<b>Bijsluiter</b>. De hyperlink naar het originele document werkt niet meer. Daarom laat Woogle de tekst zien die in dat document stond. Deze tekst kan vreemde foutieve woorden of zinnen bevatten en de opmaak kan verdwenen of veranderd zijn. Dit komt door het zwartlakken van vertrouwelijke informatie of doordat de tekst niet digitaal beschikbaar was en dus ingescand en vervolgens via OCR weer ingelezen is. Voor het originele document, neem contact op met de Woo-contactpersoon van het bestuursorgaan.<br><br>====================================================================== Pagina 1 ======================================================================

<pre>Insectenresistente en herbicide-tolerante
maïs (1507)
Beoordeling van de veiligheid voor de consument, volgens de Europese
verordening 258/97 betreffende nieuwe voedingsmiddelen en nieuwe
voedselingrediënten
Insect-resistant and herbicide-tolerant
maize (1507)
Assessment of safety for the consumer, in accordance with European
Regulation 258/97 concerning novel foods and novel food ingredients
</pre>

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<pre></pre>

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<pre>Gezondheidsraad                             Vice-voorzitter
Health Council of the Netherlands
Aan de Minister van Volksgezondheid, Welzijn en Sport
Onderwerp          : aanbieding advies Insectenresistente en herbicide-tolerante maïs (1507)
Uw kenmerk         : GZB/VVB 2160036
Ons kenmerk        : 2003/04VNV, U-1582/CvR/cv/622-CP
Bijlagen           :1
Datum              : 4 november 2003
Mijnheer de minister,
Hierbij bied ik u een advies aan in het kader van de door u mede namens de Minister van
Landbouw, Natuur en Voedselkwaliteit aan de Gezondheidsraad voorgelegde adviesaanvraag over
de veiligheid van nieuwe voedingsmiddelen en nieuwe voedselingrediënten. Het advies is een
zogenoemde eerste beoordeling, conform de Europese verordening 258/97, van Insectenresistente
en herbicide-tolerante maïs (1507). Deze beoordeling is verricht door de Commissie
‘Veiligheidsbeoordeling nieuwe voedingsmiddelen’ van de Gezondheidsraad. Ik heb dit advies
heden ook aangeboden aan de Minister van Landbouw, Natuur en Voedselkwaliteit.
Hoogachtend,
Prof. dr JGAJ Hautvast
Bezoekadres                                                              Postadres
Parnassusplein 5                                                         Postbus 16052
2511 VX Den Haag                                                         2500 BB Den Haag
Telefoon (070) 340 7520                                                  Telefax (070) 340 75 23
E-mail: gr@gr.nl                                                         www.gr.nl
</pre>

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<pre></pre>

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<pre>Insectenresistente en herbicide-tolerante
maïs (1507)
Beoordeling van de veiligheid voor de consument, volgens de Europese
verordening 258/97 betreffende nieuwe voedingsmiddelen en nieuwe
voedselingrediënten
Gezondheidsraad:
Commissie Veiligheidsbeoordeling nieuwe voedingsmiddelen (VNV)
aan:
de minister van Volksgezondheid, Welzijn en Sport
de minister van Landbouw, Natuur en Voedselkwaliteit
Nr 2003/04VNV, Den Haag, 4 november 2003
</pre>

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<pre>De Gezondheidsraad, ingesteld in 1902, is een adviesorgaan met als taak de regering en
het parlement “voor te lichten over de stand der wetenschap ten aanzien van vraagstuk-
ken op het gebied van de volksgezondheid” (art. 21 Gezondheidswet).
    De Gezondheidsraad ontvangt de meeste adviesvragen van de bewindslieden van
Volksgezondheid, Welzijn & Sport; Volkshuisvesting, Ruimtelijke Ordening & Milieu-
beheer; Sociale Zaken & Werkgelegenheid en Landbouw, Natuur en Voedselkwaliteit.
De Raad kan ook eigener beweging adviezen uitbrengen. Het gaat dan als regel om het
signaleren van ontwikkelingen of trends die van belang kunnen zijn voor het overheids-
beleid.
    De adviezen van de Gezondheidsraad zijn openbaar en worden in bijna alle gevallen
opgesteld door multidisciplinaire commissies van – op persoonlijke titel benoemde –
Nederlandse en soms buitenlandse deskundigen.
Deze publicatie kan als volgt worden aangehaald:
Gezondheidsraad. Commissie Veiligheidsbeoordeling nieuwe voedingsmiddelen. Insec-
tenresistente en herbicide-tolerante maïs (1507). Den Haag: Gezondheidsraad, 2003;
publicatie nr 2003/04VNV.
auteursrecht voorbehouden
Een openbare versie van dit advies is in te zien via de website van de Gezondheidsraad:
www.gr.nl.
</pre>

