the safety assessment of proteins introduced into crops ... · thus, the safety evaluation of...

259

11 The Safety Assessment of Proteins Introduced into Crops Developed through Agricultural Biotechnology:A Consolidated Approach to Meet Current and Future Needs

Bruce Hammond and Andrew Cockburn

Contents

11.1 Introduction.................................................................................................26011.2 BiochemicalDifferencesbetweenProteinsandLow-Molecular-Weight

Chemicals:ImpactonSafetyAssessmentofProteins................................26011.3 AbsorptionofProteinsfromtheGITract................................................... 26211.4 SummaryofSafetyAssessmentsonProteins............................................. 26211.5 SafetyAssessmentStrategyforProteinsIntroduced

intoFood/FeedCrops.................................................................................26811.5.1 ModeofActionandFunctionality.................................................26811.5.2 Bioinformatics................................................................................26911.5.3 Digestibility....................................................................................26911.5.4 ConfirmatorySafetyStudies..........................................................269

11.6 DietaryRiskAssessment............................................................................ 27211.7 ThresholdofToxicologicalConcern........................................................... 27311.8 TheFuture................................................................................................... 276

11.8.1 ApplicationsofProteinEngineeringforFood-ProcessingEnzymes....................................................... 277

11.8.2 ModificationofInsectControlProteinstoImprovePotencyorBroadenSelectiveActivityagainstTargetedPests................... 277

11.8.3 IntroductionofTranscriptionFactorProteinstoModifyEndogenousPlantMetabolicPathways......................................... 278

11.9 Conclusion................................................................................................... 281References.............................................................................................................. 281

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260 Food Safety of Proteins in Agricultural Biotechnology

11.1 IntroduCtIon

Theconcludingchapterofthisbookdistillsinformationfrompreviouschapterstoconsolidateanoverallriskandsafetyassessmentstrategyappropriateforproteinsintroducedintobiotechnology-derivedfoodandfeedcrops.Thestrategybuildsontheinformationfromsafetyassessmentsofproteinsusedinfoodproduction(enzymesand animal somatotropins), proteins used as therapeutic agents, proteins that arecomponentsofmicrobialpesticidesappliedtoagriculturalcrops,andproteinsintro-ducedintobiotechnology-derivedcrops.Thesafetyassessmentschemeadoptsthewell-establisheddietaryexposureproceduresusedforlow-molecular-weightchemi-calsaddedtofoods,butdiffersfundamentallyinsomerespectsregardingtheoverallhazardidentification.Thesedifferencesareaconsequenceofuniquestructural,func-tional,andbiochemicalpropertiesofproteinsthatdifferinmanyrespectsfromlow-molecular-weightchemicalsusedasfoodadditivesorpesticides.Thesedifferenceshaveaprofoundimpactonthehazardpotentialofproteinsscreenedforintroduc-tionintofoodcrops,whichisgenerallylessthanthatofmanylow-molecular-weightchemicalsthatenterthehumanfoodchain.Thereare,ofcourse,proteinsknowntobetoxictohumansorpharmacologicallyactiveinman,buttheyhaveintentionallynotbeenselectedforintroductionintofoodandfeedcrops.

Thischapterwillalsolookintothefuturetoexploretheanticipateduseofpro-teinstodevelopnewandimprovedfoodandfeedcrops.Theproposedriskassess-mentstrategyisconsideredtoberelevanttobothexistingandnewproteinsthatwillensurethatfutureimprovedfoodandfeedcropvarietiesaresafeforconsumption.Potentialhazardsthatmightresultfromanunexpectedorunintendedchangetotheplantfromtheintroductionoftheproteinarenotthefocusofthischapterbutareneverthelessaddressedinsubsequentdiscussions.

11.2 BIoChemICaldIfferenCesBetweenProteInsandlow-moleCular-weIghtChemICals:ImPaCtonsafetyassessmentofProteIns

Aspointedoutinthefirstchapterinthebook,therearesomefundamentalstructuralandbiochemicaldifferencesbetweenproteinsandlow-molecular-weightchemicals.Examplesareasfollows:

Low-Molecular-Weight Chemicals

1. Chemicalstructuresvaryconsiderablyandmaybenovel(notfoundinnature)orrelatedtobiochemicalsfoundinnature.Forexample,thechemicalstructureoftheinsecticidechloropyriphoswouldbeconsiderednovel,whereastheherbicideglyphosate is structurally related to the aminoacidglycine.Examplesof foodadditives with novel structure could include the artificial sweetener saccharin,whereas another artificial sweetener, aspartame, is structurally related to theaminoaciddipeptideaspartate-phenylalanine.

2. Low-molecular-weightchemicalfoodadditivesandcontaminantshavemolecularweightsgenerallyrangingfromapproximately200–800MW.

3. Absorptionfromthegastrointestinal(GI)tractvariesdependingonthestructuralproperties of the low-molecular-weight chemical. For example, lipid solubility

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The Safety Assessment of Proteins 261

cansignificantlyenhancesystemicabsorptionfromtheGItract.Approximately47% to 69% of an oral dose of two different lipophilic low-molecular-weightchemicalinsecticideswereabsorbedintactfromtheGItractoftheratwithinanhouroforaldosing.1Othermorepolarlow-molecular-weightchemicalsthatareionizedatthepHoftheintestinaltractoraremorewater-solublearelesslikelytobe absorbed systemically, such asglyphosate (~30%absorbed).2Plants alsometabolizefoliar-andsoil-appliedpesticidestomorepolarderivativesthataremuchlesslikelytobeabsorbedsystemicallythantheparentcompound.Acaseinpointistheherbicideacetochlor,whichisabsorbedsystemicallyat>80%whenfedtorats.Itstwomajorplantmetabolites,t-ethanesulfonicacidmetaboliteandt-oxanilicacidmetabolite,whicharemorepolarthanacetochlor,arelessreadilyabsorbed,upto12%and39%,respectively.3

Proteins

1. Virtuallyallproteinsarepolymerscomposedofdifferentcombinationsandper-mutationsofthesame20commonaminoacidmonomers.Therearemillionsofproteinsofdiversestructureandfunctionfoundinnatureandtheyaremadeupofsomeorallofthese20aminoacids.Aminoacidspersehaveloworaltoxicityandareessentialtohumanlifeandnutrition(Chapter1).

2. Molecularweight(MW)ofproteinscanvaryfrom10,000(~50aminoacids)tomorethanamillion(>3000aminoacids,seeChapter1).Proteinsareordersofmagnitude larger than low-molecular-weight chemicals, which greatly reducestheirpotentialsystemicabsorptionacrossGIcellmembranes.

3. Ingested proteins are subjected to degradation to polypeptides, peptides, andaminoacidsby thecombinedactionof lowpHandpepsin in thestomachandassortedproteasessecretedintotheintestinaltract.Lossofquaternaryandter-tiarystructureoftheproteinduringdigestionresultsinlossofstructuralintegrityandusuallylossofbiochemicalfunction.

4. Proteins produced in mammalian cells can have important physiological andpharmacologiceffectswheninjectedintravenouslyfortherapeuticapplications,buttheseeffectsarenotgenerallyapparentwhentheseproteinsareingestedduetorapiddenaturationanddegradationwithintheGItract(Chapters6,10).

Asaconsequenceof thefundamentalstructuralandsizedifferencesbetweenproteinsandlow-molecular-weightchemicals,theprobabilityforsystemicabsorp-tionof themajorityof intactproteins from theGI tract is exceedingly lowwhencomparedtolow-molecular-weightchemicals.Theneedfortoxicologicalassessmentoflow-molecular-weightchemicalsislargelydrivenbyobservationsofpharmaco-logicalortoxicresponsesinoraldosingstudies.

Aswillbeshownlater,thevastmajorityofproteinsinvolvedinfoodusethathavebeenselectedandsubjectedtosafetytestingdonotcausesystemictoxicity.Thereisalonghistoryofsafeconsumptionofplantandanimalproteinsinthediet.Asdis-cussedabove,dietaryproteinsaregenerallydegradedandthuspoorlyabsorbedintactfromtheGItract(seediscussionbelow);hence,thereisverylowsystemicexposure.Thus,thesafetyevaluationofproteinsintentionallyselectedandsubsequentlyintro-ducedintofoodgenerallyrequireslesstoxicologytestingthanthatcarriedoutforlow-molecular-weightchemicalsinfoodorfeedwheresystemicabsorptionofbio-logicallyactiveparentcompoundormetabolite(s)generallyoccurswiththepotentialforend-organtoxicitypriortoandorduringexcretion/elimination.

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11.3 aBsorPtIonofProteInsfromthegItraCt

Astudyofthesystemicabsorptionofpeptides(3to51aminoacidsinlength)foundthatpeptidesgreaterthan10aminoacidsinlengthwerepoorlyabsorbedintactfromtheGItract.4Othershavereportedthatgastricabsorptionisinverselyrelatedtothesizeofthemoleculesothatsmallmoleculesaremorereadilyabsorbedthanlargeones.5Anumberofanimalfeedingstudieswithbiotechnology-derivedcropshaveinvestigatedthedigestibilityandpotentialsystemicabsorptionofintactintroducedproteinsinvarioustissuesandbloodsamplesusingsensitiveimmunologicalassays.6–

15 These published reports confirm that proteins, including those introduced intobiotechnology-derivedcrops,aredigestedandhavenegligibleoralbioavailability.

