欧盟 真菌毒素

REVIEW

Regulationsrelatingtomycotoxinsinfood

PerspectivesinaglobalandEuropeancontext

HansP.vanEgmond&RonaldC.Schothorst&MarcoA.Jonker

Received:3March2007/Revised:12April2007/Accepted:20April2007/Publishedonline:17May2007#Springer-Verlag2007

AbstractRegulationsrelatingtomycotoxinshavebeenestablishedinmanycountriestoprotecttheconsumerfromtheharmfuleffectsofthesecompounds.Differentfactorsplayaroleinthedecision-makingprocessofsettinglimitsformycotoxins.Theseincludescientificfactors,forexampletheavailabilityoftoxicologicaldataandoccurrencedata,detailedknowledgeaboutpossibilitiesforsamplingandanalysis,andsocio-economicissues.Bytheendof2003,approximately100countries(coveringapproximately85%oftheworld’sinhabitants)hadspecificregulationsordetailedguidelinesformycotoxinsinfood.Theregulationswererelatedtoaflatoxins(B1,B2,G1andG2),aflatoxinM1,trichothecenes(deoxynivalenol,diacetoxyscirpenol,T-2tox-inandHT-2toxin),fumonisins(B1,B2,andB3),agaricacid,ergotalkaloids,ochratoxinA,patulin,phomopsins,sterig-matocystin,andzearalenone.InEurope,andinparticularintheEU,regulatoryandscientificinterestinmycotoxinshasundergoneadevelopmentinthelastdecadefromautono-mousnationalactivitytowardsmoreEU-drivenactivitywithastructuralandnetworkcharacter.HarmonizedEUlimitsnowexistfor40mycotoxin–foodcombinations.Itisexpectedthisnumberwillgrowin2007toapproximately50.ThedirectorindirectinfluenceofEuropeanorganiza-tionsandprogramsontheEUmycotoxinregulatorydevelopmentsissignificant.TheyincludetheEuropeanFoodSafetyAuthority,theScientificCooperationonQuestionsrelatingtoFood,theRapidAlertSystemforFoodandFeed,thecreationofanEUCommunityReferenceLaboratoryforMycotoxinsandamandateoftheECtotheEuropeanStandardizationCommitteeinmethodsforanalysisformyco-H.P.vanEgmond(*):R.C.Schothorst:M.A.JonkerLaboratoryforFoodandResidueAnalysis,

NationalInstituteforPublicHealthandtheEnvironment,P.O.Box1,3720BABilthoven,TheNetherlandse-mail:hp.van.egmond@rivm.nl

toxinsinfood.Largepan-Europeanresearchandnetworkingprojectsas“BioCop”and“MoniQA”arealsoimportant.KeywordsMycotoxin.International.Regulation.Analysis.Foodsafety

Introduction

Mycotoxinsaremetabolitesoffungicapableofhavingacutetoxic,carcinogenic,mutagenic,teratogenic,immuno-toxic,andoestrogeniceffectsinmanandanimals.Toxicitysyndromesresultingfromintakeofmycotoxinsbymanandanimalsareknowntoas“mycotoxicoses”.Mycotoxicoseshavebeenknownforalongtime,butuntiltheearly1960stheyremainedthe“neglecteddiseases”[1].Sincethediscoveryoftheaflatoxinsin1960andsubsequentrecognitionthatmycotoxinsareofsignificanthealthconcerntobothhumansandanimals,regulationsgraduallydevelopedformycotoxinsinfoodandfeed.Intheearlydaysofmycotoxinregulationsthesemeasuresfocusedmainlyontheaflatoxins.Theywereestablishedbyindustrializedcountries,andlimitsoftenhadanadvisoryorguidelinecharacter.Overtheyears,thenumberofcountrieswithknownspecificmycotoxinregulationshasincreasedfrom33in1981[2]to56in1987[3],77in1995[4],and100in2003[5].Currentregulationsencompass13differentmycotoxinsorgroupsofmycotoxins,andspecificlimitshavebeenestablishedformanyfoodandfeedcommoditiesandproducts.

Untilthelate1990ssettingofmycotoxinregulationswasmostlyanationalaffair.Gradually,severaleconomiccommunities(e.g.EU(EuropeanUnion),MERCOSUR(MercadoCómundelSur),AustraliaandNewZealand)harmonizedtheirmycotoxinregulations,therebyoverruling

148existingnationalregulations.Currentregulationsarein-creasinglybasedonscientificopinionsofauthoritativebodies,forexampletheFAO/WHOJointExpertCommitteeonFoodAdditivesoftheUnitedNations(JECFA)andtheEuropeanFoodSafetyAuthority(EFSA).Atthesametime,requirementsforadequatesamplingandanalyticalmethodsputhighdemandsonotherprofessionalorganizations,forexampleAOACInternationalandtheEuropeanStandard-izationCommittee(CEN).Inthisarticlethefactorsaffectingtheconstitutionofmycotoxinregulations,andtheroleofdifferentinternationalorganizationsintheprocessofestablishingandenforcingregulations,willbediscussed.Somegeneralobservationsarepresentedaboutthecurrentsituationonworldwideregulationsformyco-toxinsinfood.ThefocuswillthenbedirectedontheEuropeanUnionandthemanyEuropeanactivitiesrelatedtotheseregulatorydevelopments.

