum (Leopold and Lendl 2003).449 450 3.3.Quantitative analytical techniques451 Quantitative andelemental analyses of migratednanomaterials take place mainly452 MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT17withinductivelycoupledplasmamass(ICP-MS),atomicemission(ICP-AES)and453 opticalemissionspectrometry(ICP-OES).Thehighselectivity,sensitivityand454 accuracymakethemthemostefficienttechniquesdeterminingtracemetalions.455 Sampleconcentrationisnotalimitation,thelimitofdetectionisnormally0.110456 ppm. Results highly depend on the sample complexity. Because of its high selectivity457 andsensitivity,ICP-MSismorefavouredthanICP-OESandICP-AES.Whenthe458 nanomaterialisintroducedintotheICP,theatomsoftheanalyteproduceaflashof459 gaseous ions in the plasma, which are measured as a single pulse by the detector and460 appearasaspikeinthegraph(EchegoyenandNern2013).Thepresenceof461 nanomaterialscouldclogthesampletipswithinthespraychamber,andalsothe462 existenceoffoodcomponentsmayhindercompletesampleatomization,adigestion463 process of the sample is therefore needed before the sample is pipetted and analysed.464 Differentiating metal in the samples whether present in nanoparticulate form or ionic465 form are key aspects that should be investigated.466 InthecaseofICP-MS,samplescannotonlybeinjecteddirectlyintotheion467 sourcebutviaacombinedtechnique,suchashydrodynamicchromatography468 (HDC-ICP-MS)(Tiedeetal.2010)andflowfieldfractionation(FFF-ICP-MS)469 (Loeschneretal.2013).HDCseparatesparticlesonthebasisoftheparticles470 diffusioncoefficients,whichareinverselyrelatedtotheirhydrodynamicdiameters471 throughtheStokes-Einsteinequation.Minimalsamplepreparationisrequiredand472 minimalsampleperturbationoccursduringthepassageofthesamplethroughthe473 HDC column (Proulx and Wilkinson 2014). In FFF an external field force or gradient474 causes differential retention of nanoparticles according to their hydrodynamic radius.475 Following the time- and size-resolved elution, the analyte particles are carried to one476 orseveraldetectors.FFFiswell-suitedfordeterminingthesizedistributionof477 particles suspended in the food simulant used for the migration study(Schmidt et al.478 MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT182009).However,bothHDCandFFFareunabletodealwithverylownanoparticle479 concentrations at the ng/L level. Single particle ICP-MS (SP-ICP-MS) is an emerging480 analyticaltechniquethatoperatesbyintroducingmetal-basednanoparticlesintothe481 ICP-MSindividuallyandthenrecordingthetime-resolvedpeakforeachparticle482 within each short dwell time. The intensity of the respective peaks gives information483 abouttheparticlesize,thenumberofpeaksgivesinformationabouttheparticle484 concentration. At the same time, dissolved metal is detectedas a constant signal and485 canthereforebedistinguishedfromtheparticlesignal(Telgmannetal.2014).486 SP-ICP-MScanintheorybeusedtomeasureparticlesizesof10nmorlowerwith487 very low concentration detection limits, normally at part per trillion levels.488 Atomic absorption spectrometry (AAS) is an effective technique, alternating ICP,489 todetectnanomaterials,becauseofitshighspeed,precision,sensitivity,alsothe490 relativecheapness.Itisusedmostlyforquantifyingtheconcentrationofaparticular491 metal element within samples like food and biomaterials by measuring the amount of492 energyintheformofphotonsoflightthatareabsorbedbythechemicalelementof493 interest. This is done byreading the spectra produced when the sample is excited by494 radiation.Typically,thetechniquemakesuseofflame(FAAS)orgraphitefurnace495 (GFAAS)toatomisethesample.Adetectormeasuresthewavelengthsoflight496 transmittedbythesample,andcomparesthemtothewavelengthswhichoriginally497 passedthroughthesample(GarcaandBez2012).Nevertheless,AAShaslimited498 linearrange,andisfailedformulti-elementanalysis(Liuetal.2012).Another499 particularchallengeisaccuratelydistinguishingrelativelysmallnanoparticlesfrom500 dissolved ion signals.501 502 4.Migration assessment of nanocomposite packaging503 Migration is the mass transfer process by which low molecular mass constituents504 MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT19initiallypresentinthepackagearereleasedintothecontainedproduct.Since505 packaging materials are not chemically inert and, direct contact between the package506 andtheproductpackagedcanleadtosubstancemigrationintotheproduct.Infood507 packaging,thisprocessiscriticalsincethenon-intendedtransferofundesirable508 packagingconstituentsmayaffectthefoodsafetyfortheconsumer(Torresetal.509 