It is recognized that forproteinsstable todigestion,minutequantitiescanbetakenupintactbyPeyerspatchesliningtheGItract,ormaypassthroughintestinalcellsviaphagocytosisorpermeationbetweenepithelialcelljunctions.Anexampleistheeggallergenovalbumin,whichisstabletodigestioninsimulatedgastricfluidforatleast60minutes.Mostcommonplantproteins,incontrast,aredigestibleinlessthan15secondsinsimulatedgastricfluid(SGF).16Eggovalbuminwasadministeredto ratsasanoralbolusdose (50mg/rat).Bolusdosing increases thepotential forabsorptionduetoadministrationofaconcentratedsolutionstraightintothestomach.Asaresult,higherpeakbloodlevelsareachievedcomparedtolowerdosesresultingfromconsumptionofalbuminasacomponentoffoodinthediet.Nevertheless,evenafterbolusdosingofthestableeggovalbuminprotein,only0.007%to0.008%oftheadministereddosewasabsorbedfromtheGItract.17

Similar resultswerereportedforotherproteinallergens thatarealsostable todigestion,suchas thesoybeanallergenGlymBd30k,whereonlyapproximately0.004%ofalargebolusdosewasabsorbed.18Therearealsohumanstudiesreportingverylowbloodlevels(generallylessthan0.0001%ofingestedprotein)ofstablefoodproteins suchasovalbumin,ovomucoid, andβ-lactoglobulin after consumptionoffoodscontainingtheseproteins.19–21Theseproteinsareallhighlyabundantallergenicproteinsinfoodsthatarecomparativelystabletodigestion.16Forproteinsthatarenotstabletodigestion,thepotentialforsystemicabsorptionofintactproteinwouldbeexpectedtobeordersofmagnitudelowerthantheverylowlevelsofabsorptionforstableproteinsalludedtoearlier.ThisgenerallackofsystemicbioavailabilityfromtheGItractforintactproteinswouldminimizeanypotentialfortoxicitycomparedwithsinglelow-molecular-weightchemicalsubstancesfollowingoraladministration.

11.4 summaryofsafetyassessmentsonProteIns

Asdiscussedearlier,theoralbioavailabilityofdigestibleproteinsisnegligible,thustheirpotentialtoexertsystemicadverseeffects,ifsuchactivityweretobecharac-teristic,isalsoverylow.Asaconsequence,thereisnotnormallythescientificcasetosubjectproteinsscreenedforintroductionintofoodandfeedcropstothesameextensivebatteryof safety tests required for low-molecular-weightchemicals thatendupinfoodorfeed.Asdiscussedinprecedingchapters,nosystemictoxiceffectshavebeenidentifiedinthemanydietarytoxicitystudiesthathavebeencarriedoutwithproteinsofvariablestructureandfunctionthatareusedinfoodproduction.

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AlistofacuteandsubchronicoraltoxicitystudiesconductedwiththeseproteinsispresentedinTables11.1and11.2.Thesetableslistthe“no-observed-adverse-effect-levels”(NOAELs)which,foralltheproteinslisted,representsthehighestdosagesthatweretested.Manyoftheseproteinsareenzymesthathavebeenproducedbymicrobial fermentation and areused in foodprocessing. It hasbeen a regulatoryrequirementthattheseenzymepreparationsbetestedforpotentialacuteandsub-chronictoxicity.AsdiscussedinChapter5,thistestinghasnotbeenundertakentoresolvequestionsaboutsafetyoftheenzymesthemselves.Rather,testinghasbeen

taBle11.1summaryofnoaelsinacutehigh-dosestudieswithdifferentProteins

Protein function noaela,b reference

Cry1Ab Insectcontrol 4000mg/kg 22

Cry1A.105 Insectcontrol 2072mg/kg 23

Cry1Ac Insectcontrol 4200mg/kg 22

Cry2Aa Insectcontrol 4011mg/kg 22

Cry2Ab Insectcontrol 1450mg/kg 22

Cry3A Insectcontrol 5220mg/kg 22

Cry3Bb Insectcontrol 3780mg/kg 22

Cry1F Insectcontrol 576mg/kg 24

Cry34Ab1 Insectcontrol 2700mg/kg 25

Cry35Ab1 Insectcontrol 1850mg/kg 25

Vip3a Insectcontrol 3675mg/kg 26

ACCdeaminase Enzyme 602mg/kg 27

Alkalinecellulase Enzyme 10,000mg/kg 28

Dihydrodipicolinate-synthase(cDHDPS) Enzyme 800mg/kg 29

β-galactosidase Enzyme 20,000mg/kg 30

Enolpyruvyl-shikimate-3-phosphatesynthase(CP4-EPSPS)

Enzyme 572mg/kg 31

β-glucanase Enzyme 2000mg/kg 32

Glutaminase Enzyme 7500mg/kg 33

Hexoseoxidase Enzyme 2000mg/kg 34

Laccase Enzyme 2700mg/kg 35

Lactase Enzyme 10,000mg/kg 36

Lactoseoxidase Enzyme 900mg/kg 37

Lipase Enzyme 2000mg/kg 38

Lipase Enzyme 5000mg/kg 39

Neomycinphosphotransferase Enzyme 5000mg/kg 40

Phosphinothricinacetyltransferase Enzyme 2500mg/kg 41

Phosphomannoseisomerase Enzyme 3030mg/kg 42

Pullulanase Enzyme 10,000mg/kg 43

Xylanase Enzyme 239mg/kg 44

Xylanase Enzyme 2000mg/kg 45

aHighestdosagetestedthatcausednoadverseeffects.bActualdelivereddosagemaybelowerbasedonthepurityoftheenzymepreparationstested.

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taBle11.2summaryofnoaelsinsubchronicfeedingstudieswithdifferentProteins

Protein function study noaela reference

Bovinesomatotropin Hormone 13weeks 50mg/kg 46

DipelBtmicrobialCryproteinmixture

Insectcontrol 13weeks 8400mg/kg 22

DipelBtmicrobialCryproteinmixture

Insectcontrol 2years 8400mg/kg 22

TeknarBtmicrobialCryproteinmixture

Insectcontrol 13weeks 4000mg/kg 22

BtBerlinermicrobialCryproteinmixture

Insectcontrol 5days(human) 1000mg/adult 22

Cry1Ab Insectcontrol 28days 0.45mg/kg/day 22

Amylase Enzyme 90days 17.5mg/kg/day 47

Amylase Enzyme 90days 890mg/kg 48

Amyloglucosidase Enzyme 14days 1640mg/kg 49

Aminopeptidase Enzyme 90days 2000mg/kg 50

Arabinofuranosidase Enzyme 14days 103mg/kg 49

Chymosin Enzyme 90days 1000mg/kg 51

Chymosin Enzyme 90days 11.9mg/kg 51

β-galactosidase Enzyme 6months(rat)30days(dog)

4000mg/kg1000mg/kg

30

Glucanase Enzyme 90days 1258mg/kg 52

Glutaminase Enzyme 90days

365days

9000mg/kg/day(yeastCK)1200mg/kg/day(yeastCKD10)10,000mg/kg/day(yeastTK)

13,000mg/kg(yeastCK)

33

Hexoseoxidase Enzyme 90days 5000HOXunits/kg 34

Laccase Enzyme 90days 1720mg/kg 35

Lactase Enzyme 28days 1540mg/kg 36

Lactoseoxidase Enzyme 90days 900mg/kg 37

Lipase Enzyme 90days 658mg/kg 39

Lipase Enzyme 90days 1680mg/kg 38

LipaseG Enzyme 90days 1516mg/kg 53

LipaseAY Enzyme 90days 2500mg/kg 54

Pectinmethylesterase Enzyme 14days 133mg/kg 49

Phosphodiesterase Enzyme 28days 165mg/kg 55

Phospholipase-A Enzyme 90days 1350mg/kg 49

Phytase Enzyme 90days 1260mg/kg 49

Pullulanase Enzyme 28days 5000mg/kg 56

Tannase Enzyme 91days 660mg/kg 57

Xylanase Enzyme 90days 1850mg/kg 49

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considerednecessarytoconfirmtheabsenceofpossibletoxiccontaminants(myco-toxins,bacterialtoxins)fromthefermentationmediumthatmightbepresentintheenzyme preparation. Such testing, also applied to protein based vaccines, is alsoknownas“freedomfromabnormaltoxicity”(FAT)testing.