Factorsaffectingthepromulgationofmycotoxinregulations

Avarietyoffactorsmayaffectthepromulgationofmycotoxinlimitsandregulations.Theseinclude:––the–theavailabilityknowledgeavailabilityofoftheoftoxicologicalexposuredistributiondatadataofofofmycotoxinsmycotoxinsmycotoxins

concen-trationswithincommodityorproductlots––the–

legislationavailabilitytheneedforinotherofsufficientcountriesanalyticalmethods

foodwithsupply.

whichtradecontactsexistThefirsttwofactorsprovidetheinformationnecessaryforhazardassessmentandexposureassessment,respec-tively,themainbasesofriskassessment.Riskassessmentisthescientificevaluationoftheprobabilityofoccurrenceofknownorpotentialadversehealtheffectsresultingfromhumanexposuretofood-bornehazards.Itistheprimaryscientificbasisforpromulgationofregulations.Thethirdandfourthfactorsareimportantfactorsenablingpracticalenforcementofmycotoxinregulations,throughadequatesamplingandanalysisprocedures.Thelasttwofactorsaremerelysocio-economicinnaturebutareequallyimportantinthedecision-makingprocesstoestablishmeaningfulregulationsandlimitsformycotoxinsinfoodandfeed.Riskassessment

Regulationsareprimarilybasedonknowntoxiceffects.Forthemycotoxinscurrentlyconsideredmostsignificant(aflatoxinsB1,B2,G1andG2;aflatoxinM1;ochratoxinA;patulin;fumonisinsB1,B2andB3;zearalenone;T-2andHT-2toxins;anddeoxynivalenol)theJointExpertCom-

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mitteeonFoodAdditives(JECFA—ascientificadvisorybodyoftheWorldHealthOrganization(WHO)andtheFoodandAgricultureOrganization(FAO))hasevaluatedtheirhazardinseveralsessions[6–12].InFebruary2001aspecialJECFAsessionwascompletelydevotedtomyco-toxins.Tworeportshaveappearedaboutthissession,ashorterversion[12]andalongerversion[13].Thesereportsprovidegoodanddetailedinsightintotheprocessofriskassessmentofmycotoxins.Thereportsaddressedseveralconcernsaboutthemycotoxinsconsidered—theirpropertiesandmetabolism,toxicologicalstudies,andfinalriskevaluation.WiththemycotoxinevaluationstheCommitteediscussedgeneralconsiderationsonsampling,analyticalmethods,associatedintakeissues,andcontrol.Risksassociatedwithmycotoxinsdependonbothhazardandexposure.Thehazardofmycotoxinstoindividualsisprobablymoreorlessthesameallovertheworld(althoughotherfactorsare,sometimes,alsoimportant,e.g.hepatitisBvirusinfectioninrelationtothehazardofaflatoxins).Exposureisnotthesame,becauseofdifferentlevelsofcontaminationanddietaryhabitsinthevariouspartsoftheworld.Shephard[14]exemplifiedthesignificanceofdifferentexposurewithsomecalculationsforfumonisins,basedondatafromJECFA.JECFAestablishedagroupprovisionalmaximumtolerabledailyintake(PMTDI)forfumonisinsB1,B2,andB3togetherof2μgkg−1bodyweightperday[12].ThisPMTDIiseasilyexceededbyindividualsonamaize-baseddiet,whenmaizeconsump-tionisapproximately400gperpersonperday,asituationfoundinsomepartsofSouthernAfrica.Shephardcalculat-edthatatacontaminationlevelforfumonisinsinmaizeof2000μgkg−1(alevelwithintherangeofcommonregulatorylimits)−1dietaryexposurefora60-kgadultwouldbe13μgkgbodyweightperday,or650%ofthePMTDI.Inthedevelopedworldmaizeintakeisusuallylessthan10gperpersonperday[15]andfoodcontaminatedatlevelsashighas12mgkg−1canbeconsumedbeforedietaryexposureexceedsthePMTDIsetbyJECFA.

OnthebasisofthesedataFig.1hasbeenconstructedtoshowhowconsumptiondatamayaffectthesettingofregulatorylimits.Assumingatolerabledailyintake(TDI)forfumonisinsof120μgperperson(2μgkg−1bodyweight,60kg),Fig.1showswheretheregulatorylimitshouldbeset,dependingonconsumption.TheareaundertheTDIcurveisthe“safearea”,incontrastwiththeareaabovethecurve—theundesiredarea.Asanexample,inacountryinwhichmaizeconsumptionisapprox.15gpercapitaperdayalegallimitof8mgkg−1wouldsufficetopreventtheTDIbeingexceeded.Inanothercountrywheremaizeconsumptionisapprox.125gpercapitaperday,however,alegallimitof1mgkg−1wouldberequiredtogivethesamelevelofprotection.Inrealitythespreadofmaizeconsumptionthroughouttheworldisevenbroader.

AnalBioanalChem(2007)3:147–157Fig.1Effectofdifferentdailymaizeconsumptionbypopula-tionsworldwideonthesettingoflimitsforfumonisins

Limit [mg FUM/kg maize]149

201816141210820TDI curveregulatory limitregulatory limit10 100 Daily maize consumption [g]300400Note,however,thattheexampleaboveissimplified.Forexample,itignoresotherpossiblefoodsourceswhichmaycontributetofumonisinsexposure.Itmayhelp,however,torealizethatharmonisationofregulationspersedoesnotnecessarilyleadtoequalhealthprotection.Italsostressestheimportanceofreliableconsumptiondataifmeaningfulmycotoxinregulationsaretobedeveloped.

Exposureassessmentisanimportantaspectofriskassessment,andreliabledataontheoccurrenceofmycotoxinsindifferentcommoditiesanddataonfoodintakeareneeded.Quantitativeevaluationofintakeofmycotoxinsisquitedifficult.Duringits56thmeetingJECFAstressedtheimpor-tanceofusingvalidatedanalyticalmethodsandapplicationofanalyticalqualityassurance(seealsothesectiononanalysis)toensurethattheresultsofsurveysprovideareliableassessmentofintake[12].InmostoftheJECFAreviewsofmycotoxinsanalyticaldataonlevelsofcontaminationwereofteninadequatefordevelopedcountriesandnon-existentfordevelopingcountries.Becausemostmycotoxincontaminationisheterogeneouslydistributed,adequatesamplingisanotherimportantconsiderationforobtaininginformationonlevelsofcontamination(seealsothesectiononsampling).Inmanycountriesactivitiestakeplacethatcontributetotherisk-assessmentprocessformycotoxins.IntheEU,forexample,animportantroleisplayedbytheEuropeanFoodSafetyAuthority(EFSA).EFSAmakesuse,wherepossible,ofexposureandconsumptiondata,generatedbytheEuropeanSCOOPactivities(seethesection“TheEuropeandimension”formoredetailsaboutEFSAandSCOOP).Sampling

Thedistributionoftheconcentrationofmycotoxinsinproductsisanimportantfactortobeconsideredwhenestablishingregulatorysamplingcriteria.Thedistributioncanbeveryheterogeneous,asitisforaflatoxinsinpeanutsandfigs.Thenumberofcontaminatedpeanutkernelsinalotisusuallyverylowbutthelevelofcontaminationwithinakernelcanbeveryhigh.Ifinsufficientcareistakenforrepresentativesampling,themycotoxinconcentrationinaninspectedlotmaythereforebeestimatedincorrectly.Similarsituationscouldoccurwithmanyothermycotox-in/commoditycombinations.Adequatesamplingisneces-sarytomakejustifiedmanagementdecisionsaboutwhattodowithlotsthatmaybecontaminatedwithmycotoxins.Becauseofvariabilityamongsampletestresults,mycotox-inconcentrationsinlotscannotbedeterminedwith100%certaintybytakingsamplesfromthelot.