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<pre>    Inhoud
    Samenvatting, conclusies en aanbevelingen 9
1   Inleiding 11
2   Volledigheid en juistheid van het dossier 13
2.1 Administratieve gegevens 13
2.2 Algemene beschrijving van het voedingsmiddel 14
2.3 Classificatie van het voedingsmiddel voor beoordeling 14
2.4 Informatievergaring over het voedingsmiddel 14
2.5 Beknopt overzicht door de aanvrager 15
2.6 Overige beoordelingen van het gewas 15
2.7 Etiketteringsvoorstel van de aanvrager 15
3   Interpretatie en evaluatie van voorgelegde gegevens 17
3.1 I Specificatie van het nieuwe voedingsmiddel (NV) 17
3.2 II Effecten van het gevolgde productieprocédé op het NV 17
3.3 III Achtergrondinformatie over het als bron voor het NV gebruikte organisme 18
3.4 IV Effect van de genetische modificatie op de eigenschappen van het gastheerorganisme 18
3.5 V Genetische stabiliteit van het als bron voor het NV gebruikte genetisch gemodificeerde organisme
    (GGO) 20
3.6 VI Specificiteit van de expressie van het nieuwe genetisch materiaal 21
3.7 VII Overdracht van genetisch materiaal vanuit het GGO 22
    Inhoud                                                                                           7
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<pre>3.8  IX Verwachte opname en gebruiksfrequentie van het NV 22
3.9  X Informatie op basis van eerdere blootstelling van de mens aan het NV of zijn bron 23
3.10 XI Informatie over de voedingswaarde van het NV 23
3.11 XII Microbiologische informatie over het NV 25
3.12 XIII Toxicologische informatie over het NV 25
     Literatuur 29
     Bijlagen 33
A    De adviesaanvraag 35
B    De commissie 37
C    EU-procedure 39
D    Samenvatting van het dossier 41
E    Schematische weergave van de genetische modificatie 49
     Engelse vertaling 51
8    Insectenresistente en herbicide-tolerante maïs (1507)
</pre>

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<pre>Samenvatting, conclusies en
aanbevelingen
De firma’s Pioneer Hi-Bred en Mycogen Seeds dienden gezamenlijk als aanvrager een
veiligheidsdossier in over de genetisch gemodificeerde maïslijn 1507. Het dossier bevat
moleculair-biologische, voedingskundige en toxicologische informatie. Als referentie
dient een conventionele maïslijn met een geschiedenis van veilig gebruik in de Europese
Unie.
    De gemodificeerde maïslijn verschilt van een conventionele lijn door de aanwezig-
heid van de genen cry1F en pat en de expressieproducten hiervan, het Cry1F- en het
PAT-eiwit. De ingebrachte genen zijn afkomstig uit bacteriën en aangepast voor een
optimale expressie in de plant. Hierdoor is de maïsplant bestand tegen vraat door
bepaalde plaaginsecten en tegen behandeling met het herbicide glufosinaat-ammonium
op het veld. Uit de moleculair-biologische analyse van het nieuwe gewas is gebleken dat
één volledige kopie van deze beide genen aanwezig is op één enkele plaats in het DNA
van 1507-maïs. Er zijn geen aanwijzingen dat de nieuwe eiwitten in de voorkomende
concentraties toxisch of allergeen zijn voor de mens.
    De beoogde verandering is gepaard gegaan met onbedoelde herschikkingen van
DNA aan de rand van de insertie, die ook in detail zijn geanalyseerd. Er zijn geen aan-
wijzingen dat de veranderingen in het genoom van de maïsplant leiden tot de onbe-
doelde productie van andere nieuwe eiwitten.
    De samenstelling van de gemodificeerde maïslijn is vergeleken met die van een vrij-
wel isogene, niet-gemodificeerde maïslijn door chemische analyse van een groot aantal
bestanddelen: micro- en macro-nutriënten, antinutriënten en secundaire plantenstoffen.
Daartoe werden op meerdere locaties veldproeven gedaan, waarvan de resultaten per
Samenvatting, conclusies en aanbevelingen                                               9
</pre>