Thesestudiesconfirmtheabsenceoforaltoxicityevenwhentheproteinprepara-tionswereadministeredatveryhighdosagelevels.ThestudieslistedinTables11.1and11.2havebeenpublished,buttherearemanyothersthathavebeencompletedand have not been published. According to a recent review,63 as of 2001 almost800 toxicity tests have been conducted on approximately 180 enzymes by mem-bercompaniesof theEuropeanAssociationofManufacturersandFormulatorsofEnzymeProducts(AMFEP).AccordingtoAMFEP,thesestudiesraisednoissuesoftoxicologicalconcern.63Giventhehistoryofsafeuseforcertainmicroorganismstomakeenzymepreparations,ithasbeenproposedthatroutinetoxicologytestingofhighlycharacterizedspecificenzymepreparationspreparedfromthesemicroorgan-ismsisnolongerscientificallyjustifiedandisinhumanebecauseofitsunnecessaryuseoflaboratoryanimalsfortoxicologytesting.63

Although the vast majority of subchronic feeding studies with food enzymeshaveconsistentlyfoundnoevidenceoftreatment-relatedadverseeffectsintestani-mals,acoupleofstudiesreportedlocalirritationtothestomachcausedbyfeedinghigh levelsofproteaseenzymes to rats.Sucheffectsmightbeanticipateddue toproteolyticeffectsof theenzymeson thestomachmucosaathighexposures.64Afewothersubchronicfeedingstudiesreportedadverseeffectsusuallylimitedtothehighestdosagestested,andatlowerdosagesnoadverseeffectswerereported.Sincelowerdosageswerestillmanytimeshigherthanpotentialhumandietaryexposures,a very large safety margin existed for the use of these enzymes in food produc-tion.Theadverseeffectswerenotattributedtotheenzymesthemselves,butrathertootherconstituentsintheenzymepreparation.Forexample,enzymepreparationswithhighlevelsofash(saltsandminerals)fromthefermentationmediumproducednephrocalcinosis43orincreasedwaterconsumptioninrats.64Othereffects,suchasslightanemia32orreducedurinepH,foundinotherstudieswereeithernotcorre-latedwithanymicroscopicevidenceofpathologicchangesorwerenotreproducible

taBle11.2(ContInued)summaryofnoaelsinsubchronicfeedingstudieswithdifferentProteins

Protein function study noaela reference

Xylanase Enzyme 90days 4095mg/kg 49

Lactoferrin(human) Irontransport 90days 2000mg/kg/d 58

Lactoferrin(bovine) Irontransport 90days 2000mg/kg/d 59

Silkwormpupaeprotein

Notdefined 30days 1500mg/kg/d 60

Thaumatins Sweetner 90days 2696mg/kg/d 61

Ice-structuringprotein

Cryopreservation

90days 580mg/kg/d 62

a Inallcases,theNOAELswerethehighestdosetested.

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(salivaryglandenlargementwhenratswerefedtheenzymeinthedietbutnotbystomachtube).65Atarecent(2005)EuropeanToxicologyForumconferenceonthesafetyassessmentoffoodenzymes,aEuropeanregulatorwasaskedwhetherhehadeverseenevidenceofadverseeffectsinsubmittedsubchronictoxicologystudiesthatweredirectlyattributabletotheenzymefedtorats.66Herespondedthatinhismanyyearsofexperience,hehadnot.

Noevidenceofpre-neoplasticmicroscopicchangeshavebeenreported in thetissuesoflaboratoryanimalsfedproteins(enzymes,etc.)insubchronicfeedingstud-ies.AsdiscussedinChapters5and6,proteinsarenotconsideredtobecapableofmutagenic interactionswithDNA,and thiswouldbeeven less likelyforproteinsconsumedinthediet.Mutagenicitystudieshavebeencarriedoutwithmanyenzymepreparationstoconfirmtheydidnotcontaingenotoxiccontaminants(e.g.,mycotox-ins)fromthefermentationmedium.MembersoftheUnitedStatesEnzymeTechni-calAssociation(ETA)reportedthat,asof1999,102bacterialmutagenesistestsand63mammalianchromosomalaberrationmutagenesistestshadbeencarriedoutwithenzymepreparationsthatwerefromconventionalandgeneticallymodifiedmicroor-ganisms.67Thevastmajorityofthesetestsfoundnoevidenceofmutagenicactivity;thefewteststhathadpositiveresultswereconsideredtobelargelyattributabletoartifactsinthetestsystem(e.g.,presenceoffreehistidineintheenzymepreparationgavefalsepositiveresultsinthehistidinereversionbacterialmutagenicitytests).67Itwasconcludedthattestingenzymesforpotentialgenotoxicitywasnotnecessaryforsafetyevaluation.67

SimilarconclusionswerestatedinChapter6regardingInternationalConferenceonHarmonization (ICH)guidelines for safety testingofproteinpharmaceuticals.TheICHguidelinesforgenotoxicitytestingcommentthatbiologicals(whichincludeprotein therapeutics) are not expected to interact directly with DNA. They aredegradedtopeptidesandaminoacidswhicharenotconsideredtohavegenotoxicpotential.Routinegenotoxicitytestingofproteinpharmaceuticalsisnotconsiderednecessarytoconfirmsafety.

Thereareafewpublishedexamplesofenzymepreparationsbeingtestedinratteratologyand/oronegenerationratreproductionstudiestoconfirmtheabsenceoffermentationcontaminantsthatmightexertadverseeffects.Noevidenceofadverseeffectsattributabletotheenzymesonprogenydevelopmentorreproductiveperfor-mancewerereportedinthesestudies.28,30,64,68

Afewchronicfeedingstudieshavebeencarriedoutwithproteinpreparationsproducedbyfermentation.22,69Thiswasdonetodeterminewhethertherewereanychronicadverseeffectsattributabletopotentialcontaminantsfromthemicroorgan-ismsusedinthefermentationproduction.Thesestudiesdidnotreportthatproteinpreparationscausedcancerinlaboratoryanimals.Thereisnoevidencetothatpro-teinsdirectlyinducedcancer,birthdefects,ormutageniceffectswhenfedinthedietoflaboratoryanimals.67

Inthe1980stherewassomecontroversyregardingthechroniceffectsoftrypsininhibitorproteinsontheratpancreasandtherelevanceofthesefindingstohumans.Trypsininhibitorsareconsideredtobeantinutrientsandmembersofalargerfamilyofproteaseinhibitorsfoundnaturallyinavarietyoffoodcropssuchaslegumes,cere-als,andpotatoes.70Asthenameimplies,trypsininhibitorsblocktheproteaseactivity

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oftrypsininthegut,interferingwithproteindigestion.Proteaseinhibitorsmayplayaroleinplantdefensebyinterferingwithinsectdigestionandreducinginsectfeedingonthecrop.ThesafetycontroversybeganintheUKwhenratsthathadbeenfedadietcontainingraw(unprocessed)soybeanmealweredosedwithazaserine,a low-molecular-weight chemical that inducespancreatic cancer.71Soybeanmealmustbesubjectedtothermalprocessingtoinactivatetrypsininhibitorsbeforethemealisusedasfood/feedorthetrypsininhibitorswillinterferewithproteindigestion.Theafore-mentionedstudyfoundthattrypsininhibitorsinsoybeanspromotedthedevelopmentofpancreaticcancerinducedbyazaserine.Inaddition,controlanimalsthathadnotbeentreatedwithazaserine,butmaintainedchronicallyonunprocessedsoybeanmealalsodevelopedhypertrophicandhyperplasticchangesinthepancreas.

Itwassubsequentlyshownthatthisresponsewasnotduetoadirecteffectoftrypsin inhibitors on the pancreas but, rather, to negative hormone feedback bycholecystokinin (CCK), a hormone produced in the stomach. CCK is released inresponsetoundigestedproteinandfeedsbackonthepancreastoincreaseproduc-tion of proteases for release into the digestive tract to increase protein digestion.Thecontinuedpresenceoftrypsininhibitorpreventedproteindigestion;moreCCKwasreleasedtostimulatethepancreasandthecyclecontinued.RatschronicallyfedunprocessedsoybeanmealhadveryhighlevelsofbloodCCKlevelsduetoimpairedproteindigestion,resultinginchronicstimulationofpancreaticgrowthwhicheven-tuallyledindirectlytothedevelopmentoftumors.72

Questionswereraisedabouttherelevancetohumanfoodsafety72–74sinceitwasreportedthattheaverageadultintakeoftrypsininhibitorsfromconsumptionofnor-malfoodsintheUKdietwasapproximately330mg/person/day.74Feedingstudieswithrawsoybeanmealinotherspecies(dog,pig,calf)didnotdemonstratehyper-trophic or hyperplastic changes in the pancreas,74 suggesting that rats were moresensitive thanother species andmaynot be a relevantmodel for humans. Itwasrecognizedthattrypsininhibitorsmediatedtheireffectsontheratpancreasthroughtheendocrinesystem.Moreover,accordingtoGumbmannetal.in1986,“[T]hereisnoevidenceofabsorptionfromthegastrointestinaltract,directneoplasticactionor tumor induction,genotoxicity, interactionwithcellulargeneticmaterialorepi-demiological indicationofapotential risk inman.”75 Itwasultimatelyconcludedthat“humansarenotatincreasedriskforpancreaticneoplasiaforfoodscontainingnaturaltrypsininhibitoractivity.”72Thus,theearlierobservationoflackofevidencefordirectcarcinogeniceffectsofproteinsfedinthedietremainstrue.