Detailedstudiesonthevariabilityassociatedwithsampling,samplepreparationandlaboratorytestingoffeedandfeedcommoditieshavebeenconductedintheUSAbyWhitaker[16].Thisresultedinaseriesofsamplingplansfordifferentmycotoxin–commoditycombinations,outlinedinFig.2[17].Whitakerdevelopedso-calledoperatingcharacteristiccurves(OCcurves)foreachofthese.AnOCcurveisaplot,representingacertainsamplingplan,whichshowsthedependenceoftheprobabilityofacceptingalotwithaparticulartoxinconcentrationonthetoxinconcen-trationinthelot.OCcurvesshowtheriskofacceptanceoflotsthatdonotfulfilregulatoryrequirements(falsenegatives—buyer’srisk)and,atthesametime,theriskofunjustifiedrejection(falsepositives—seller’srisk).Itseemsthatsamplingerrorsareusually(much)largerthansample-preparationerrorsandanalyticalerrors;thisresultsinmuchuncertaintyaboutthetruemycotoxinconcentrationininspectedlots.Increasedsamplesizeshavethepositivebenefitsofreducingbothbuyers’andsellers’risksassociatedwithclassifyinglotsaboveandbelowaspecified

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CommodityMycotoxinmaizepeanutscotton-almonds hazel-wheatbarleycoffeeseednuts aflatoxin fumonisin DON ochratoxinFig.2Samplingplansformycotoxinsindifferentcommodities,asdevelopedbyWhitaker[17]

tolerance,e.g.aregulatorylimit.Achangeofaccept–rejectlimitsalsohasamarkedeffectontheserisks.Regulatoryauthoritiesmustconsiderthesebuyers’andsellers’risks(sometimesalsocalledasconsumers’andproducers’risks)whenestablishingsamplingcriteriaforproductsinwhichmycotoxinsareheterogeneouslydistributed.

Severalcountrieshavegraduallyestablisheddetailedscience-basedsamplingplans,whichtakeintoaccounttheheterogeneousdistributionofmycotoxinsinagriculturalcommodities.ExamplesofofficialsamplingplansformycotoxinsarethoseappliedbytheUSAforaflatoxinsinpeanuts[18]andbytheEUforseveralmycotoxins[19].Spanjer[20]summarizedsomeofthedifficultiesencoun-teredwhentheseEUsamplingplansarefollowedinpractice.ThiswasillustratedbyconsideringanEUsamplingdirectiveforimportcontrolonnutspackedinsacks—twofoodinspectorswouldneedhalfaworkingdaytosampleonlyonecontaineronjustoneship.Spanjerindicatedthatinmanyothercircumstancesthesituationisevenmoreunmanageable—becausethecommodityisloadedinbulkor,justtheopposite,whenitconsistsofconsumerorvacuumpackages.TheEUsamplingdirectiveseemstoofferthepossibilityofalternativesasitstates:“Ifitisnotpossibletocarryoutthemethodofsamplingdescribedabovebecauseoftheunacceptablecommercialconsequencesresultingfromdamagetothelot(becauseofpackagingforms,meansoftransport,etc)analternativemethodofsamplingmaybeappliedprovidedthatitisasrepresentativeaspossibleandisfullydescribedanddocumented”.Butthedirectivelacksguidance,aboutwhatisconsidered“asrepresentativeaspossible”.Spanjerrecommendedsimplifiedsamplingproce-dures,e.g.checkingatimportcontrolonly,toovercomesomeoftheproblems.Analysis

Legislationcallsformonitoringmethods.Reliableanalyt-icalmethodsmustbeavailabletoenableenforcementofthe

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regulationsindailypractice.Inadditiontoreliability,simplicityisdesired,asitwillaffecttheamountofdatageneratedandthepracticalityoftheultimatemeasurestaken.Thereliabilityofmycotoxinanalysisdatacanbeimprovedbyuseofinterlaboratory-validatedmethodsofanalysis(e.g.themethodsofAOACInternationalandmethodsstandardizedbyCEN).Thesemethodshavebeenlargelydevelopedinresponsetoplannedregulationsformycotoxinsorregulationsthatcameintoforce.Therequirementsforthesemethodsweredictatedbytheneeds,i.e.theyhadtobesuitableforthe(planned)regulatedmycotoxin–matrixcombination(s).Thelimitsofdetermi-nationofthemethodshadtobedemonstratedtobelowenoughforpreciseandaccuratedeterminationofthemycotoxinsofinterestatregulatorylevels.Methodswerealsodevelopedandvalidatedfortoxin–matrixcombinationsforwhichtherewerenoregulations(yet),butforwhichthescientificcommunitysawaneed,e.g.forsurveillancepurposes.Thesedevelopmentseasedtheestablishmentofspecificmycotoxinregulations.