2012).In active packaging, nanomaterials maymigrate into food once present in the510 foodpackagingmaterials.Themigrationofnanomaterialscouldeventuallyinduce511 organolepticchangesoffoods.Forexample,TiO2couldcauserancidityresulting512 fromlipidoxidationinlipidfoods(deAzeredo2013).Thepotentialriskfrom513 nanotechnologyhastobedefinedbetterintermsofthepropertiesofthe514 nanomaterialsandtheirtransferrate.Becauseofpoorpackagingperformanceand515 subsequentmigrationofnanomaterialsfromthepackaging,ingestionoffoods516 previouslyincontactwithnano-packagingcanbeanexposureroute(Cushenetal.517 2012).Forinvestigatingthepossibilityofapplyingnanomaterialstothefood518 packagingusageandassessthesafetyofapackageincontactwithfoodstuffs,itis519 necessary to carry out migration analysis under controlled conditions, which depends520 directly on the foodstuffs or the type of food simulant tested.521 Thissectionreviewscriticallythecurrentstateofexperimentalandtheoretical522 investigationsofmigrationfromfoodpackagingcontainingnanomaterial.Table2523 providesasummaryofcurrentlyavailablemigrationstudiesforfoodpackaging524 nanomaterials, including study conditions and key observations.525 526 4.1.General aspects of nanomaterial migration527 Themigrationprocessinvolvestwostages.Theinitialmigrationmustbedueto528 those nanomaterials, which are encapsulated within the surface layers. The subsequent529 releaseofnanomaterialfromtheinteriorpartofthespecimenhastopassthrough530 MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT20voids and other gaps between the polymer molecules, which will depend on polymer531 propertiessuchasdensity,crystallinityanddegreeofcrosslinkingandbranching.In532 somecases,thenanomaterialsencapsulatedwellinsidethefilmneedtooxidiseand533 migrateoutthroughthepolymermatrices.Thesenanomaterialsarepredominately534 responsible for the release at later times (Huang et al. 2011).535 Howfastandtowhatextentmigrationofnanomaterialintofoodwilloccur536 depend on the chemical and physical properties of food and polymer. Various factors537 suchasoriginalconcentration,particlesize,molecularweight,solubilityand538 diffusivity of the specific substance in the polymer, as well as pH value, temperature,539 polymerstructureandviscosity,mechanicalstress,contacttime,andfood540 compositionarethemaincontrollingparametersinmigration.Thesolubilityof541 metallicnanoparticleinaqueoussolutionincreaseswithincreasingtemperatureand542 decreasing pH value, which will increase migration of metal in the system (Song et al.543 2011).Migrationofnanomaterialwithahigherratecouldbeattributedtoan544 increasingdiffusivitythatmaybeexpectableinpolymericmatriceswithlower545 molecular weight and thus larger free volume (Schmidt et al. 2011). Migration rate of546 asystemincreaseswhennanoparticlesizeandpolymerdynamicviscositydecrease547 (Cushen et al. 2012). If the food itself has a high affinity for the polymer, then it may548 be absorbed into the package, which causes swelling or plasticisation of the polymer549 matrix,therebyenlargingthegapsandincreasingadditivemigrationrates.For550 instance,foodcomponents,particularlyfat,thatmigrateintoplastics,like551 polyethylene(PE)orpolypropylene(PP),willconsiderablyincreasethemobilityof552 plasticcomponents,thus,enhancingthemigrationintothecontainedfood.Organic553 chemicaladditives,suchasplasticisers,stabilisers,antioxidants,etc.,intheplastic554 may also migrate into food, which would reduce the solubility of nanomaterials in the555 foodmatrixandfurtherimpedetheirmigrationintofood(Songetal.2011).In556 MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT21addition, the migration of nanomaterials is sensitive to food preservation/sterilisation557 methods.EchegoyenandNern(2013)reportedthatmicrowaveheatingwould558 acceleratethemigrationofsilverionduetostructuralmodificationoftheplastic559 when exposed to microwaves.560 Somenanomaterialshavehighsurfaceareaandactivesurfacechemistrywhich561 may cause chemical reactions. It is thus problematic that the use of nanomaterials may562 give rise to formation of unexpected reaction products during the packaging material563 fabrication(Bradleyetal.2011),orcouldpotentiateorslowdownthemigrationof564 non-nanoingredients.deAbreuetal.