AsdiscussedinChapter2,certainproteinsareknowntobetoxic tohumans.76Someof these toxinsareproducedbypathogenicbacteria thatelaborate the toxinsintheGItractwheningested.Somepathogenicbacteriaarepresentinfoodandformproteintoxinsinfood.Understandingeachstepinthelifecycleofproteintoxinscanhelptodefinetheirmodeofactionandexplainwhysomearetoxicwheningestedandothersarenot(Chapter2).Therearealsoproteinantinutrients,suchasproteaseinhibi-torsandlectins,thatarenaturallypresentinanumberoffoodsthataretraditionallyconsumed(legumes,grain,potatoes,etc.).70,77Althoughthereisahistoryofsafecon-sumptiontomanyoftheseproteins,afewofthemaretoxic,particularlywhenthefoodisnotproperlycookedtoinactivatethetoxin(e.g.,kidneybeanlectin).78Theareotherexamples,suchasthecastorbeanplant,whichisnotconsumedforfoodbutitsoilhas

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beenusedasacathartic.Castorplantsproducesricin,ahighlytoxiclectinthatcausespoisoninginhumansandanimalsthataccidentallyconsumethebean.79

Lastly,thereistheexampleofauniqueclassofproteinsknownasprionsthatarecomponentsofmammalianneurons.Prionstructurecanbemodifiedbyspontane-ousmutationsinthepriongenetoformstable,pathogenicformsthatcauseneuro-degenerativediseases.Themodifiedprionscauseunmodifiedprionsinneuronstoassume thealteredstructuralconfiguration that inducesneuropathologicchanges.Modifiedprionscancontaminatesurgicalequipmentorbloodandbetransmittedtoothers.Ruminantswithbovinespongioformencephalopathy(BSE)causedbymodi-fiedprionsmay“infect”thosewhoconsumemeatfromtheseanimals.80Modifiedprionproteinsareunusuallystableastheyareresistanttoproteases,standardsteril-ization,anddisinfectionagents.

Aswillbediscussedbelow,developersofimprovedcropvarietiesinitiallyscreentheproteinsthatarebeingconsideredforintroductionintoagriculturalcropsforarangeofattributes. Inparticular, theefficacyof the trait tobeconferred (e.g., insecticidalactivity),andtheydonothavepropertiesthatwouldposearisktoconsumersorfarmanimals.Subsequently,followingselectionandfirstproofofconcept,theyundergosys-tematicbioinformatics,in vitroandin vivotestingonacase-by-casebasis.Todate,noneoftheproteinsintroducedintoagriculturalcropshasshownanyevidenceofadverseeffects,confirmingtherigorousnessofthescreeningsystemthathasbeendeveloped.

11.5 safetyassessmentstrategyforProteInsIntroduCedIntofood/feedCroPs

InChapter10,asafetytestingapproachwasoutlinedforproteinsintroducedintobiotechnology-derived crops. This strategy was based on guidelines provided bytheOrganisationforEconomicCo-operationandDevelopment(OECD),theWorldHealthOrganization(WHO),theEuropeanFoodSafetyAuthority(EFSA),etc.Thebasicelementsofthistestingstrategyare:

HistoryofSafeUse(HOSU):Proteinsintroducedintobiotechnology-derivedcropsthathaveahistoryofsafeuse/consumptioninfood,orarestructurallyandfunctionallyrelatedtoproteinswithaHOSU,aregenerallyconsideredsafetoconsume.TheHOSUconcept iswidelyused in a regulatory context to provideguidanceon the level offamiliaritywithrespecttoprobablesafetyofchemicalsorproteinsinfood.Safetytest-ingguidelinesdevelopedbyEFSAstate,“Thestudiesrequiredtoinvestigatethetoxic-ityofanewlyexpressedproteinshouldbeselectedonacase-by-casebasis,dependingontheknowledgeavailablewithrespecttotheprotein’ssource,function/activityandhistoryofhuman/animalconsumption.InthecaseofproteinsexpressedintheGMplantwhereboththeplantandthenewproteinshaveahistoryofsafeconsumptionbyhumansandanimals,specifictoxicitytestingmightnotberequired.”81

11.5.1 ModeofActionAndfunctionAlity

Understandingthemodeofactionand/orbiologicalfunctionoftheintroducedpro-teinwillinformthesafetyassessmentsothatappropriatetestingcanbeundertaken

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toaddressanysafetyconcernsthatmayexist.Ifthemodeofactionisspecificforacertainbiologicalfunction(forexample,enzymaticconversionofsubstrateAtoproductB)andtheproductsoftheenzymaticreactionposenosafetyconcerns,thennoadditionalsafetytestingmaybewarrantedbeyondthebioinformaticsanddigest-ibilityassessmentspreviouslydiscussedinChapter10.

If the mode of action is not established (control insect pests by an unknownmechanism) or the function is related to the mode of action of known mamma-lianproteintoxinsorpharmacologicallyactiveproteins[antifungalprotein(AFP)example,Chapter10],thenadditionalsafetytestingiswarrantedtoassesswhethertheproteincanbesafelyused.

11.5.2 BioinforMAtics

Theproteinintroducedintobiotechnology-derivedcropsshouldnotshowaminoacidsequence similarity to known mammalian toxins, allergens, or pharmacologicallyactiveproteins.Ifsimilaritytothoseproteinsisfound,additionalsafetyevaluationswillbeneededtodeterminewhethertheseproteinscanbesafelyconsumedinthediet.

11.5.3 digestiBility

Proteinsthatarereadilydigestedin vitro usingsimulatedgastricand/orintestinalflu-idswouldnormallybecapableofbeingdigestedordegradedwhenconsumedinthediet.AsdiscussedinChapter10,digestibleproteinswould,inthemajorityofcases,belesslikelytoactasfoodallergenswhicharegenerallymorestabletodigestion.

11.5.4 confirMAtorysAfetystudies

AsdiscussedinChapters3and10,high-doseacutetoxicologystudiesarerequiredbytheU.S.EnvironmentalProtectionAgency(EPA)toassessthepotentialhazardsofplant-incorporatedprotectants (PIPs).This testing requirement isbasedon theneedtodemonstratethatthetoxicmechanismoftheplantprotectantisnotrelevanttoanimalsandman.Forexample,theknowledgethatexistingcommercialinsecti-cidalCryproteins(derivedfromBacillus thuringiensis bacteria)actthroughacutemechanismsatlowdosestocontrolinsectpests(Chapter3)andthatdoesnotoccurinmanisimportantandreassuringfromthesafetyperspective.TheEPArequiresthatPIPsbetestedathighdosagelevels(generallyg/kgbodyweightwherefeasible)toconfirmtheirsafety.Further,althoughmostconsumedproteinsarenottoxic,thosethataretoxicgenerallyexerttheireffectsthroughacutemodesofaction.82

Theproceduresforcarryingouthigh-doseacutetestingofproteinswerepresentedinChapter10.Todate,notreatment-relatedadverseeffectshavebeenobserveduptothehighestdosagestested(Table11.1).Aswillbeshownlater,thehighdosagesofproteinsadministeredtomiceareordersofmagnitudehigherthanpotentialhumandietaryexposuresfromconsumingfoodfrombiotechnology-derivedcrops.ForPIPsthathaveahistoryofsafeuseanddefinedmodeofaction,theEPAdoesnotrequireadditionaltoxicologytestingbeyondacuteoralmaximumhazarddosetesting.22

Acute toxicology studies are generally conducted via the oral route becausethedietisthemostlikelyrouteofhumanexposuretotheproteinsintroducedinto

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biotechnology-derived crops. Mice are generally used instead of rats as they areapproximately1/10thebodyweightofratsandrequiremuchlessproteinfordosing.Micearealsoknowntobesensitivetotheadverseeffectsofknownproteintoxinsandaremostcommonlyusedtoassesstheirtoxiceffects.83

Intravenous(IV)dosinghasalsobeenusedtoassesstheintrinsicsafetyofproteinsintroducedintobiotechnology-derivedcrops.41Generally,lowdosages(~10mg/kg)oftheintroducedproteinareadministeredasitisassumedthatonlysmallamountsofingestedproteinscouldbeabsorbedintact,andIVdosingposesthemostconserva-tivetestofpotentialtoxicity.However,dosingbythisroutemaynotsimulatewhatoccurs locallyin theGItract,andthusitsrelevancetodietaryexposurecouldbequestioned.Forexample,thepotentialtoxicityofantinutrientproteinsthatinterferewithproteindigestion anduptake (protease inhibitors, lectins)maynot bemani-festinthesamewayiftheywereadministeredintravenouslyinsteadofbytheoralroute.ForIVdosing,proteinsproducedinbacteriawouldneedtobehighlypurifiedto removebacterial/fermentation contaminants (e.g., lipopolysaccharides) that arethemselvestoxicwhenadministeredparenterally.84IftherewasevidenceoftoxicityfollowingIVdosingof theprotein,acuteoral toxicologystudieswouldstillneedtobeconductedtoresolvewhethertheseeffectswererelevanttodietaryexposure.RepeatIVdosingisalsonotrecommendedasplant-derivedproteinswouldberec-ognizedasforeigntorodents,leadingtothedevelopmentofneutralizingantibodiesinthebloodthatwouldconfoundinterpretationofstudyfindings.Thisphenomenoniswelldocumentedfortherepeatedadministrationofprotein-basedpharmaceuti-calsthatarenotnativetothetestspecies(Chapter6).

EFSAguidelines for testing the safetyofbiotechnology-derivedcropsdonotrecommendacutehigh-dosetestingforinsecticidalproteinsorforothernonpesti-cidalproteins.81Rather,EFSAproposesacase-by-caseassessmentofthesafetyofintroducedproteins,andifthebiologicalprofile/activityoftheproteinraisesques-tionsaboutsafetyortheproteinisconsideredtobe“novel,”thena28-dayfeedingstudy with the protein is recommended. This recommendation is appropriate forcertain classes of potentially toxic proteins such as lectins or protease inhibitorswhosetoxicityismanifestafterashort-termfeedingstudy.85–86Thecharacteristicsthat define an introduced protein as novel have not been elaborated and are bestdeterminedonacase-by-caseassessment.