AOACcurrentlyhasapproximately45analyticalmethodsfordeterminationofmycotoxins[21].Allhaveundergoneextensivetestingininterlaboratoryvalidationstudies,andsubsequentreviewbytheAOAC’srigorousapprovalprocess.AOACmethodsarereferredtoasofficialmethodsinmycotoxinlegislationinafewdozencountries[5].InEurope,CENmethodsarebecomingincreasinglyimportant.TenmycotoxinmethodshavebeenstandardizedbytheCEN,andthisnumberwillgrowsubstantiallyintheyearstocome(seealsothesection“TheEuropeandimension”).AlthoughCENmycotoxinmethodsarenotmandatoryforofficialfoodcontrolintheEU,allCENmycotoxinmethodscanbeusedintheEUforofficialfood-controlpurposes,becausetheirperformancecharacteristicsfulfilthecriteria,laiddownintheEUregulationforsamplingandanalysis[19].Figure3showsthatworldwideofficialmycotoxinmethodsarestilllargelybasedonchromatographicproce-dures.Thin-layerchromatography,anestablishedseparationtechniqueinmycotoxinmethodology,isstillofmajorimportanceinregulatoryanalysis.

Theuseofgood,validatedmethodsofanalysisisnoguaranteethatreliableanalyticalresultswillbeobtainedinmycotoxindetermination.Analyticalqualityassurance(AQA)isanotherprerequisiteforadequatefood-lawenforce-ment.AQAincludes,wherepossible,theuseof(certified)referencematerials(e.g.CRMssuppliedbytheEuropeanCommission’sJointResearchCentre/InstituteforReferenceMaterialsandMeasurements;JRC/IRMM,seehttp://www.irmm.jrc.be).CRMscurrentlyavailableformycotoxins,andthoseexpectedtobecomeavailableinthenearfuture,arelistedinTable1[22].Veryimportant,especiallyinfreetradeareas,ishowenforcementbodieshandletheissueofmeasurementuncertainty.Europeanlegislationformycotox-

AnalBioanalChem(2007)3:147–157Fig.3Analyticaltechniquesusedformycotoxinregulatoryanalysis.Thesizeoftheballisproportionalwiththefrequencyofuseofthetechnique(in2004)[5]

insnowrequiresthat“Theanalyticalresultmustbereportedasx±U,wherexistheanalyticalresultandUistheexpandedmeasurementuncertainty”[19,23],butdetailedguidancehowtoestimatemeasurementuncertaintyisnotyetprovided.TheCENhasbeenmandatedbytheECtoprepareanewdocumentoncriteriaforstandardizedmethodstodeterminemycotoxinsinthenearfuture.Thisdocumentwillincludediscussionofmeasurementuncertainty(seealsothesection“TheEuropeandimension”).Tradecontactsandfoodsupply

Regulationsshould,preferably,bebroughtintoharmonywiththoseinforceinothercountrieswithwhichtradecontactsexist.Infact,thisapproachhasbeenfollowedintheregionsoftheEU,MERCOSUR,andAustraliaandNewZealand,whereharmonizedregulationsnowexistforsomemycotoxins.Strictregulatoryactionsmayleadcountriestobanorlimittheimportofcommodities;thiscanmakeitdifficultforexportingcountriestofindormaintainmarketsfortheirproducts.Distortionofthemarketbecauseofmycotoxinregulationsinimportingcountriescouldleadtoexportofthelesscontaminatedfoodandfeed,leavinginferiorfoodandfeedforthelocalmarket.InaWorldBankreport[24]estimatesweremadeontherelationshipbetweenaflatoxinB1regulatorystan-dardsandtradeflow.Studiesforcerealsandnutspredictsignificantlossesforexportingcountries(Africa)ifstrin-gentstandards,suchasthoseinforceintheEU,werebeadopted.Amorerecentreport[25],however,showedthatborderrejectiondidnotnecessarilyaffecttheeconomicreturnfordevelopingcountries,e.g.theAfricanshareoftheEUmarketfordriedfruitincreased.Althoughborderrejectionswereirritatingtoexporters,someproducingcountriesincreasedtheirEUmarketshare.

Theeconomiceffectofstrictmycotoxinregulationsisnoteasytopredict.Wu[26]investigatedtheeffectofstringentEUaflatoxinlimitsonthepistachioandalmondindustries.ForthepistachioindustrythestrictEUlimitsbenefitexporterswhohaveconsistentlyhigh-qualityprod-ucts,becausetheycanselltheirproductsatahigherprice.

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Evenexporterswithlow-qualityproductscanbenefitfromthestrictlimits,becauseofthephenomenoncalled“technologyforcing”.Theyadopttechnologiesenablingbettercontrolofaflatoxins.ThishappenedwiththeIranianpistachioindustry.Thealmondindustry,incontrasthasmainlylosers.TheUSalmondindustryhassufferedfromthestrictEUregulations,despitehavingahigh-qualityproduct.EUfoodindustriesalsoaresufferingfromthestrictlimits.Thelimitsreducesupply.Thiscausespricestorise,onlysomeofwhichcanbepassedontotheconsumer.WurecommendedthatEUpolicymakersshouldconsidertheeconomicimpactofstrictmycotoxinlimitsontheirownfoodindustriesandcitizens.Interestingly,thisisalreadyoneoftheobjectivesofthenewEUprojectMoniQA(seealsothesection“TheEuropeandimension”).

Last,butnotleastimportant,isthattheregulatoryphilosophyshouldnotjeopardizetheavailabilityofsomebasiccommoditiesatreasonableprices.Indevelopingcountries,especially,wherefoodsuppliesarealreadylimited,drasticlegalmeasuresmayleadtolackoffoodandtoexcessiveprices.Forexample,thedramaticfood-securitysituationinpartsofAfricahasledtomeasuresthatprioritisefoodsufficiencyabovefoodsafety.Mycotoxinsareanimportantproblem,asisapparentfromoccasionaloutbreaksofhumanmycotoxicoses,forexampleacuteaflatoxicosisinKenyain2004and2005[27],theroleofaflatoxinsinlivercancer,thestuntedgrowthinchildreninwesternAfrica[28],andfumonisinsinoesophagealcancerinSouthAfrica[14].Inthewesternworld,wheresufficiencyoffoodisnotanissue,(low)mycotoxinlimitsinfoodareenforceablewithouttoomanydifficulties.Synopsis

Weighingthevariousfactorsattheinterfaceofscience,foodsecurity,andregulationsisnotatrivialactivityand

Table1BCRandIRMM(certified)referencematerialsdevelopedformycotoxins(February2007)[22]Certifiedreferencematerial