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<pre>   afzonderlijke locatie werden verwerkt. Waargenomen variaties in de onderzochte com-
   ponenten blijven binnen literatuurwaarden en hebben gezondheidskundig gezien geen
   consequenties.
       Ook uit subchronisch proefdieronderzoek zijn geen nadelige bijeffecten van de
   modificatie in 1507-maïs naar voren gekomen.
       De commissie acht de informatie in het dossier een voldoende basis voor een veilig-
   heidsevaluatie. De ingediende gegevens zijn in het dossier correct geïnterpreteerd. Op
   basis van de huidige stand van de wetenschap luidt het oordeel van de commissie dat
   consumptie van 1507-maïs en daaruit geproduceerde voedingsmiddelen en
   voedselingrediënten voor de mens even veilig is als consumptie van gangbare niet gene-
   tisch gemodificeerde maïs en maïsproducten.
10 Insectenresistente en herbicide-tolerante maïs (1507)
</pre>

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<pre>Hoofdstuk 1
          Inleiding
          Op 28 februari 2001 vroeg de minister van VWS het oordeel van de commissie Veilig-
          heidsbeoordeling nieuwe voedingsmiddelen (VNV), hierna te noemen ‘de commissie’,
          over de veiligheid voor de consument van voedingsmiddelen en voedselingrediënten die
          gemaakt zijn uit een nieuwe maïsvariant. Deze maïs, aangeduid als lijn 1507, is afkom-
          stig van een plant (Zea mays L.) die door genetische modificatie twee nieuwe eiwitten
          produceert: het CRY1F- en het PAT-eiwit. Het CRY1F-eiwit beschermt de plant tegen
          vraat door bepaalde insecten, zoals de Europese maïsboorder (Ostrinia nubilalis). Het
          PAT-eiwit maakt de plant bestand tegen het herbicide glufosinaat-ammonium. Daardoor
          is het mogelijk om tijdens de groei van deze planten onkruid op de akkers te bestrijden
          met het herbicide, zonder dat de maïsplanten hiervan te lijden hebben. De maïsvariant is
          ontwikkeld door Pioneer Hi-Bred en Mycogen Seeds, die tevens de aanvraag indienden
          (Pio01). De aanvraag bevatte een veiligheidsevaluatie met een aantal bijbehorende
          onderzoeksrapporten. In juni 2001 verzocht de commissie de aanvrager het dossier aan
          te vullen met extra informatie over de moleculaire karakterisering en over de analyse
          van de samenstelling van de nieuwe maïslijn. Tevens vroeg de commissie de aanvrager
          om resultaten van aanvullend proefdieronderzoek te overleggen en om het dossier te
          completeren door toezending van alle aangehaalde gegevens uit de literatuur. In februari
          2002 verstrekte de aanvrager een deel van de gevraagde nieuwe informatie (Pio02),
          gevolgd door een aanvulling in februari 2003 (Pio03). Hierop vroeg de commissie in
          maart 2003 nog verduidelijking over de moleculair-biologische analyse, het onderzoek
          naar de samenstelling van de gemodificeerde maïs en het toxicologisch onderzoek aan
          de nieuwe eiwitten. De aanvrager verstrekte een deel van de gevraagde aanvullende
          Inleiding                                                                                11
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<pre>   informatie in mei 2003 (Pio03a). In juli 2003 volgde aanvullende informatie, nadat de