Itmaynotbepossibletocarryoutrepeat-dosingstudiesforcertainmembrane-boundenzymesiftheyareconsideredtobenovel.Purificationandisolationofcertainmembrane-boundenzymescan lead to their immediate inactivationasmembranelipidsand thecofactorsneeded forcatalytic functionof theenzymeare removedduringpurification.87Asapracticalmatter,therecouldbenegligibledietaryexpo-suretofunctionallyactivemembrane-boundenzymesinfoodsifsolventextractionandheatprocessing(e.g.,foodsderivedfromsoybeans)resultsintheirinactivation.Thismayobviate theneedforconfirmatorysafety testingofproteins inanimals,giventhenegligiblepotentialforhumanandanimaldietaryexposure.

Whenanintroducedproteinisfunctionallyorstructurallyrelatedtoproteinsthataretoxictomammals(AFPexample,Chapter10),thenanacutehigh-dosetoxicitystudymaynotbesufficienttoconfirmsafety.Otherhypothesis-drivenstudies(basedonknowledgeoftheprotein’smodeofaction)maybenecessary,asoutlinedforthe

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AFPexample.Thesestudiescouldincludea28-daydietarystudywiththepurifiedproteininrodents,assumingitcouldbepreparedinsufficientquantitiestotest.

Not all introduced proteins have pesticidal properties, as some impart otherdesiredtraitsintocropssuchasherbicidetolerance,virusresistance,improvementsinnutrientcontent,etc.Oftentheseproteinsareenzymesthatcatalyzespecificbio-chemicalreactions.Basedontheirknownmodeofaction,specificity,lackoffunc-tionalorstructuralsimilaritytoproteintoxins,digestibility,historyofsafeuse,etc.,theweightofevidencewouldsuggesttheseproteinswouldnotraisefoodsafetycon-cerns.However,incertaincountriesoutsidetheUnitedStatesorEurope,regulatorshaverequestedhigh-doseacutestudiestoprovidefurtherconfirmationofsafety,andproteinsthathavebeensotestedarealsolistedinTable11.1(seealsoChapter10).AswiththecaseofPIPs,therehasbeennoevidencetodateofadverseeffectsinmicedosedwithhighlevelsofnonpesticidalproteins.

Proteins introduced into biotechnology-derived crops are also components ofgrainorseedthatareformulatedintodietsandfedtoratsforapproximately90daystoconfirmthelackofanyunintendedeffectsinthebiotechcrop.Thus,theirsafetyistestedasacomponentofthegrain/seedfedtorats.Otherstudies,suchasmolecularcharacterization of the gene insert, the nutrient/antinutrient composition of food/feed,thephenotypicandagronomiccharacteristicsoftheplantgrownindifferentenvironmentalconditions,andanimalperformancestudieswithfeedwillalsohavebeencarriedouttoassessthepotentialforunintendedeffects.

The study design for a 90-day rat feeding study is adapted from OECD 408guidelines for subchronic studies that includemeasurement a comprehensivebat-teryoftoxicologyparameters.Commercialrodentdietsusedbytoxicologytestingfacilitiesoftenincludeprocessedsoybeanmealandcornmealindietformulationsasa sourceofdietaryprotein.Whennewbiotechnology-derivedcornor soybeancropsaredeveloped,theycanbeincorporatedintocommercialrodentdietstosub-stituteforconventionalcorngrainorprocessedsoymeal,andtheirsafetycanbeassessed.Sincetheratsarefedlevelsofcorngrainapproximately100timeshigherthanhumanswouldconsumeinEurope(assumesconservativelythat100%ofthecorngrainisderivedfromthebiotechnology-derivedcrop),thesestudiescanprovideconfirmation of an acceptable safety margin for the biotechnology-derived cropsincludingtheintroducedprotein(s).Iftriggered,forexample,byresultsfromcompo-sitionalanalysisordifferencesinphenotypicoragronomicperformance,subchronicfeedingstudiesmaybeconductedtodeterminewhetherthebiotechnology-derivedfood is“assafeas”conventional,nonbiotechcomparators inaccordancewith thegeneralprinciplesofsubstantialequivalence.88–90

Subchronicfeedingstudiesareoftenrequiredtoobtainregistrationofthebio-technology-derived crop in the EU even though the aforementioned triggers didnotoccur. Itwas recentlyacknowledged inadraftEFSAguideline91 that “In thesituationwheremolecular,compositional,phenotypicandagronomicanalysishavedemonstratedequivalencebetweentheGMplantderivedfoods/feedandtheirnearisogeniccounterpart,exceptfortheinsertedtrait(s),anddonotindicatetheoccur-renceofunintendedeffects,theperformanceof90-dayfeedingtrialswithrodentsorwithtargetanimalspecieswouldbeconsideredtoaddlittleifanythingtotheoverallsafetyassessment.…Thesestudiesdidnotshowanyindicationfortheoccurrence

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ofunintendedeffects.”Thishasbeendemonstratedin90-dayratstudiesconductedtodate,someofwhichhavebeenpublishedinpeer-reviewedjournals.92–96

11.6 dIetaryrIskassessment

Riskassessmentsareroutinelyperformedtoassessthesafetyimplicationsfortheintentional or unintentional presence of low-molecular-weight chemicals in foodand feed. The procedures and mathematical models used to predict risk haveevolvedovertheyearsandhavebeenextensivelyreviewed.97–99Thedietaryassess-mentincludesbothacuteandchronicexposureassessments.Acuteexposureassess-mentsaddressshort-termexposuresusingapproximately95th-or97.5th-percentilefood consumption data (where available) and acute toxicity data generated withthelow-molecular-weightchemical.Some,however,mayquestiontheuseofacutedietaryriskassessmentsforproteinswhenthereisnoevidencethattheyareacutelytoxic.Chronicexposureassessmentsusemean(50th-percentile)foodconsumptiondata anduse the lowest no-effect level from thebatteryof toxicology studies toestablishanacceptabledailyintake(ADI)forthelow-molecular-weightchemicaladdedtofood.CalculationofanADIhasnotbeenconsiderednecessaryforcertainproteins such as the Cry insecticidal proteins. Cry proteins, whether introducedintobiotechfoodcrops,orsprayedonfoodcropsascomponentsofcommercialmicrobialpesticideformulations,havegenerallybeenexemptedfromtherequire-mentofatolerance.

Thesameprocedureshavebeenusedforpreparingdietaryriskassessmentsforproteins introduced into biotechnology-derived food and feed crops. The dietaryintake of the introduced protein can then be estimated by multiplying the intakeestimatesbytheconcentrationoftheintroducedproteininthefood.Chapter9pro-vides listsoffoodconsumptiondatabasesthatareavailableforvariouscountries.Somefoodconsumptiondataisbasedontheannualdisappearanceoffoodwithinthebordersof thecountry,whichisdividedbytheoverallpopulationtoestimatedailyintakeofthefoodcommodity.Thesedatabasesoverestimatedailyintakeofthefoodbyadults.Themoreaccurateconsumptiondatabasesarebasedonsurveyinfor-mationofindividualsover24to48hours.Thisinformationcanbecollectedforbothadultsandchildren.Thereisaneedforcountriestodevelopmorecomprehensivefoodsurveydataontheirrespectivepopulationssothatdietaryriskassessmentscanbemoreaccuratelyperformed.Atpresent,95th-or97.5th-percentilefoodconsump-tiondataareonlyavailableforcertaincountriessuchastheUnitedStates,theUK,andAustralia.However,asshowninChapter9,anumberofcountrieshavebeencarryingoutfoodconsumptionsurveysanditishopedthatthiswillbemorepub-liclyavailableforthosethathaveaneedforthisinformationtocarryoutdietaryriskassessments.Anexample foradietary riskassessment forYieldGard®Cornborer(MonsantoTechnology,LLC.), an insect-protected,biotechnology-derivedcrop isprovidedbelow.

Cry1Ab protein derived from Bacillus thuringiensis (Bt) was introduced intocornplantstoprovideprotectionagainstcornborerpeststhatdamageboththestalkandears.ThelevelsofCry1Abproteininleafandstalksisaround12ppm,andingrain,0.3ppm.100AsshowninTable11.1,miceweredosedupto4000mg/kgwithCry1Abproteinandexperiencednoadverseeffects.

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1. Acute Dietary Exposure Assessment

• The97.5th-percentilecornendosperm�fractionconsumptionintheUKforadultsis113g/person/day÷70kgbodywt/person=1.6g/kg.

• The97.5th-percentileadultdietaryintakeofCry1Abproteinwouldbe:1.6g/kg/day×0.3mg/gcorn=0.48mg/kgforanadult(0.00048mg/kg).

• The margin of safety for acute exposure to Cry1Ab protein is 4000 mg/kg ÷0.00048mg/kg=8,333,333X.

Putanotherway,a70-kg-bodyweighthumanadultwouldneedtoconsume>900,000kg(900metrictonnes)ofgraininonedaytoattainthesameacutedosage(4000mg/kg)ofCry1Abproteingiventomicewhichproducednoadverseeffects.

2. Chronic Dietary Exposure Assessment

• Theaverage(50th-percentile)cornconsumptionintheUKforadultsis~16gcorn/person/day÷70kgbodywt/person=0.23g/kg.

• TheaverageadultdietaryintakeofCry1Abproteinwouldbe:0.23g/kg/day×0.3mg/gcorn=0.07mg/kgforanadult(0.00007mg/kg).