Available

(Re-)

development

AflatoxinM1inmilkpowderxAflatoxinM1calibrantxAflatoxinsinpeanutbutterx(x)

AflatoxinB1inpeanutmealxAflatoxinB1infeedstuff

xAflatoxinsB1,B2,G1,G2calibrantsxOchratoxinAinwheatx

OchratoxinAcalibrantx

DONinmaizeandwheatx

T-2andHT-2calibrantsx

ZearalenoneinmaizexZearalenonecalibrant

x

152commonsenseisamajorfactorinreachingadecision.Publichealthofficialsareconfrontedwithacomplexproblem—mycotoxins,particularlythecarcinogenicmyco-toxins,shouldbeexcludedfromfoodasmuchaspossible.Becausethesubstancesarepresentinfoodsasnaturalcontaminants,however,humanexposurecannotbecom-pletelyprevented,andexposureofthepopulationtosomelevelofthemycotoxinshastobetolerated.Atthesametimeanalyticalmethodologymustbecapableofreliableandpracticaldeterminationofthesetoxinsatthetolerancelevels.Despitethedilemmas,inrecentdecadesmycotoxinregulationshavebeenestablishedinmanycountriesandnewerregulationsarestillbeingdrafted.IntheEuropeanUnion,inparticular,manydevelopmentsareoccurringinmycotoxinregulationwhicharescientificallyunderpinnedandsupportedbyavarietyofEuropeanorganisationsandactivities.

TheinternationalmycotoxinregulatorysituationWorldwidemycotoxinregulations

Severaltimesinrecentdecades(1981,1987,1995,2003)internationalinquirieswereheldandpublishedaboutregulationsformycotoxinsinfoodandfeed[2–5].Themostrecentenquiryin2003wasconductedbytheNationalInstituteforPublicHealthandtheEnvironment,undercontracttotheFAO,amongtheAgriculturalServicesofDutchEmbassiesaroundtheworld,withtherequesttogatherup-to-dateinformationfromlocalauthoritiesonthesituationregardingmycotoxinregulationsinasmanycountriesoftheworldaspossible.Wherethisproceduredidnotleadtothedesiredinformation,personalcontactswereused.Bytheendof2003datahadbeenreceivedfromcountries.Togetherwithinformationgatheredinpreviousenquiries,detailedinformationbecameavailableabouttheexistenceorabsenceofspecificmycotoxinlimitsandregulationsinfoodandfeedin119countries.Allthedatareceivedweresortedbycountryandbyeconomiccommunity(ASEAN,AustraliaandNewZealand,EU,MERCOSUR).Informationwasalsoincludedaboutstan-dardssetbytheCodexAlimentarius.Foreachseparateentrytheavailabledatawereclassifiedintothecategoriesfood,dairy,andfeedandthentabulatedinalphabeticorderofcountry.TheresultsoftheenquirywerepublishedindetailbytheFAOinFoodandNutritionPaperinEnglish,French,Spanish,andChinese[5].

Atleast99countrieshadmycotoxinregulationsforfoodand/orfeedin2003,anincreaseofapproximately30%comparedwith1995.Whereasin199523%oftheworld’sinhabitantslivedinaregionwherenoknownmycotoxin

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regulationswereinforce,thispercentagehasdecreasedto13%in2003,withthebenefitofslightincreasesincoverageinLatinAmericaandEuropeandmoresignificantincreasesinAfrica,Asia,andOceania.Allcountrieswithmycotoxinregulationsin2003hadregulatorylimitsforatleastaflatoxinB1orthesumofaflatoxinsB1,B2,G1,andG2infoodand/orfeed,asituationthatwasalsotruein1995.Rangesandmajorlimitsfortotalaflatoxinsinfoodin2003aredepictedinFig.4forthedifferentregionsoftheworld.Specificregulationsalsoexistforseveralothermycotoxins(aflatoxinM1;thetrichothecenesdeoxynivale-nol,diacetoxyscirpenol,T-2toxinandHT-2toxin;thefumonisinsB1,B2,andB3;agaricacid;theergotalkaloids;ochratoxinA;patulin;phomopsins;sterigmatocystin,andzearalenone).Mostofthelimitsaresetforhumanfoods.Typicallyhigherregulatorylevelsareusedforanimalfeed.Informationaboutalltheregulatorylimitsinforcearoundtheworldforvariouscommodities,withinformationabouttheirlegalbasis,theresponsibleauthorities,themethodsusedforsamplingandanalysis,andaseriesofgraphicalpresentationsoftheregulatorysituations,arepublishedindetailinFAOFoodandNutritionPaper81[5].Thenumberofcountriesregulatingmycotoxinshasincreasedsignifi-cantlyovertheyears.Ifthesituationsin1995and2003arecompareditseemsthatin2003moremycotoxinswereregulatedinmorecommoditiesandproductsandthattolerancelimitsusuallyremainedthesameortendedtodecrease.Regulationshavebecomemorediverseanddetailedwithnewerrequirementswithregardtoofficialproceduresforsamplingandanalyticalmethodology.TheEuropeandimension

InEurope,andinparticularintheEU,regulatoryandscientificinterestinmycotoxinshasundergoneadevelopmentinthelastdecadefromnationalactivitiesandapproachestowardsmoreEU-drivenactivitieswithastructuralandnetworkcharacter.Thiscanbeseennotonlyinthe

Fig.4Ranges(bars)andmajorlimits(triangles)fortotalaflatoxinsinfood[5]

AnalBioanalChem(2007)3:147–157harmonizationof(stringent)legallimitsformycotoxins,butalsointheincreasedsignificancethatEuropeanprogramsandorganizationsplay,indirectorindirectsupportoftheseregulatorydevelopmentsintheCommunity.