(2010)revealedthattheincorporationofclay565 nanoparticles in a polyamide (PA) film causes a decreasing migration rate of triclosan,566 caprolactam and trans-,trans-1,4-diphenyl-1,3-dibutadiene up to six times. It could be567 attributed not only to the tortuous path created by the nanoparticles but to the potential568 adhesion phenomena.569 Nanocompositefilmcannotonlybeusedforpackagingbutalsoas570 two-dimensional delivery systems, from which migration of nutrient supplements into571 thefoodcanbeintended.Forexample,packagingisabletoreleasefunctional572 additives depending on the nutritional needs and tastes, including mineral, probiotics,573 vitamins,phytochemicals,marineoils,prebioticsandotheractivematterontothe574 foodsurfacesinacontrolled,systematicmanner(Rodriguezetal.2013).Thesewill575 beregardedasfoodadditivesunderlegislation,andcouldreducetheamountof576 chemicaladditiveneeded,comparedtohavingtodistributetheadditivethroughout577 thewholefood(Bradleyetal.2011).Theseinnovativetechnologiesgenerallydeal578 withfunctionalingredientswhichcanbedirectlyincludedinthepackageorcoating579 materialstogeneratehealthierfoodswithupgradingnutritionalvaluethrough580 migration.581 582 MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT224.2.Experimental approaches583 Migrationtestsareanimportantaspectofsafetyassessments.Whetherornota584 newpackagingmaterialcontainsnanomaterialsissubjecttomigrationtestas585 establishedbyCommissionRegulation(EU)No10/2011(TheCouncilofthe586 EuropeanCommunities2011)onplasticmaterialscontactingwithfoodstuff.This587 regulation also involves The Union List: an useful annex for reference associated with588 migrating substances (Cushen et al. 2014b). Although the best approach is to perform589 migrationtestwithrealfoodmatrices,sincemostfoodstuffshavecomplex590 compositions,itisanalyticallydifficult,tediousandtimeconsumingtodirectly591 measure the migration into a food. The typical valid route to assess the mass transport592 process is to evaluate the specificand overall migration of targeted substances using593 foodsimulantsaccordingtothespecificationsofeachcase(BusoloandLagaron594 2012). Food simulants are selected model systems that produce similar interactions to595 thoseofthefood.Thisimpliesthatboththeextentandkineticsofmasstransport596 should be similar (Hernndez-Munoz et al. 2002). According to the norm UNE-ENV597 13130-1(SpanishAssociationforStandardisationandCertification2005),wateris598 considered as a food simulant for a wide range of food products, such as bread, fresh599 fruits and vegetables, meat and fish, among others. The use of slightly acidified water600 solutions(e.g.containing3%aceticacid)isalsousedasanevenmoreaggressive601 foodsimulantforacidicaqueousfoodssuchascolaandcarbonatedbeveragesthat602 haveapHlowerthan4.6(Farhoodietal.2014).Alcoholicfoodsandbeveragescan603 be simulated by a solution of 10% ethanol or actual alcoholic strength if concentration604 exceeds 10%. Oils probably yield mass transfer data very similar to those occurring in605 real fatty foods, but analysis is very complicated, due to the numerous oil components606 and their non-volatility. Instead, other pure liquids are used, which vary considerably607 inchemicalnature,rangingfromnon-polarn-heptaneorisooctanetopolar608 MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT23isopropanol.Inthecaseofwater-sensitivematerialsintendedonlytocontaindry609 foodstuffs (e.g. paper, board and bioplastic), alternative tests consider the use of some610 solidsimulants,suchasTenax(modifiedpolyphenyleneoxide)and611 polysaccharide-based gels (Mauricio-Iglesias et al. 2010).612 The extractability of a component from the package can be measured by making it613 contact with the food or food simulant at specific conditions of time, temperature and614 static/dynamicmode(Avellaetal.2005).Time-temperatureconditionsarechosen615 accordingtotheintendeduseofthefinalfoodcontactmaterialorarticle.Council616 Directive97/48/EC(TheCounciloftheEuropeanCommunities1997)establishes617 whatcontacttimes,andtemperaturesaretobeusedinmigrationtestsperformed618 understandardisedconditions.Italsoallowsagreaternumberofpossible619 combinationsoftimesandtemperaturesforvariousprocessingandstorage620 conditions.621 Migration contact can be carried out by different approaches as described in Part 1622 ofEN1186(BritishandEuropeanStandards2002).Totalimmersionistheeasiest623 waytotestmigration.Innerandoutersurfacesofmaterialareincontactwiththe624 simulant,andmigrationoccursfrombothsides.Thinmaterialwillbenearlytotally625 extractedduringmigrationcontact.Thesingle-sidedtestisthemostrealisticwayto626 testpackagingmaterialswhicharewithonlyonesideincontactwiththefoodand627 especiallyrecommendedforasymmetricmultilayerconstructions(Langowski2008).628 For articles in container form, it is often most convenient