   commissie de betreffende vraag nader had gespecificeerd (Pio03b). De commissie
   wijdde enkele bijeenkomsten aan de bespreking van het dossier en rondde in november
   2003 haar beoordeling af. Dit rapport is de weergave van haar bevindingen.
12 Insectenresistente en herbicide-tolerante maïs (1507)
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<pre>Hoofdstuk 2
          Volledigheid en juistheid van het dossier
2.1       Administratieve gegevens
          De namen en adressen van de aanvragers, hierna te noemen ‘Pioneer/Mycogen’ of ‘de
          aanvrager’, zijn als volgt:
          Pioneer Hi-bred International Inc.
          400 Locust Street, Suite 800
          Des Moines, IA 50309, USA
          Mycogen Seeds
          c/o Dow Agroscience LLC
          9330 Zionsville Road
          Indianapolis, IN 46268-1054, USA
          Bij deze aanvraag worden deze firma’s vertegenwoordigd door:
          Pioneer Overseas Company,
          Avenue Tedesco 7
          B-1160 Brussel, België.
          Volledigheid en juistheid van het dossier                                         13
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<pre>2.2 Algemene beschrijving van het voedingsmiddel
    De aanvraag betreft het op de Europese markt brengen en verhandelen van
    maïslijn 1507 voor directe consumptie en voor verdere verwerking tot voedingsmidde-
    len en voedselingrediënten. De aanvraag omvat ook kruisingen van maïslijn 1507 met
    niet genetisch gemodificeerde maïslijnen.
2.3 Classificatie van het voedingsmiddel voor beoordeling
    Het dossier bevat een argumentatie voor indeling in klasse 3.1, één van de zes hoofd-
    klassen en subklassen van nieuwe voedingsmiddelen zoals genoemd in tabel 1 in deel I
    van Aanbeveling 97/618 van de Europese Commissie (EG97a). Het gaat hier om een
    genetisch gemodificeerde plant, waarvan de conventionele variant een geschiedenis van
    veilig gebruik heeft in de Europese Unie. De commissie is het eens met deze indeling.
2.4 Informatievergaring over het voedingsmiddel
    De aanvrager specificeert de informatie die essentieel is bij het beoordelen van de
    geschiktheid voor de consumptie van een voedingsmiddel in klasse 3.1. Dit gebeurt aan
    de hand van de in EC-aanbeveling 97/618 voorgeschreven thema’s:
    I.    Specificatie van het nieuwe voedingsmiddel (NV)
    II. Effecten van het gevolgde productieprocédé op het NV
    III. Achtergrondinformatie over het als bron voor het NV gebruikte organisme
    IV. Effect van de genetische modificatie op de eigenschappen van het gastheerorga-
          nisme
    V. Genetische stabiliteit van het als bron voor het NV gebruikte genetisch gemodifi-
          ceerde organisme (GGO)
    VI. Specificiteit van de expressie van het nieuwe genetisch materiaal
    VII. Overdracht van genetisch materiaal vanuit het GGO
    IX. Verwachte opname en gebruiksfrequentie van het NV
    X. Informatie op basis van eerdere blootstelling van de mens aan het NV of zijn bron
    XI. Informatie over de voedingswaarde van het NV
    XII. Microbiologische informatie over het NV
    XIII. Toxicologische informatie over het NV.
    De aanvrager doorloopt de stroomschema’s bij elk van deze thema’s en verwijst voor de
    gebruikte gegevens naar bijlagen of naar de literatuur.
14  Insectenresistente en herbicide-tolerante maïs (1507)
</pre>