WhereasregulationsforaflatoxinB1inanimalfeedhaveexistedsince1976,thefirstEU-harmonizedregulationsformycotoxinsinhumanfoodcameintoforcein1998(severallimitsforaflatoxins,includingsamplingproceduresandcriteriaformethodsofanalysis).Theyhavegraduallyexpandedtoavarietyofmycotoxinsindifferentfoodstuffs,includingbabyandchildrenfoods.Atthetimeofwritinglimitshavebeensetforapproximately40toxin–foodcommoditycombinations[29]andlimitsforafurthertentoxin–commoditycombinationsarecomingintoforceforseveralFusariumtoxinsduring2007.Newlimitswillprobablyalsobeestablishedintheyearstocomeforergotalkaloidsinfoods,andsomelimitsmayberevisedforaflatoxins,dependingonEFSAscientificopinions.TheEFSAisanindependentbodyoftheEuropeanCommission,establishedin2002,andcharged,amongothertasks,withthedevelopmentofriskassessmentsonissuesofconcerninthefoodandfeedsupply.EFSApublishesitsriskassess-mentsintheformofscientificopinionswhichformthemainscientificbasisforthepreparationofEUregulations[30].OpinionsaboutrisksofmycotoxinsinfoodandfeedaredevelopedinEFSA’spaneloncontaminantsinthefoodchain.Asanexample,thispanelhasrecently(March2007;http://www.efsa.europa.eu)publishedanopiniononthepotentialincreaseintherisktoconsumerhealthofapossibleincreaseincurrentmaximumlevelsforaflatoxinsinalmonds,hazelnuts,pistachios,andderivedproducts.Thepanelconcludedthatchangingthemaximumlevelsfortotalaflatoxinsinalmonds,hazelnuts,andpistachiosfrom4to8or10μgkg−1wouldhaveminoreffectsonestimatesofdietaryexposureandcancerrisks.ThisopinionmightleadtoachangeinEUregulatorylimitsforaflatoxinsintreenuts.AnotherimportantEUactivityisSCOOP(ScientificCo-operationonQuestionsrelatingtoFood),fundedbytheEuropeanCommission,andtargetedtomakethebestestimatesofintakeofcontaminantsbyEUinhabitants.Inthe1990stheseactivitiesresultedinareportassessingexposuretoaflatoxins[31].SCOOPreportswerelaterpublishedforseveralothermycotoxinsincludingochra-toxinA[32],patulin[33],andseveralFusariumtoxins(trichothecenes,fumonisins,andzearalenone)[34].TheobjectivesofthisSCOOPactivityistoprovidethescientificbasisforevaluationandmanagementofrisktopublichealtharisingfromdietaryexposuretomycotoxins,takingintoaccountrecentlyavailabledataonoccurrenceandconsumption.SpecialemphasisisplacedonevaluationofdietaryintakeofmycotoxinsineachoftheEUmemberstatesandinhigh-risksub-groupsofthepopulation.DataprovidedbyparticipantsintheSCOOPexerciseswere

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collected,harmonised,andreportedwiththefollowingobjectives:–

toprovideadescription,harmonisedatEuropeanlevel,ofthestatusofmycotoxincontaminationoffoodstuffsineachparticipatingmemberstate;

–togroupavailableinformationoneachrawmaterialand/orfoodproduct;

–toevaluatethebestestimatesofthemycotoxindietaryintakefromfood,bothforeachparticipatingmemberstateandatEuropeanlevel;and

–

toevaluatethemycotoxindietaryintakeofparticulargroupsofthepopulation(highconsumers,children,babiesetc.).

Carehadtobetakenwithinterpretationofthedietaryintakeresults,however,becauseforalimitednumberofpositivesamplesmeanoccurrencelevelsarestronglyaffectedbythelimitofdetectionoftheanalyticalmethodsusedandthiscaneasilycontributetooverestimationofcalculatedtotaldietaryintake.OnemajoroutcomeoftherecentSCOOPactivityonFusariumtoxinswasthatconsumptiondataforspecificpopulationgroups(forexamplechildren)shouldbecollected,toassessmoreaccuratelytheexposureoftheEuropeanpopulation.AnotherwasthatacommonapproachtoassessingdietaryintakeinEuropeancountriesshouldbeelaborated.Figure5showsestimateddietaryintakeofdeoxynivalenolfordifferentpopulationgroupsinseveralEUcountries[35],comparedwiththetolerabledietaryintake(TDI)ofdeoxynivalenol,asestablishedbytheJECFA[13].Figure5makesclearthatinsomecountriestheTDIisexceededforsomeorallintakegroupsofthepopulation.

SCOOPexercisesmakeitclearthatmycotoxinsareamongthechemicalcontaminantsinfoodtowhichEUinhabitantsmaybeexposed.Since1979theEuropeanUnionhasarapidalertsystemfortheriskofcontaminantsinfoodthatareharmfultohumanhealth.TheintroductionoftheGeneralFoodLawin2002[36]wasanimportantstepintheevolutionoftherapidalertsystemforfoodandfeed(RASFF)asitexistsnowadays.Thesysteminvolvesrapidinformationexchangebetweenthecompetentauthor-itiesofthememberstates(includingEFTA/EEAcountries),theEuropeanCommissionitself,andtheEFSA.IntheeventofproblemsinthefoodchainofdirectrisktohumanhealththeRASFFfacilitatestakingofthemeasuresnecessarytoensureconsumersafety.Differentkindsofnotification—newsnotification,informationnotification,andalertnotification—canbearranged,dependingontheextentofriskandtheneedfordirectaction.Newsnotificationisrelatedtoinformationaboutfoodorfeedsafety.Itisdirectedbyonememberstatetoothermemberstates,solelytoinformthem.Informationnotificationdescribesanidentifiedriskoffoodorfeedattheouter

1

300

AnalBioanalChem(2007)3:147–157

Mean intake200% of the TDIHigh level intake100

0

AustrBeialgium; All;De 13-1nm8arkFin;lan AlldFra; Aduncltse; AdFraultsnce; FraFranMcenc;e; Ch FGeildGermrenanrmy; anAy; duYoltsungGe chrmThilane Ny; deBtThhee Nrlaabyndeths;Therle Nan Alldseth: 1erl-4ands 1NoNorw-6rwayay: M: M, conNoNorws.rwayay F F, coNonsrw.ay: BPoabyrtugSwal; edenAll 1UK8: M-74,UK 16-6: F4, 16-UK: 1,5-UK4,5: BabUK: My >65UK: F>6UK5: 4UK-6: 7UK-10: 11UK-14: 15-18Fig.5EstimateddietaryintakeofdeoxynivalenolbydifferentpopulationgroupsinseveralEUcountries,andcomparisonwiththeJECFAtolerabledailyintake[35]

borderoftheEU,butrequiresnoimmediateactionofothermemberstates,althoughtheproblemalsomightexistfortheircountries.Analertnotificationrelatestoproductsthatareonthemarketalready.ItisimmediatelytransmittedbytheCommissiontoothermembersofthenetworkandleadstowithdrawaloftheriskyproductsfromthemarket.