to test them by filling with629 food. Sealable films can be sealed to pouches and the contact side inside can be filled630 withsimulant.Asanalternativetousingapouch,areversepouchmaybeused.In631 thiscasethesurfaceintendedtocomeintocontactwiththefoodstuffistheouter632 surface and the pouch is exposed to the food simulant by total immersion.633 At the end of the test, an accurate analytical method (see Section 3) is conducted634 MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT24to measure the target substance amount in the food/simulant, and the migration level635 canthusbeestimated.However,sincethereisnostandardisedanalyticalmethodto636 characteriseandquantifynanoparticlesinfoodsimulantsormorecomplicated637 matrices,usingacomplimentarysuiteofaquantitativelyanalyticaltechnique,e.g.638 ICP,withmassconcentrationlevelsdowntotheng/L,modifiedtothefoodsystem639 containingnanoparticlesisthebestapproach(seeTable2)(Cushenetal.2014b).640 Sincespecificmigrationonlyconcernsagivenmigrant,thetotalinteractionof641 packages with foodstuffs is better reported by overall migration, which measures the642 totalamountofallcompoundsmigratingintofoodsindependentlyofmigrant643 composition (Avella et al. 2005).644 645 4.3.Theoretical approaches646 Migrationisafunctionofthemasstransportparametersandthethermodynamic647 equilibria between the contact materials and food (Torres et al. 2012). To estimate the648 magnitude of a migration process from a packaging film into a food it is necessary to649 knowtheconcentrationchangeofmigratingspecieswithtime,ineither thepackage650 or the food. The key point of modelling migration in food contact materials is the way651 ofobtainingtwofundamentalparametersfromthemigrationkinetics:thediffusion652 andpartitioncoefficientsthatarespecificforeachcombinationofmigrant,package653 andfood.Inmostcases,migrationofasubstancefromanelatomericpolymeric654 packagingfilmabovetheglasstransitiontemperatureisoftencontrolledbythe655 moleculardiffusionofthemigrantinthefilm,whichcanbedescribedbyFicks656 second law657 22p pC CDt x = (1) where Cp refers to the concentration of the migrant in the packaging material at time t658 MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT25and position x, and D is the diffusion coefficient which measures the rate at which the659 diffusionprocessoccurs,andmaybeeitherconstantorconcentrationdependent.660 Migrationkineticsisgivenbytherateatwhichthetransferredsubstancesmove661 throughthesystem,whichcanbecharacterisedbythediffusioncoefficient.The662 uni-directional migrant transport from package to food takes place at the contact side663 and the mass balance is described as:664 f f ppfV C CK DA t x = (2) where Vf is the volume of food and A is the contact area. Kpf is the partition coefficient,665 definedastheratiooftheconcentrationofthemigrantinthepackagingfilmandits666 concentrationinthefoodsystem(Cf)atequilibrium.Theextentofthemigrationis667 given by the thermodynamic equilibrium and can be measured by partition coefficient668 (Hernndez-Munozetal.2002),whichdependsonthesolubilitycoefficient,669 indicating the polymer-food compatibility. For food safety, a large K limits migration670 from packaging material to food; conversely, a lower K indicates that more migrant is671 adsorbedintothefood.Tosimplydescribethemigrationprocess,thefollowing672 assumptionsareoftenconsideredintheliterature(Roduitetal.2005;Stoffersetal.673 2005; Torres et al. 2012): (1) the migrant is initially distributed homogeneously in the674 packagingfilm;(2)theliquidfoodiswellmixedsothatthereisnomigrant675 concentrationgradientinthefood;(3)themigrationfollowsaFickiandiffusive676 processinthepackagingfilmandisnotcontrolledbyotherkineticssteps;(4)677 diffusioncoefficientandpartitioncoefficientareconstantduringmigrationand678 depend only on temperature; and (5)equilibrium exists all the time during migration679 attheinterfaceofpackagingfilmandfoodandisgovernedbythepartition680 coefficient.681 Aconsiderableamountofworkhasbeendevotedtomodellingthetransferof682 MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT26substances used in the production or conversion of packaging materials (Roduit et al.683 2005;Stoffersetal.2005;Torresetal.2012),suchasmonomersandparticularly684 additives like antioxidants and stabilisers. But to date, to the authors' best knowledge,685 therehasbeenrelativelylittleworkreportedonthepredictionofmigrationin686 nanocompositepackaging.AtheoreticalmodellingbasedonFicksdiffusionwas687 developedbyHuangetal.