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<pre>2.5 Beknopt overzicht door de aanvrager
    Het dossier bevat een beknopt overzicht dat aan de lidstaten is toegestuurd, conform
    artikel 6, lid 2 van de Europese verordening (EG) 258/97 (EG97).
2.6 Overige beoordelingen van het gewas
    In het kader van de richtlijn 2001/18/EC (EG01) inzake de doelbewuste introductie van
    genetisch gemodificeerde organismen in het milieu worden moleculair-biologische
    aspecten van dit nieuwe voedingsmiddel in Nederland beoordeeld door de Commissie
    Genetische Modificatie (COGEM) op verzoek van het ministerie van Volkshuisvesting,
    Ruimtelijke Ordening en Milieu.
         De beoordeling van de veevoederveiligheid van genetisch gemodificeerde gewassen
    wordt in Nederland uitgevoerd door het Rijkskwaliteitsinstituut voor Land- en Tuin-
    bouwproducten (RIKILT).
         De toestemming voor het in Nederland in het veld behandelen van deze maïs met
    glufosinaat-ammonium ligt bij het College voor de Toelating van Bestrijdingsmiddelen
    (CTB).
2.7 Etiketteringsvoorstel van de aanvrager
    Het dossier bevat een etiketteringsvoorstel. Onlangs is de Europese verordening (EG)
    nr. 1830/2003 gepubliceerd over traceerbaarheid en etikettering van genetisch gemodifi-
    ceerde organismen en de traceerbaarheid van daaruit geproduceerde levensmiddelen en
    diervoerders (EG03a). Na de inwerkingtreding van deze verordening zullen echter nog
    niet alle artikelen hiervan direct van toepassing zijn. Totdat de nieuwe verordening van
    kracht wordt, zal de etikettering in ieder geval in overeenstemming moeten zijn met
    EG-verordeningen 258/97 (EG97), 1139/98 (EG 98) en 49/2000 (EG00). Een etikette-
    ringsvoorstel wordt in Nederland evenwel in het Regulier Overleg Warenwet besproken
    en wordt niet beoordeeld door deze commissie.
    Volledigheid en juistheid van het dossier                                                15
</pre>

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<pre>16 Insectenresistente en herbicide-tolerante maïs (1507)</pre>