In2005theRASFFreceivedatotalof22informationnotifications[37]ofwhich906relatedtoriskstohumanhealthofmycotoxins.Thismeansthataveryhighpercentagehadtobeascribedtoanidentifiedproblemwithmycotoxins,asillustratedinFig.6.Itcan,moreover,beconcludedfromthefigurethatproblemswithmycotox-insweremuchgreaterthanthoseofothermenaces.Almost90%ofthesemycotoxinnotificationswererelatedtohumanhealthrisksfromaflatoxinsinnuts(pistachionuts,peanuts,hazelnuts,almonds)andnutproducts(peanutbutter)whichwereimportedintotheEUin2005.Othernotificationsconcernedrisksofaflatoxinsinfruitandvegetables(driedfigsandderivedproducts,andmelon),andherbsandspices(chilli,paprika,curry,andnutmeg).NotificationswerealsosentforochratoxinAinthesameproductsasaflatoxinsand,moreover,incoffeeandcoffeeproductsandincerealsandcerealproducts.Finally,amuchsmallernumberofthesenotificationsweresenttothepartnersintherelevantnetworkinrelationtorisksof

fumonisinsincerealproductsandofpatulininbabyfoodandfruitjuices.

RASFFnotificationsprovideusefuldataforthedevelop-mentofnewEUmeasuresasnewCommissionregulationsandsafeguardmeasuressuchasthespecialconditionsforimportofpistachionutsfromIran[38].Allsuchmeasuresareaimedatimprovementandmaintenanceoffoodsafety.SinceJuly2006,theEuropeanCommission’sJointResearchCentre/InstituteforReferenceMaterialsandMeasurements(Geel,Belgium)hasfulfilledtheroleofEUCommunityReferenceLaboratory(CRL)formycotoxins.

vet. medicineproducts6%path. micro-organisms14%Components7%foodadditives6%rest27%mycotoxins40%Fig.6InformationnotificationsdistributedtotherelevantnetworkmembersoftheEUrapidalertsystemforfoodandfeedin2005[37]

AnalBioanalChem(2007)3:147–157Thetasks,duties,andrequirementsofCRLsinrelationtofood,feed,andanimalhealthhavebeenpublished[39].Figure7givesanimpressionofthemainCRLtasksanditspositionintheEU.AmongotherdutiestheCRLformycotoxinshasbeencreatedtotakeinitiativesandtoco-ordinateactivitiesrelatedtothedevelopment,improvement,andapplicationofsamplepreparationandmethodsofanalysisforofficialcontrolofmaximumlevelsformycotoxinsinfoodandfeed.Amongthetasksmentionedisthefunction“toprovidetechnicalassistancetotheCommissionand,uponitsrequest,toparticipateininternationalforarelatingtotheareaofcompetence,concerninginparticularthestandardizationofanalyticalmethodsandtheirimplementation”.So,itisevidentthatthelinkwiththeEuropeanStandardizationCommittee(CEN)willbeimportant.

ThepositionoftheCENwithregardtomycotoxinregulationswillalsobestrengthenedasaconsequenceoftheEuropeanCommission’s“MandateforstandardizationaddressedtoCENinthefieldofmethodsofanalysisformycotoxinsinfood”[40].ThenewmandatefallswithintheframeworkofregulationECno.882/2004[39].ThisregulationstipulatesthatmethodsforsamplingandanalysisusedinthecontextofofficialcontrolshallcomplywithrelevantCommunityrulesor,ifnosuchrulesexist,withinternationallyrecognizedrulesorprotocols,forexamplethosethattheCENhasaccepted.TheviewoftheEuropeanCommissiononCENstandardsisclear:“TheestablishmentofstandardizedmethodsofanalysisisofutmostimportancetoguaranteeauniformapplicationandcontroloftheEuropeanlegislationinallMembersStates.Standardizedmethodsofanalysisareanindispensableelementinguaranteeingahighleveloffoodsafety”.

Inthetextofthemandateastatementismadethatelaborationofthestandardsshouldbeundertakeninco-operationwiththebroadestpossiblerangeofinterestedgroups.

Fig.7TasksandpositioningoftheEUCommunityReferenceLaboratoryformycotoxins.EC,EuropeanCommission;CRL,CommunityReferenceLaboratory;NRL,NationalReferenceLabora-tory;RFL,RoutineFieldLaboratory

155

Twelvesuch(mainlyEuropean)organizationsarespecificallymentionedinthemandate,whichshowstheimportancethattheEuropeanCommissionattachestoabroadsupportforthestandardizedmethods.Intheannexofthemandatethemethodsofanalysisarespecificallymentioned.Theserelatetopatulin,deoxynivalenol,zearalenone,aflatoxinB1,ochra-toxinA,andfumonisinsB1andB2inavarietyoffoodstuffs.Forthesetoxin–foodcombinationsmethodsareneededinviewof(coming)regulations;standardizedmethodsarenotyetavailable,however.Oneofalistoftaskstobeundertakenisthepreparationofareviewofupdatedandextendedperformancecriteriaformethodsofanalysisofmycotoxins.TheobjectiveistoproduceanupdatedandextendedversionofthecurrentCENcriteriadocument[41],whichhasbeenthebasisforstipulatingperformancecriteriaformycotoxinmethodsincurrentEUlegislation.ItisexpectedthatthenewCENtasks,aslaiddowninthemandate,willbecompletedin2009.