(2015)thatpermitsthepredictionoftheamountof688 nanoclaymigrationfromamultilayernanocompositepackagingfilmtoaqueousand689 fattyfoodsimulants.Themodelwassolvedusingafiniteelementmethodandits690 accuracywassuccessfullydemonstratedbyexperimentalvalidation.Themodelalso691 providedreliableestimatesofthediffusionandpartitioncoefficientsinthesystem,692 whichshowedanArrheniusbehaviour.Thereisatheoreticalmodelputforwardby693 Simonetal.(2008),capableofpredictingandquantifyingnanoparticlesmigrating694 from nanocomposite packaging to food based on physical and chemical properties of695 bothnanoparticlesandpackagingpolymermaterials.Theresultindicatedthatany696 significant migration, even for a long-time contact with foods, may takeplace solely697 inthecaseofverysmallnanoparticleswitharadiusintheorderof1nmfrom698 polymer matrices that have a relatively low dynamic viscosity, and that do not interact699 with the nanoparticles, such as polyolefines (PE, PP). However, further tests on other700 materials are required to establish a better understanding and to validate the modelled701 migrationbehavioursforothernanocomposites.Therefore,thethresholdsizefor702 different migrating nanoparticles may be different since it is affected by nanoparticle703 typeandpolymermatrixchemistry(Schmidtetal.2011).Cushenetal.(2014a)704 developedamigrationandexposuremodelbasedonmathematicallydefined705 migratabilityandsubsequentmigratablesusingtheWilliams-Landel-Ferryequation.706 Themodelaccuratelypredictedthenanosilveramountsobtainedfromthemigration707 tests.vonGoetzetal.(2013)describedthesilverreleaseovertimeusingapower708 MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT27functionandaLagrangianparticletrackingmodelthatsimulatesFickiandiffusion709 through the commercial plastic container. Bott et al. (2014) carried out a mathematical710 modellingofthemigrationofcarbonblacknanoparticlesfromLDPEandPSinto711 different food simulants using the commercial software Migratest Lite 2001 software,712 whichisbasedontheanalyticalsolutionofFickssecondlaw.Theresultsof713 migration modelling based on theoretical considerations were in agreement with their714 experimental findings.715 716 5.Development of regulations for nanocomposite packaging717 Whilenanotechnologyhasshownitspotentialtorevolutionisethereal-world718 applications,anumberofproductshavealreadyemergedonthemarketinthepast719 few years. Data showed that, the commercial nanomaterial market consisted of >1000720 manufacturer self-identified products as of 2011 (Fabrega et al. 2011). A review was721 publisheddealingwithemergingnanotechnologiesthatthereappearedtobemore722 food contact products for packaging, storage, or cooking. They accounted for 17 food723 andbeverageproductsinthedatabase(WoodrowWilsonInternationalCenterfor724 Scholars2008).Besides,nearly9,800productscontainingnano-scalecomponents725 havebeenidentifiedbytheEnvironmentalWorkingGroup(2006).Despitethe726 incrediblesocialandeconomicpotentialofnanotechnology,theglobalmarketfaces727 numeroushurdlesintheregulationoftheseproducts.Currently,regulationsof728 nanotechnologyforfoodindustryapplicationsarelimited.Thissectionreviewsthe729 current worldwide regulatory framework used to assess the safety of nanotechnology,730 specifically in regards to food contact materials.731 732 5.1.European and US legal perspective on food nanotechnology733 Mostapplicationsoffoodnanotechnologyshallbesubjecttosomeformof734 MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT28permissionprocessbeforebeingapprovedforuse.Therearemainconcernsabout735 nanotechnology based on fears regarding the reduced particle size. The small size of736 manynanomaterialscausesthemtoexhibitdifferentchemicalandphysical737 propertiesfromtheirmacroscalechemicalcounterparts,implyingthatfindingsof738 toxicity studies on conventional form of large particles cannot be easily extrapolated739 fordeterminingtheirtoxicityprofiles(Munroetal.2009).Thisisaparticular740 problembecausethegenerallyrecognisedassafe(GRAS)determinationswere741 likelytohavebeenmadeintheabsenceofnanoscalesafetyevaluations.One742 exampleistheuseofnano-TiO2topreventfoodfromcontactingairandmoisture.743 The weight-based limits of the Foodand Drug Administration (FDA) onTiO2 may744 notadequatelyconsiderthepotentialhazardofnano-TiO2(Sandoval2009).The745 limitingfactorinconductingriskassessmentsofnanotechnologiesisthelackof746 bioavailabilityandtoxicokineticsofnanomaterials.Althoughsomeengineered747 nanomaterialshavethepotentialtocauseharmtohumanhealth,itiscurrently748 unclearwhetherthereisenoughscientificevidencetoapplytheprecautionary749 