• TheaverageratdietaryintakeofCry1Abproteinina90-dayfeedingstudyis25gcorn/kgBW×0.3mg/gcorn=7.5mg/kg

• ThemarginofsafetyforchronicdietaryexposuretoCry1Abproteinis7.5mg/kgdividedby0.07mg/kg=107X

Thisdietaryexposureassessmentmakessomeveryconservativeassumptions.Itassumesthat100%ofthecornconsumedinthedietisYieldGard®CornborerthatcontainstheCry1Abprotein.Inreality,manyvarietiesofcornaresoldcom-mercially,sothatYieldGard®Cornborerrepresentsonlyafraction(~20%)ofthetotalcornvarietiesconsumedinthediet(asof2002).101Italsoassumesthat theCry1Ab protein is not denatured by thermal processing of corn grain into foodproducts.Soybeansarebothheat-processedtoinactivatetrypsininhibitorsandsol-vent-extractedtoremoveoil.ProcessingdenaturesproteinslikeCP4EPSPS,whichhavebeenintroducedintosoybeanstoimparttolerancetoglyphosateherbicide.

ThedietaryriskassessmentshownaboveusescornconsumptiondataforadultsintheUK.IfadietaryriskassessmentwaspreparedforCentralAmerica,thesafetymarginwouldbesomewhatlower,ascornconsumptionishundredsofgramsperpersonperday.102However, the safetymarginwould still bevery large since thelevelofCry1Abincorngrainisverylow.Thus,riskassessmentscanbetailoredforindividualcountrieswhenthereareaccuratefoodconsumptiondataavailable.

11.7 thresholdoftoxICologICalConCern

Introducedproteinsaregenerallypresentatlowlevelsinthegrain/seedofbiotechnol-ogy-derived crops commercialized to date (Table11.3). One could assume that thepresenceinfoodoflowlevelsofintroducedproteinsposesminimalrisksandshouldnotrequirecomprehensivesafetyassessment.Thereisaregulatorymandateinmost

� Humandietaryexposures areestimatedusing thecornendosperm fraction.This fractioncontainsmostoftheproteinwhichwouldincludetheintroducedprotein.Othercornfractionssuchasbran,sweeteners,andoilcontainverylittleprotein.ItalsoassumesthattheCry1Abproteinhasnotbeenintroducedintosweetcorn.DataderivedfromtheDEEM-UKdatabase(Exponent,Inc.).

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countriestoassessthesafetyofthemanysubstancesfoundinfood,whethertheyoccurnaturallyorareaddedinsomemannertofood.Withoutsomemeanstoprioritizeallsubstancesthatneedfurtherevaluation,regulatorswouldbeutilizingscarceresourcestoassesssafetyformanysubstancesthatmaynotrequireacomprehensivesafetyeval-uation.Moreover,withoutprioritization, the costswouldbeenormous to carryoutindiscriminatesafetytestingandmanyresearchanimalswouldbeusedunnecessarily.Thereisagrowingdemandtoreduceanimalexperimentationwherepossible.112

A risk assessment strategy has been proposed for evaluating low-level expo-suretolow-molecular-weightchemicalsinthediet.Ifadequatesafetymarginsexistfor human exposure to these substances, then no further safety testing would berequired.Thiswouldenable regulators to focus resourcesonhigher-priority foodsafetyissues.112Thisriskassessmentstrategyisdescribedasthethresholdoftoxi-cologicalconcern(TTC).112–114AccordingtoKroesetal.,theTTC“isapragmaticriskassessmenttoolthatisbasedontheprincipleofestablishingahumanexposurethreshold value for chemicals, below which there is a very lowprobability of anappreciable risk tohumanhealth.Thisconcept…is inherent in settingacceptable

taBle11.3levelsofIntroducedProteinsinthegrain/seedofBiotechnology-derivedCrops

Crop IntroducedProtein Concentrationa(ppm) reference

Corn

RoundupReady® CP4EPSPS 10–14 103

YieldGard®Cornborer Cry1Ab 0.3 100

YieldGard®Rootworm Cry3Bb1 70 94

YieldGard®Plus Cry3Bb1Cry1Ab

20(range15–26)0.38(range0.2–0.47)

104

YieldGard®RootwormPlus

Cry3Bb1Cry1AbCP4EPSPS

32(range22–48)0.56(range0.48–0.67)9.6(range7–14)

105

Herculex1®InsectProtection

Cry1F 71–115 106

LysineMaize Dihydrodipicolinate-synthase(cDHDPS)

24(range13–43) 107

Cotton

RoundupReady® CP4EPSPS 47–117 108

Bollgard® Cry1Ac 1.62 106

BollgardII® Cry2Ab2/Cry1Ac 34–60/1.3–1.6 109

RoundupReadyFlex® CP4EPSPS 67–580 110

soy

RoundupReady® CP4EPSPS 186–395

a fwt,freshweight.® Registeredtrademark,MonsantoTechnology,LLC.

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dailyintakes(ADIs)forchemicalswithknowntoxicologicalprofile.”113Thisconceptcouldalsobeappliedtoproteinsintroducedintofoodandfeedcrops.

TheTTCvaluesforlow-molecular-weightchemicalsareaslowas1.5μg/person/dayforthosethathavenotbeentestedforcarcinogenicitybuthavestructuralprop-erties(alerts)similartoknownchemicalcarcinogens.Exposuresbelowthe1.5μg/person/daylevelareconsideredtoposeaverylowrisk(<1inamillion)ofproducingcancer inman.Other low-molecular-weightchemicals thatdonothave structuralproperties or alerts that raise questions about potential toxicity have TTC levelsmuchhigher,rangingupto1800μg/person/dayinthediet.113

ProteinswerenotinitiallyincludedindeterminingTTClevelsbecause,againcitingKroesetal.,“[T]hereareinsufficientdose–responsedataregardingallergenicityofpro-teinsandlow-molecular-weightchemicals,onwhichaTTC(oranyotherassessment)canbebased.”113However, asdiscussed inChapter8,developersofbiotechnology-derivedcropsrigorouslyavoidintentionallyintroducingpotentiallyallergenicproteinsintofoods,forobviousreasons.AsindicatedinChapter8,thereisabatteryoftestsundertakentoconfirmthatintroducedproteinsdonotfittheprofileforknownaller-gens.Basedontheverylowprobabilitythatproteinsintroducedintobiotechnology-derivedcropsposeanallergenicrisk,theTTCriskassessmenttoolcouldbeappliedtolow-levelexposuretointroducedproteinsinbiotechnology-derivedfoodcrops.

One fundamental difference between proteins introduced into foods and low-molecular-weightchemicalsisthegenerallackofevidencefortoxiceffectlevelsinanimalsafetystudieswithselectedproteins(Tables11.1and11.2).Forlow-molecular-weightchemicals,TTCvalueswerecalculatedusingthe5thpercentileofthedistri-butionoftheNOELs(basedonanimaltoxicologystudies)dividedbyanuncertaintyfactorof100,andassuminganaveragehumanbodyweightof60kg.114Low-molecu-lar-weightchemicalsweredividedintothreedifferentclassesbasedontherelatednessoftheirchemicalstructurestothosethateitherposedminimalsafetyconcernsorthosethatsuggestedpotentialfortoxicity.Proteinscouldlikewisebecataloguedintothreestructuraldivisionsbasedontheirrelatedness,orlackthereof,toproteinsknowntobetoxic.Relatednessisalreadyevaluatedbybioinformaticssearches,asdiscussedinChapter10.Themosttoxicproteinstohumansaregenerallythosederivedfrommicro-organismsthatcausefoodpoisoning,andthesecouldrepresentoneclass.Thenextclassofproteinscouldincludethosegenerallyfoundinplantsthatactasantinutrients(lectins,proteaseinhibitors).Asapracticalmatter,proteinswithpotentialmammaliantoxicityareobviouslynotconsideredforadditiontofoodorfeedcrops,althoughthereisahistoryofconsumptiontomanyendogenousantinutrientproteinsfoundinfood(lectins,proteaseinhibitors,etc.).Thelastcategoryofproteinswouldincludeproteinsbeingintroducedintofoodandfeedcropsthatarestructurallyandfunctionallyrelatedtothosecurrentlypresentinfoodorhavebeensafelyusedinfoodproduction(e.g., CryproteinsfromBacillus thuringiensismicrobialspraysandfoodprocessingenzymes).

Asanexercise,NOAELsforallofthenon-toxicproteinslistedinTables11.1and11.2wereaveragedforeitheracuteorsubchronictoxicity.Sincetheenzymecon-centrationpresentinfermentationpreparationscanvaryfrom2%to70%,63anarbi-traryassignmentof10%enzymeconcentratewasappliedtoallNOAELsforthoseenzymespreparedbycustomaryfermentationtechniques(somepublicationslistedtheconcentrationofenzymeinthepreparation,whereasmanyothersdidnot).This10%

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correctionfactorwasapplied toall theNOAELspresentedinTables11.1and11.2.TheadjustedNOAELswereusedindeterminingtheoverallaveragesforacuteandsubchronictoxicitystudies.Themeanvaluesweredividedbya100-folduncertaintyfactortoestimateTTClevelsforacuteandchronicexposures.