InadditiontotheguidingorsupportingroleoftheEuropeanorganisationsandstructuralactivitiesdescribedaboveinestablishmentofmycotoxinregulationsintheEU,resultsfromseveralEC-fundedresearchprojectsmayalsoaffectEUmycotoxinregulations.WithoutintentionallyignoringotherrelevantECprojects,examplesofprojectswithsignificantpotentialimpactinclude“BioCop”and“MoniQA”.ThesetwoprojectsarefundedintheEC’s6thFrameworkProgramme.

“BioCop”(http://www.biocop.org)isanintegratedproject(IP)withmorethan30partnersfocussingon“newtechnologiestoscreenmultiplechemicalcontaminantsinfoods”.Thispan-Europeanprojectrunsfrom2005until2010.BioCopmethodsarebasednotonchromatographybutonnewbiotechnology,forexampletranscriptomics,proteo-mics,andbiosensors.Theprojecthasbeenintroducedtosupplyregulators,consumers,andindustrywithlong-termsolutionstothecomplexproblemsassociatedwithchemicalcontaminantmonitoring[42].Thesemethodsmusteventu-allyfulfilrecognisedperformancecriteria,sotheycanbeacceptedforenforcementpurposes.Oneoftheworkpack-agesofBiocopistodealwithmycotoxins,morespecificallywiththedevelopmentofnewmethods,includinguseoftranscriptomics(identificationofchemicalresiduesbygenomicfingerprints)andsensors(bothlow-cost(electro-chemical)portabledevicesandsurfaceplasmonresonance(SPR)sensors)foranalysisofaselectionoftrichothecenesofEuropeaninterest(deoxynivalenol,nivalenol,andT-2andHT-2toxins).Ifsuccessfulinthefirstphase,themethodswillbevalidatedininterlaboratorytestsandstandardizedbytheCENworkinggroupBiotoxins,possiblyintheperiodaftertheprojecthasbeenfinalized.BecausemycotoxinmethodspermittedbyEUlegislationarebasedonperfor-mancecriteria,thepotentialofBioCop-developedmethodsforregulatoryuseissignificant.

156“MoniQA”(http://www.moniqa.org)isanetworkofexcellence(NoE),atthetimeofwritingjuststartedtorunintheperiod2007–2011.MoniQAhasmorethan60partners.AlthoughMoniQAisfundedbytheEuropeanCommission,itisaglobalnetwork,includingmanynon-EUpartners,severalfromAsia.Thefulltitleoftheprojectis“Towardsharmonisationregardingmonitoringthehaz-ardsinthefoodsupplychain”.InMoniQAthecoreconsortiumseekstoestablishmechanismsforcoordinatingandfinallymergingresearchactivities,personnel,andinfrastructure[43].MoniQAisdirectedtowardmultidisci-plinarythemes,named“clusters”.Oneofthemainclustersis“mycotoxins”.Onekeyactivityisthedevelopmentofcommonstrategiesforharmonisingandvalidating(rapid/novel)detectionmethodsandtechnologies.ThiscouldpossiblyincludemethodssuchasthosedevelopedinBioCop.AspecificworkpackageofMoniQAwillevaluatetheeconomiceffectofimplementationoftheanalyticalmethodsrequiredbythenewregulationsinthefieldoffoodqualityandsafety(e.g.effectiveness,efficiency,andconsistency),andtocomparetheeffectofdifferentoptionsinqualitative,quantitative,andmonetaryterms.Risk–benefitanalysisisobviouslyreceivingmoreattention.WiththefundingofBioCopandMoniQAtheEuropeanCommissionhasnotceaseditsinterestinadvancingthedevelopmentandstandardizationofanalyticalmethodsandsamplingmethodsforcontaminants,includingmycotoxins.Inthenewlylaunched7thFrameworkProgramme(rangingfrom2007to2013)largecollaborativeprojectsareforeseeninwhich(rapid)methodsformycotoxins,sampling,andstandardizationissueswillreceiveattention.

Conclusions

Bytheendof2003approximately100countriesintheworldhadspecificregulationsinfoodandfeed.Thenumberofcountriesregulatingmycotoxinshassignificant-lyincreasedovertheyears.Comparisonofthesituationsin1995and2003revealsthatin2003moremycotoxinswereregulatedinmorecommoditiesandproductsandthattolerancelimitsgenerallyremainedthesameortendedtodecrease.Regulationshavebecomemorediverseanddetailedwithnewerrequirementswithregardtoofficialsamplingproceduresandanalyticalmethods.Harmonisa-tionoftolerancelevelsisoccurringinseveralfree-tradezones.IntheEU,especially,thenumberofharmonizedregulationsformycotoxinsinfoodhasrapidlygrown,andwillgrowfurtherintheyearstocome.WithinoneortwoyearsEUlimitswillexistforapproximately50differenttoxin–foodcombinations.Thesedevelopmentsarescientif-icallyunderpinned,supported,andflankedbythecomple-mentaryactivitiesofavarietyofEuropeanorganizationsor

AnalBioanalChem(2007)3:147–157

programmes(e.g.EFSA,SCOOP,RASFF,CRL,andCEN).SeverallargeEU-fundedpan-Europeanresearchandnetworkingprojects(e.g.BioCop,MoniQA)alsohaveaneffect.Itis,therefore,expectedthatinthefuturethescientificbasisandtransparencyofmycotoxinregulationsandstandardisationandharmonisationofthecorres-ponding(bio)analyticalmethodologywillbenefitfromthesedevelopments.

AcknowledgementTheauthorswishtothanktheFoodandAgricultureOrganizationoftheUnitedNationsforpermissiontomakeuseofmaterialpublishedin2004inFAOFoodandNutritionPaper81:“Worldwideregulationsformycotoxinsinfoodandfeedin2003”.ProfessorT.B.Whitaker(NorthCarolinaStateUniversity,USA)isgreatlyacknowledgedforpermissiontoreproduceFig.2.Figure5wasreprintedfromToxicologyLetters153,SchothorstRCandVanEgmondHP.ReportfromSCOOPtask3.2.10“CollectionofoccurrencedataofFusariumtoxinsinfoodandassessmentofdietaryintakebythepopulationofEUmemberstates”Subtask:trichothecenes,133–143.Copyright(2004),withpermissionfromElsevier[35].

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