principle to all applications of food nanotechnology. Sufficient new knowledge has750 tobeestablishedonhownanomaterialbasedprocessescouldaffecthumanhealth751 beforegaining insight in their potential hazard and associated risks.Information on752 thetypeofnanomaterialsappliedandtheirclaimedaddedvaluefortheproductis753 essentialforestablishinganyregulationinthisfieldtoprovideconsistentand754 comprehensive screening and protection for consumers.755 Nowadays, there are no internationally agreed research protocols or standards. The756 provisionofdatahasnotbeenrequiredonparticlesizeandsomecommon757 nanomaterials, such as nanoclaysand metal oxides, may thus beauthorised although758 notpreciselyinnano-sizedforms(Bradleyetal.2011).TheUSAandtheEUare759 examples of administrative authorities first to adapt to regulating nanotechnologies in760 MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT29theareaoffood.IntheEuropeanfoodlaws,therewasnoprovisionforthe761 developmentofspecificregulationstoregardnanomaterialsasaseparateclassof762 materialsin2009,buttheEuropeanParliamentrespondedtotheCommissions763 communicationonnanomaterialregulations,andcontestedtheCommissionsclaim764 thatexistingregulationwassufficient(EuropeanCommission2007).AnInstituteof765 Food Science and Technology (IFST) report has recommended that nanomaterials be766 dealtwithasnew,potentiallyharmfulmaterials,untiltheirsafetyisproved.The767 EuropeanFoodSafetyAuthority(EFSA)statedthataprudent,case-by-caserisk768 assessmentisrequiredforsubstancesdeliberatelyengineeredtoparticlesizewhich769 exhibitfunctional,physicalandchemicalpropertiesthatsignificantlydifferfrom770 thoseatalargerscaleuntilmoreinformationisavailableaboutnanoscienceand771 nanotechnologies (EFSA 2009). The EFSA has also provided a guidance document on772 thepotentialriskassessmentofnanomaterialsforfood-relateduses,whichreports773 requirementsforthedetection,identificationandcharacterisationofnanomaterials774 (EFSA2011).Studiesoninvitroandinvivotoxicityarerequiredifitcannotbe775 provedthatthereisnonanomaterialmigrationafterformulation,andalso776 nanomaterial transforms neither before nor during digestion (Llorens et al. 2012b).777 IntheUSA,variousfederalagenciesareinchargeofnanotechnology-based778 products. In 2006, the FDA formed the Nanotechnology Task Force, which is charged779 withdevelopingregulatoryapproachestonanobasedproductsthatwillensuresafety780 andefficacy,whilealsofacilitatingbeneficialtechnologicalinnovation.Itissueda781 reportrecommendingevaluationofagencyguidancethatcouldpointoutwhatdata782 manufacturerhastoreporttotheFDAonnano-products(Silvestreetal.2011).783 However, the regulatoryframework of the FDA is challenged by the complexities of784 nanotechnologiesanditisconsideredthatresearchonriskassessmentisnot785 advancing at a sufficient rate to handle progresses in nanotechnologies, since the use786 MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT30of nanomaterials could change the legislative status of products. Developing a scheme787 to integrate nanotechnology into the FDA-regulated products is a problem (Sandoval788 2009).Thereisagapexistingbetweenconsumersexpectationsandcommercial789 developmentsaboutregulatingnanotechnologies,despitemostmanufacturersof790 industry,notsurprisingly,believethattheFDAsexistinglegislationisadequatefor791 nano-basedproducts.ToaddressthisshortcomingthenatureoftheUSsystemof792 regulatoryauthoritycompartmentalisesthelegislationofproducts.ItistheFDAs793 responsibilitytoensurethesafetyofnano-basedproductsandupdateitschemistry794 guidance documents to consider substances in which size is technically critical (FDA795 2011).Case-by-caseassessmentofproductshasbeenthestandardsincetheFDAs796 inception, and this will continue to be the case. Nonetheless, as there are currently no797 labellingrequirements,itcouldbedifficultfortheFDAtomonitornano-based798 products and assess whether they have safety concerns (Sandoval 2009).799 800 5.2.Risk assessment and compliance of food nano-packaging801 Thecomponentsofplasticpackagingmaterialsrequirepre-marketpermission802 withasafetyassessment,accordingtochemistryandtoxicitydatasubmittedbythe803 manufacturerbasedonguidelinesondatarequirements(Bradleyetal.2011).804 Europeanlegislationcontrolsthecompoundsthatcanbeusedinthemanufactureof805 packagesintendedtoholdfood.ThemainEUregulatoryframeworkstemsfromthe806 Regulation1935/2004/EC(TheCounciloftheEuropeanCommunities2004).This807 regulationonthecomplianceoffoodcontactmaterialsisforanyofitscomponents808 