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<pre>Hoofdstuk 3
          Interpretatie en evaluatie van
          voorgelegde gegevens
3.1       I Specificatie van het nieuwe voedingsmiddel (NV)
          Het gaat in deze aanvraag om een maïslijn, waarin twee nieuwe genen zijn geïntrodu-
          ceerd. Het ene gen is het pat-gen, dat codeert voor het eiwit phosphinothricin-N-acetyl-
          transferase (PAT). Dit is een enzym dat van nature voorkomt in de bodembacterie
          Streptomyces viridochromogenes en dat ervoor zorgt dat de gemodificeerde maïsplant
          ongevoelig is voor het herbicide glufosinaat-ammonium. Het tweede toegevoegde gen is
          het cry1F-gen. Dit gen codeert voor een eiwit dat overeenkomt met het biologisch
          actieve gedeelte van het CRY1F-eiwit uit de bodembacterie Bacillus thuringiensis,
          ondersoort aizawai. Ondanks dit onderscheid wordt het nieuwe eiwit in de transgene
          plant ook aangeduid als CRY1F. Door de productie van dit eiwit in de gemodificeerde
          maïsplant wordt de plant beschermd tegen vraat door bepaalde insecten, zoals de larven
          van de Europese maïsboorder.
3.2       II Effecten van het gevolgde productieprocédé op het NV
          Hele maïskorrels worden op grote schaal gebruikt als diervoeder, maar slechts in
          geringe mate voor directe menselijke consumptie (namelijk in het geval van suiker-
          maïs). Uit maïskorrels geproduceerd meel, zetmeel en olie vormen echter belangrijke
          grondstoffen voor de productie van voedingsmiddelen. Daarbij wordt een groot deel van
          het maïszetmeel weer omgezet in siropen of in ethanol. De modificatie in maïslijn 1507
          is van agronomische betekenis en heeft geen invloed op de productieprocessen bij de
          Interpretatie en evaluatie van voorgelegde gegevens                                      17
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<pre>    verwerking van maïs tot voedingsmiddelen of voedselingrediënten. De aanvrager ver-
    wijst naar de literatuur voor een beschrijving van de hierbij gebruikelijke productiepro-
    cessen (Whi95).
3.3 III Achtergrondinformatie over het als bron voor het NV gebruikte orga-
    nisme
    De bron voor het nieuwe voedingsgewas is een op conventionele wijze geteelde maïs-
    lijn, te weten Zea mays Hi-II (achtereenvolgens geslacht, soort en variëteit). Door gene-
    tische modificatie zijn aan het maïsgenoom twee genen van bacteriële oorsprong
    toegevoegd, die zijn aangepast om in de plant optimaal te functioneren. De maïsplant
    heeft een lange geschiedenis van veilig gebruik voor voedingsmiddelen en
    voedselingrediënten, en wordt verbouwd in een groot aantal gebieden, verspreid over de
    gehele wereld (Sha88).
3.4 IV Effect van de genetische modificatie op de eigenschappen van het gast-
    heerorganisme
    Deze aanvraag betreft een maïslijn, waarin door overdracht van één lineair DNA-frag-
    ment twee nieuwe genen zijn geïntroduceerd, het cry1F-gen en het pat-gen. Hiervoor is
    een ballistische techniek gebruikt, waarbij het nieuwe DNA op microscopisch kleine
    wolfraamdeeltjes in embryonale maïscellen is ingebracht. Uit plantencellen waarin het
    nieuwe DNA was ingebouwd in het genoom, werden transgene maïsplanten geregene-
    reerd. Daarbij werd eerst geselecteerd op tolerantie voor het herbicide glufosinaat-
    ammonium, en vervolgens op aanwezigheid van het CRY1F-eiwit. Uit één van deze
    planten is de maïslijn 1507 voortgekomen door kruising met een zuivere maïslijn. De
    beoogde insertie van het nieuwe DNA in deze plant is gepaard gegaan met herschikkin-
    gen in het flankerende DNA.
         Het cry1F-gen is afkomstig uit de bacterie Bacillus thuringiensis, ondersoort aiza-
    wai. Voor een optimale expressie in plantencellen is voor de transformatie een ingekorte
    versie van dit gen gebruikt, waarvan de basenvolgorde tevens is aangepast aan het
    codongebruik in planten. Het CRY1F-eiwit in de transgene plant komt overeen met de
    eerste 605 aminozuren van het bacteriële eiwit, met één aminozuursubstitutie op positie
    604. Dit gedeelte omvat het biologisch actieve deel van het eiwit, dat specifiek bepaalde
    plaaginsecten doodt door aantasting van darmcellen in deze dieren. Expressie van het
    cry1F-gen in de transgene plant wordt gecontroleerd door de ubiZM1(2)-promoter uit
    maïs en de ORF25PolyA-terminator uit de bacterie Agrobacterium tumefaciens.
         Het pat-gen is afkomstig van de bodembacterie Streptomyces viridochromogenes en
    codeert voor het PAT-eiwit. Dit eiwit katalyseert de omzetting van L-phosphinothricin
    (L-PPT), het actieve bestanddeel van het herbicide glufosinaat-ammonium, naar N-ace-
18  Insectenresistente en herbicide-tolerante maïs (1507)
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<pre>tyl-L-PPT. De stof L-PPT bindt aan het enzym glutamine synthetase en doodt gevoelige
planten, doordat de essentiële detoxificatie van ammonia wordt belemmerd. Omdat N-