For acute exposure, the average NOAEL (always the highest dosage tested)across30acutestudieswas1790mg/kg,andwhendividedbya100-folduncertaintyfactor,wouldprovideaTTCof17.9mg/kg,or1074mg/adultperson/dayforacutedietaryexposure(assumesadultbodyweightof70kg).Forchronicexposure,theaverageNOAEL(alwaysthehighestdosagetested)across40subchronicstudieswas249mg/kg,whichdividedbya100-folduncertaintyfactorwouldprovideaTTCof2.49mg/kg,or149mg/adultperson/day.

Thechronicdietaryexposurestovariousintroducedproteinshavebeencalculatedinpublicationsforthreebiotechnology-derivedcornproducts[RoundupReady®corn;YieldGard®Rootwormcorn,andYieldGard®Cornborercorn;(MonsantoTechnology,LLC.)]thatwerefedtoratsinsubchronictoxicologystudies.92–94Theintakeofintro-ducedproteinswas0.27mg/person/dayforCP4EPSPSprotein,1.3mg/person/dayforCry3Bb1protein,and0.005mg/person/dayforCry1Abprotein.Thesedietaryexposureswerebasedontheveryconservativeassumptionsthat100%ofthecornconsumedwasderivedfromeachbiotechvarietythatwastested,andtherewasnolossoftheintroducedproteinsduringthermalprocessingofcorngrainintofoodproducts.Evenatthe95th-percentileU.S.cornconsumption level (which isapproximately4× themeandietaryexposure),themg/person/dayintakeswouldstillbefarbelowtheTTC(149mg/person/day)forchronicdietaryexposuretointroducedproteins.ForpartsofMexicoandAfrica,where thepercapitacornconsumption is approximately20 times that in theUnitedStates,themg/person/dayintakeswouldstillbewellbelowthecalculatedTTClevel.

The levels of the aforementioned introduced proteins in the grain from threebiotechnology-derivedcornproductsarequite low:14ppm(CP4EPSPS),70ppm(Cry3Bb1),and0.3ppm(Cry1Ab).Toachievealevelofproteinconsumptionequiva-lenttothe149mg/person/dayTTClevel,andusinga50th-percentiledailyU.S.adultcornendospermconsumptionfigureof0.27g/kg/day(DEEMdatabase,Exponent,Inc.),thelevelsofanintroducedproteinwouldhavetobeapproximately7800ppminthegrainfordietaryconsumptiontoreachtheTTClevel.IfthedietaryexposureforanintroducedproteinexceededtheTTC,thiswouldnotmeanthattherewasasafetyconcern. Appropriate toxicology studies could be done to assess safety at dietarylevelsabovetheTTC,asdiscussedpreviously.AdoptionoftheTTCconceptforriskassessmentwouldmeanthatdietaryexposurestoproteinsbelowtheTTCwouldnotrequireconfirmatoryanimalsafetytestingbasedonthefollowingconditions:(1)thesourceoftheproteinraisesnosafetyconcerns;(2)themodeofactionoftheproteinisknownandposesnosafetyconcerns;(3)theproteinisnotstructurallyorfunctionallyrelatedtoproteinsthatareknownmammaliantoxinsorantinutrients;(4)theproteinisdigestible;and(5)theproteindoesnotfittheprofileofknownfoodallergens.

11.8 thefuture

As thenextgenerationofbiotechnology-derivedcropsapproachescommercializa-tion,it is importanttoconfirmwhethertheexistingsafetyassessmentparadigmisappropriateforthesenewproducts.Thesafetyassessmentparadigmforintroduced

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proteins presented earlier in this chapter is aligned with existing internationallyacceptedapproachesprovidedinnumerouspublications.115–122Adiscussionofthenewkindsofintroducedproteinsthatarebeingdevelopedandtheefficacyandutilityoftheexistingsafetytestingparadigmtoconfirmtheirsafetywillbepresentedbelow.

11.8.1 ApplicAtionsofproteinengineeringforfood-processingenzyMes

The advent of biotechnology hasmade it possible tomodify proteins to increasetheirexistingfunctionalactivity,ortoimpartnewfunctionalpropertiesforadesiredapplication.Proteinengineeringincludeschangingaminoacidsatkeypositionsinthemoleculethatcanmodifytheirstructuraland/orfunctionalproperties.Thefirstapplicationshavefocusedontheengineeringoffoodenzymestoimprovetheirsta-bilityunderfood-processingconditions.Forexample,proteinengineeringhasbeenusedtomodifyproteasesbychangingkeyaminoacidstoincreasetheirstabilitytohightemperaturesandpH—conditionsthatcanoccurduringfoodprocessing.123Anotherexampleisthemodificationofα-amylasestoincreasethermostabilityforproductionofsweetenersfromcornstarch.124Biotechnologyhasalsomadeitpossi-bletoidentifyandproduceenzymesfromthermophillicandpsychrophilicmicrobesthatexhibitunique thermostableproperties,as theorganisms thatproduced themliveinextremeenvironmentalconditions(e.g.,volcanicheatedpoolsorvents).

ArecentreviewbySpokdiscussesothertoolsusedtoimproveenzymeperfor-mance:“Combinatorialapproachesofrationalproteindesignanddirectedevolutionmethodsturnouttoefficientlyalterthepropertiesofenzymes,enzymestability,cata-lyticmechanism,substratespecificityandrange,surfaceactivity,foldingmechanisms,cofactordependency,pHandtemperatureoptima,andkineticparametershavebeensuccessfullymodified.”63Othertechniquessuchasproteinshufflingcanincreasethevariabilityofenzymesthatcanbeproducedandmayyieldenzymesthatcancarryoutcatalyticactivitiesthatwereheretoforenotpossiblewithexistingenzymes.63

Biotechnologyisbeingusedtoreducethepotentialforcontaminationofenzymeconcentrateswithtoxicimpurities,whichcanbenefittheconsumer.Itisnowpos-sibletointroducethegenecodingforfoodenzymesintomicroorganismsthathavebeenwellcharacterizedandhaveanestablishedhistoryof safeusebecause theydonotmaketoxicimpurities.63Giventhisscenario,itisprobablynotnecessarytocontinuecarryingout90-dayratsafetystudieswhenthefermentationorganismsareknowntonotproducetoxiccontaminantsandtheenzymeisfullycharacterized.

11.8.2 ModificAtionofinsectcontrolproteinstoiMprovepotencyorBroAdenselectiveActivityAgAinsttArgetedpests

AwiderangeofactivityofCryproteinsagainstseveralordersofinsectshasresultedfromanaturallyoccurringrecombinationandsequencediversity.125Generally,Crypro-teinshaveadefinedspectrumofinsecticidalactivitywithinaparticularinsectorder.

Cry proteins are composed of several functional domains that have highlyconserved areas between the classes.126 For example, Cry1A proteins are highlyconservedindomainsI,II,andIII.Sequenceidentitycanindicatesimilarityinbio-logicalfunction,i.e.,activitytowardasimilarspectrumofinsects.ThesefunctionaldomainshavebeenshowntodeterminethespecificityofCryproteins:domainsI,

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II,andIIIformthetoxinportion(trypticcore),andaC-terminalprotoxindomainiscleaveduponentryintotheinsectmidgut.126DomainIisinvolvedinmembraneinsertionandporeformationanddomainIIisinvolvedinspecificreceptorrecog-nition and binding, as shown by mutagenesis studies. Domain III plays a role inreceptorbinding.ThecombinationofdomainsIandIIhasbeenshowntodetermineinsectspecificity.TheC-terminalprotoxindomainplaysaroleincrystalformation.Domainswappingisawell-knownmechanismforgeneratingdiversity.Mutagenesisanddomainswappingiswidelyusedinresearchinordertobetterunderstandfunc-tionofeachdomainandhavebeendescribedpreviously.125,127

ThesafetyassessmentoffutureCryinsecticidalproteinswithenhancedinsecticidalpropertiesdevelopedthroughdomainswappingorothertechniquescanbeconfirmedusingexistingtoxicologicalstudydesigns.Thiswouldincludethestandardbioinformat-ics,in vitrodigestibility,andhigh-doserodentacutetoxicitytestrequiredbytheEPAforregistrationofPIPs.Ifindicated,confirmationofsafetywouldalsobepossiblethrougha90-dayratfeedingstudywithgrainorseedcontainingtheinsecticidalprotein.Otherenvironmentaltoxicitytests,asoutlinedinChapter4,wouldalsobeneededtoconfirmselectivity toxicityagainst targeted insectpests andabsenceof toxicity tonontargetorganisms,asexistsforconventionalCryproteins.Ifthemodeofactionfortheinsec-ticidalproteinisnotwellcharacterized,orraisesquestionsaboutsafetyforconsumers(suchas theAFPexamplediscussedearlier), thentargetedtoxicity testsdesignedtoresolvesafetyquestionsmaybeneededbasedonacase-by-caseassessment.