which could migrate into food with intolerable results, instead of dealing with specific809 types of constituent. In Article 3, the risk of migration is stated where it requires that810 anymaterialorarticleintendedtocomeintocontactdirectlyorindirectlywithfood811 shall be sufficiently inert to avoid that their components are transferred into the food812 MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT31inunacceptablequantitiesthatcouldharmhumanhealth,changeorganoleptic813 properties of the food or deteriorate the food. To protect of the consumer health and to814 prevent the foodstuff adulteration, two migration limits have been defined for plastic815 materials.Theoverallmigrationlimit(OML)ofpermittedcompoundsfromplastics816 tobeintocontactwithfoodstuffsis60mg(ofsubstances)/kg(offoodpackagedor817 foodsimulants)inanycase(TheCounciloftheEuropeanCommunities1990).In818 addition,aspecificmigrationlimit(SML)restrainsthemigrationlevelofthose819 substanceswithpotentialhazardoustoxiceffectsintofoodsorfoodsimulants.820 Migrationlimitsareonthebasisofthetolerabledailyintake(TDI)andacceptable821 dailyintake(ADI)recommendedbytheEFSAforthesubstancestudied,andthe822 limitsareassumingthat,throughoutthelifetime,a60-kghumaneats1kgoffood823 packagedinplasticscontainingtheassociatedcompoundatthemaximumapproved824 quantityeveryday.TheEFSAregulationalsoprovidesthesimulantsandtest825 procedures under which relevant tests like overall migrations must be performed (The826 Council of the European Communities 2007). For instance, the conventional value of827 the ratio of the sample area to the simulant amount used in most cases is considered as828 6 dm2/kg (de Abreu et al. 2010).829 ThenewRegulation450/2009/EC(TheCounciloftheEuropeanCommunities830 2009)canbeconsideredasameasurethatlaysdownspecificrulesforactiveand831 intelligent materials and articles to be applied, in addition to the general requirements832 establishedinRegulation1935/2004/EC,fortheirsafeuse.Itappliestotheuseof833 new types of food packaging materials improving food quality and safety of packaged834 foods, and is broad enough to encompass nanomaterials. The regulation states that the835 activecomponentmustbeidentifiedorsupportedbyinformationontheapproved836 applications,alsothemaximumquantityofsubstancesreleasedfromtheactive837 component should be specified. The EFSA is empowered to conduct such assessments838 MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT32andtoofferarecommendationfortheEuropeanCommission(Schmidtetal.2011).839 Authorised compounds placed on a positive list are permitted to be released from the840 foodcontactmaterialwithinspecificlimitstochangethefoodcompositionorits841 sensory properties.842 Restrictionsonplasticadditivesintermsoflimitsontheirmigrationinto843 foodstuffs, in principle, are applicable also to nanomaterials. Current legislation does844 notdistinguishmaterialsproducedbynanotechnologyfromthatroutinelydeveloped845 bystandardmanufacturingmethods.Thenanomaterialsusedformanufacturing846 nano-packagingmaterialsarenotassessedasnewchemicals.Theproductsof847 nanotechnologyarethereforetreatedbyacombinationofgeneralEUfoodlawand848 moreparticularcontrolsonspecificsubstances.Nevertheless,duetopossible849 differencesinthephysicochemicalorbiologicalproperties,thesafemaximum850 migration levels determined for macro-components shall not be applicable in the case851 of their nano-equivalent material. Besides, not only the amount of elements should be852 taken into account when nanomaterials are used, the specific migration of the particles853 themselves must also be considered. Because of its highly developed surface, specific854 toxicology issues could appear, and the migration of nanomaterials, which depends on855 theirstructureandsize,shouldbequantifiedinsteadofameredeterminationofits856 constituent elements. Consequently, for Regulations 450/2009/EC and 1935/2004/EC857 tobevalidforanypotentialriskfromnanomaterials,pro-activetestingofsuch858 materials is required to identify the potential hazard and determine any dose-response.859 Ithastobedeterminedwhetherthepresenttestingprotocolsareeffectivealso860 regardingthepotentialmigrationofnanocomponentsfrommaterialsintofoods.The861 EUlegislationhasbeenamendedandnowdealswiththespecificcaseoflisted862 substancesthathavebeenengineeredasnanomaterials.Inthecaseofthe863 nanomaterialsintendedforfoodcontact,whetherlistedornotlisted,the amendment864 MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT33states that they have to be re-assessed by the EUauthorities regarding their potential865 toxicity, and hence no specific or overall migration limits are definedat the moment866 (Busolo and Lagaron 2012).867 Anexampleofpre-marketpermissionwastheuseoftitaniumnitride(TiN)868 nanoparticlesinpolyethyleneterephthalate(PET)bottles(EFSA2008).TheEFSA869 Scientific Panel on foodcontact materials adopted a positive safety opinion that TiN870 nanoparticle is totally insoluble and chemically inert in all foods and food simulants.871 Ithasbeenusedatalevelupto20mg/kginPETbottlesandhasshownnosignof872 migrationoutoftheplasticdowntothedetectionlimitof5mg/kgfortypical873 hot-fill/pasteurisation orlong-time storage atroom temperature applications. Thus, it874 isnotatoxicologicalriskforfoodandtoxicologicaldataforthisapplicationarenot875 required.876 Inthecaseofdeliberatenanomaterialintendedtobereleased(e.g.smart877 antimicrobial packaging), the nanomaterial should be treated as a food additive rather878 than a packaging component and be controlled from another perspective. Labelling of879 such packaging has to comply with the Food Additive Directive (The Council of the880 European Communities 1989).881 882 6.Conclusion883 Asthefoodpackagingindustrycontinuestoremaincompetitiveinanever884 expanding global market, it is believed that nanotechnologies show many advantages.885 Anemergingissueoffoodsafetyishowtodealwiththecomplianceofallthe886 advancednanomaterialsrecentlydevelopedfornovelpackagingapplications.The887 quality control of the packaged food and therefore the guarantee of human health are888 imperative. Thus, more research is needed to assess whether there is a potential risk of889 indirect food contamination through nanomaterials migration from food packaging. At890 MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT34present,thefollowingissuesshouldbeaddressedtofullyassessthesafetyof891 nanocompositeinfoodpackaging:(i)thoroughphysicochemicalpropertiesofthe892 developednanoparticles;(ii)methodologytodetermineandassessthemigration893 possibilityandprocessofnanoparticles;(iii)consistentandappropriate894 methodologiesforidentifying,characterisingandquantifyingnanomaterialsin895 complicated food matrices; (iv) interrelationships between nanoparticle characteristics896 andtoxicity;(v)dataonthetoxicokineticpropertiesofnanoparticlesafteroral897 consumption and toxicological dose-response relationships.898 The complete knowledge of the extent of food/packaging interactions during their899 contacttimewillaidincontrollingandlimitingmigrationofnanomaterial.The900 identificationofthefactorsimpactingonmigrationisexpectedtoenable901 manufacturerstomoreaccuratelyimprovequalityassurance.Notonlyreductionto902 zeroofmigration,butalsoreliabledataonthenanomaterialeffectsoncustomer903 safetyafterexposurewillalwayshavepriorityandmustremainthemostimportant904 criterion for optimization of packaging material design and manufacturing.905 Themigrationassessmentofnanomaterialsdoesnotimplyrestrainingtheir906 applications in the food packaging, despite legislation and market uptake are hindered907 byuncertaintiesinconsumersafety.Addressingandframingtheregulationsof908 nanomaterialsusageforfoodpackagingisunderwaythroughvariousregionaland909 international agencies. It is believed that if assessed and regulated correctly, these new910 materialsareundoubtedlyimportantforimprovingthedevelopmentsofproductand911 process,andwarrantingtheconsumerstoenjoyhi-techproductssafelyinthefood912 industry.Rigorousmigrationassessmentiscriticaltothesuccessofdeveloping913 science-based regulations that are urgently needed.914 915 Acknowledgements916 MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT35ThisworkwasfundedbytheAgencyforScience,TechnologyandResearch917 (A*STAR),SingaporegrantSERC112-117-0038.SupportfromtheNational918 UniversityofSingapore(Suzhou)ResearchInstituteunderthegrantnumber919 NUSRI2011-007 and Jiangsu Province under the Scientific Research Platform scheme920 is also acknowledged.921 922 923 MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT36References924 An,J.,Zhang,M.,Wang,S.,&Tang,J.(2008).Physical,chemicaland925 microbiologicalchangesinstoredgreenasparagusspearsasaffectedby926 coatingofsilvernanoparticles-PVP.LWT-FoodScienceandTechnology,927 41(6), 1100-1107.928 Akbari,Z.,Ghomashchi,T.,&Moghadam,S.(2007).Improvementinfood929 packagingindustrywithbiobasednanocomposites.InternationalJournalof930 Food Engineering, 3(4), 1556-3758.931 Avella, M., De Vlieger,J. 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