11.8.3 introductionoftrAnscriptionfActorproteinstoModifyendogenousplAntMetABolicpAthwAys

Modulation of regulatory control proteins and regulatory processes has occurredduringplantdomesticationthroughbothnaturalandselectedbreedingofimprovedcropvarieties.128–131Forexample,thechangesresponsibleforimprovedwheatyieldsas part of the “green revolution” involved selection for mutant Reduced height-1 genes throughconventionalbreeding.132Theproteinsencodedby thesegenesareregulatorsofendogenousgene transcription thatmakewheatplants insensitive togiberellin,aplantgrowthregulator,thusmakingtheplantsshorterandprotectingthemfromcollapsingundertheirownweight.132Asaconsequence,yieldisincreasedatharvest.WheatdomesticationalsoinvolvedtheQgene,anAP-2-liketranscrip-tionfactorthatconfersfree-threshingcharacterandreducesfragility,enablingmoreefficientgrainharvesting.133Thedomesticationofmaizefromitsancestralform,teo-sinte,hasinvolvedselectionforenhancedexpressionoftheteosinte branched 1tran-scriptionfactor134andregulatorychangesinthemaizealleleoftheteosinte glume architechture transcriptionfactor.135Anotherexampleoftheimpactoftranscriptionfactors in corn breeding is a mutation in the opaque 2 transcription factor. ThismutationledtothegenerationofQualityProteinMaize(QPM),animprovednutri-tionmaizevariety(highinlysinecontent)thatwasthewinneroftheWorldFoodPrizein2000.136ReducedgrainshatteringresultingfromasinglebasepairmutationintheDNAbindingdomainoftheputativetranscriptionfactorsh4hasbeenthoughtto be a key event in the domestication of rice.137 Tomato hybrid cultivars with amutanttranscriptionfactoryieldfruitwithalongershelflife.138

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Wearenowlearningthatthedomesticationandbreedingofmoderncropswithbeneficialtraitscarriedoutoverthepastcenturieshasinvolvedselectionforchangesinproteinsregulatingendogenousplantgeneexpression.Transcriptionfactorshaveplayedaprominentroleintheseprocesses.Thesecropvarietiesproducedasaresultof altered transcription factor expressionhaveanestablishedhistoryof safe con-sumptionastheyarestaplesinthehumandiet.Thisdemonstratesthatplantswithalterations inendogenousgeneexpressionofproteins thatmodulateother endog-enousplantgeneshavebeensafelyconsumed.

Profiling technologies such as genomics, proteomics, and metabolomics havefacilitatedidentificationofgenesthatregulateendogenousplantprocessesandthephenotypiceffectselicitedbytheirproteinproducts.139Therefore,proteinsthataffectendogenouspathwaysareamongthelikelytargetstoimprovethenextgenerationofbiotechnology-derivedcrops.Duringthelastfewyears, therehasbeenagrowingnumberofbiotechnology-derivedplantswithmodificationsinendogenoustranscrip-tionalregulatoryprocesses.140–142

A fundamentalprinciple toconsiderwhenevaluating the safetyof thesebio-technology-derived crops is that the transcription factor proteins operate throughregulationofendogenousplantprocesses.Thustheyareunlikelytoproducenovelmetabolitesnotpreviouslypresentinplants.Theseproteinswillbestructurallyorfunctionally homologous to endogenous plant transcription factor proteins. Theycould alsobeobtained from the samecrop intowhich theywill be reintroducedthroughbiotechnology.

Duringthegrowingseason,plantsarenormallysubjectedtoavarietyofbioticandabioticstressconditions.Inresponsetotheseenvironmentalconditions,avari-etyof transcription factor-mediatedchanges inendogenousplantgeneexpressionoccur. Humans and animals consume food or feed from crops that contain thecumulativegeneexpressionchangesthatoccurinplantsgrownundervariablestressconditions.

There isahistoryofconsumptionof transcription factorsas theyarepresentinalleukaryoticcells,someofwhichareconsumedasfood.Outofanestimated59,000genesinthericegenome,approximately1600(∼3%)arepredictedtoencodetranscriptionfactors.143Thesoybeangenomeispredictedtocontainapproximately1300transcriptionfactorsoutofanestimated63,500genes,representingabout2%ofthegenome.144Questionsconcerningthesafetyoffoodorfeedderivedfromcropscontainingintroducedtranscriptionfactorsshouldbeconsideredinthecontextofthehistoryof safe consumptionof food and feedderived fromplants containingthesenaturallyandregularlyoccurringchangesintranscriptionalprofiles.

Anadditionalexposureconsiderationformanyregulatoryproteinsisthattheyusuallyhaveasmallnumberofspecific targets.Moreover,although transcriptionfactorsareexpressedineverycell,theyaregenerallypresentinlowlevelsinplantandanimaltissues.InArabidopsis,forexample,thenumberofmRNAsencodinganindividualtranscriptionfactorhasbeenreportedtorangefrom0.001to100cop-iespercell,illustratingtherelativelylowlevelofthesetranscriptsinplantcells.145Thewiderangeinpotentiallevelsforagiventranscriptionfactormayresultfromspatial(celltype),temporal(cellcycle),anddevelopmental(lifecycle)regulationofgeneexpression.141Transcriptionfactorproteinsalsotendtobepresentatverylow

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amountsinplanttissue.Forexample,only50μg(80pmol)ofKAP-2transcriptionfactorwasobtainedfrom6kgofbeancells,correspondingtoabout8ngoftran-scriptionfactorproteinpergramoftissue.146

Even with large uncertainties in available estimates, it is apparent that tran-scriptionfactorsrepresentonlyatinyfractionoftotalplantproteins,andtheircon-centrations(~ppb)arelikelytobeseveralordersofmagnitudelowerthanproteinsintroduced intobiotechnology-derivedcrops (ppm) todate (Table11.3)or typicalfoodproteins thatmight constitute1% (10,000ppm)ormoreof the totalproteinpresent in the food.16 Total protein levels in food crops can range from 10% formaizeto40%forsoybeans.147Tissuesconsumedfromfoodanimalsalsoprovideadietarysourceoftranscriptionfactorsandotherregulatorycontrolproteinsastheyareubiquitousinthecellsofanimals,albeitatlowlevels.Iflevelsofthesetranscrip-tionfactorsorotherregulatorycontrolproteinsareelevatedinfoodorfeedbeyondthatnormallyobservedintheplantproduct,thisinformationwouldalsobeusedintheevaluationofthehistoryofsafeconsumptionofrelatedproteins.

The assessment of potential oral activity for introduced transcription factorsneedstotakeintoconsiderationthefollowingfactors:

1. Thelackofaspecifictransportsystemforregulatorycontrolproteinsmayprovideanexplanation,inpart,astohowGItractepitheliaarecontinu-ouslyexposedtotheseproteinsfromdietarysources(plant-andanimal-derivedfoods)withoutanyevidenceofbiologicalresponseinmammals.

2. Transcriptionfactorsandmanyotherproteinsthatregulategeneexpres-sionfunctioninthenucleus.Inorderforingestedregulatorycontrolpro-teinstobeactiveintheconsumingorganism,theproteinwouldthusneedtonotonlysurvivedigestivebarriers,gainaccesstothesystemiccircula-tion,andbetransportedtoatargettissue,butwouldalsohavetoundergocellularuptake,evadecytoplasmicdegradation,andwouldrequiresubse-quenttransportacrossthenuclearmembraneandintothenucleus.Selec-tiveimportofproteinsacrossthenuclearmembranerequiresthepresenceofanuclearlocalizationsignalwithintheproteinsequence.148Whetheranexogenoustranscriptionfactororotherregulatorycontrolproteinwouldenter the nucleus would depend partly on the interaction between thatproteinandnuclearimportmachineryincellsoftheconsumingorganism.Thespecificityrequiredforsuchinteractionsaddsyetanotherbarriertofunctionofdietaryproteinsthatregulategeneexpression.

Basedonallof theaforementionedconsiderations,onecanconclude that theexistingriskassessmentproceduresusedtoassesssafetyofproteinsintroducedintobiotechnology-derivedcropsarealsoapplicabletotranscriptionfactors.

Sinceendogenousmetabolicpathwaysmaybemodifiedtoachievethedesiredplantimprovement,theagronomicperformanceandphenotypicappearanceoftheplant will be examined under a variety of environmental conditions to confirmthattherearenodeleteriousunintendedchanges.Thecompositionofgrainorseedwill also be analyzed to confirm that endogenous nutrients or antinutrients havenot changed, unless the intended technical effect results in changes in levels of

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endogenousnutrients.Inthiscase,thesafetyandnutritionalimpactofthosechangeswillbeevaluatedindependently.

If there isevidenceofsignificantunexpected/unintendedmolecular,composi-tional,agronomic,and/orphenotypicchangesthatcouldbeadverse,thenthesafetyimplicationsofthesechangeswouldrequirefurtherstudybeforeadecisioncouldbemadewhetherthecropcouldbesafetyused.Thissafetyassessmentprocesswhichisalignedwithinternationalguidelinesdiscussedpreviouslyisconsideredtobefullyadequate toconfirmthesafetyof food/feedderivedfromplantswhosemetabolicpathwaysaremodifiedtoachieveintendedimprovementsinthecrop.

11.9 ConClusIon

Aconsolidatedriskassessmentstrategyisproposedfortheintroductionofproteinsofdiversestructureandfunctionintofoodandfeedcrops.Thestrategyisbasedon,andalignedwith,internationalguidelinesandrecommendationsandcanbeadaptedtoeval-uatethesafetyofnewandimprovedvarietiesofbiotechnology-derivedcropsthatareunderdevelopment.Basedontheoverallweightofevidencefromassessingthesafetyofproteinsofdiversestructureandfunctionusedinfoodproductionandprocessing,aswellasthoseintroducedintobiotechnology-derivedcrops,itisclearthatintroducedproteinscanbesafelyusedintheproductionoffoodandfeed.Thesafetyassessmenttoolsareinplacetoandwillcontinuebeusedasneededtoensurethatfoodandfeedderivedfromnewvarietiesofbiotechnology-derivedcropscanbesafetyconsumed.

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