acetyl-L-PPT niet aan dit enzym bindt, maakt expressie van het PAT-eiwit een trans-
gene plant tolerant voor het herbicide (OECD99). Het pat-gen in de gemodificeerde
maïs is aangepast aan het codongebruik in planten voor een optimale expressie. De
expressie van het pat-gen in de transgene plant staat onder controle van de 35S promoter
en de 35S terminator uit het bloemkoolmozaïekvirus (CaMV).
     De aanvrager analyseerde het DNA van twee verschillende generaties van maïslijn
1507 door middel van Southern blotting (verder besproken in paragraaf 3.5). Uit deze
experimenten blijkt de aanwezigheid van één volledige kopie van het nieuwe DNA, de
primaire insertie. Daarnaast werd een extra kopie van een deel van het cry1F-gen aange-
toond. Ook werd via deze techniek bevestigd dat in het DNA van maïslijn 1507 geen
andere delen aanwezig zijn van het oorspronkelijke plasmide, waaruit het voor de trans-
formatie gebruikte DNA werd geïsoleerd.
     De commissie meende dat een meer gedetailleerde analyse van de genetische modi-
ficatie gewenst was en vroeg de aanvrager daarom om aanvullende DNA sequentieana-
lyse uit te voeren, inclusief het DNA direct vóór en na de beoogde insertie. Aangetoond
werd dat de primaire insertie inderdaad bestaat uit één enkele, vrijwel volledige kopie
van het gewenste DNA. Uit de volgorde van circa 2500 basenparen vóór en circa 1600
basenparen na de primaire insertie bleek de aanwezigheid van zeven extra fragmenten
van het nieuwe DNA en van vijf fragmenten, die mogelijk afkomstig zijn van chloro-
plast DNA (schematisch weergegeven in Bijlage E). Het ontstaan van dergelijke her-
schikkingen wordt vaker waargenomen, in het bijzonder wanneer de ballistische
techniek is gebruikt voor de transformatie (Koh03).
     Op verzoek van de commissie karakteriseerde de aanvrager vervolgens de open
leesramen die zijn ontstaan door de aanwezigheid van de extra fragmenten van het
nieuwe DNA. Indien DNA-sequenties in de nabijheid hiervan transcriptie mogelijk zou-
den maken, zouden deze open leesramen potentieel in fusie-eiwitten kunnen worden
vertaald. Uit geen van de door de aanvrager uitgevoerde Northern blot-, Western blot-
en RT-PCR-experimenten zijn aanwijzingen naar voren gekomen, die duiden op werke-
lijke vorming van zulke fusie-eiwitten. Op basis van de DNA-sequentie werden 24 open
leesramen geïdentificeerd op de grensvlakken van fragmenten nieuw DNA. De denk-
bare translatieproducten hiervan werden onderzocht op eventuele overeenkomsten met
bekende toxische of allergene eiwitten. Bij een globale vergelijking van de aminozuur-
volgordes met een algemene eiwitdatabase werden geen overeenkomsten gevonden met
bekende toxische eiwitten. Tevens werden vergelijkingen uitgevoerd met een door de
aanvrager samengestelde specifieke database voor allergene eiwitten, volgens de aanbe-
velingen van FAO/WHO (FAO01). Daarbij werd gezocht naar een overeenkomst met
bekende allergenen voor elk venster van 80 aminozuren en naar het vóórkomen van
Interpretatie en evaluatie van voorgelegde gegevens                                      19
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<pre>    identieke sequenties van zes, zeven of acht opeenvolgende aminozuren. Geen van de
    mogelijke eiwitten vertoonde meer dan 35 procent overeenkomst met een bekend aller-
    geen over een lengte van 80 aminozuren. Ook werden geen identieke sequenties van
    zeven of acht opeenvolgende aminozuren gevonden. Wel werd in zes gevallen een
    opeenvolging van zes identieke aminozuren gevonden bij vergelijking met bekende
    allergenen. De aanvrager stelt dat bij de vergelijking van fragmenten van slechts zes of
    zeven aminozuren vaak toevallige overeenkomsten zullen worden gevonden, zonder kli-
    nische relevantie, en ziet daarom in dit resultaat geen indicatie voor mogelijke allergeni-
    teit. Deze stelling is volgens de commissie aannemelijk en wordt ondersteund door
    recente publicaties (Hil02, Kle02).
         Samengevat is de mening van de commissie dat een grondige moleculair-biologi-
    sche analyse is uitgevoerd, waarbij geen andere effecten van de genetische modificatie
    zijn gebleken, dan de productie van de beide beoogde nieuwe eiwitten, CRY1F en PAT.
3.5 V Genetische stabiliteit van het als bron voor het NV gebruikte genetisch
    gemodificeerde organisme (GGO)
    De overerving van tolerantie voor glufosinaat-ammonium in de nakomelingen van de
    oorspronkelijke transformant is bestudeerd na drie opeenvolgende terugkruisingen met
    een zuivere lijn. Daarbij werd de verwachte 1:1 segregatie van deze eigenschap beves-
    tigd. Hierna werd deze generatie planten nogmaals teruggekruist met een zuivere lijn,
    gevolgd door een ronde zelfbevruchting. Hieruit ontstane planten werden behandeld met
    glufosinaat-ammonium, zodat alleen tolerante planten overbleven, welke vervolgens
    weer werden gekruist met een niet-transgene zuivere lijn. Onder de nakomelingen hier-
    van werd de verwachte 2:1 segregatie van tolerante en gevoelige planten bevestigd. De
    aanvrager vermeldt dat de glufosinaat-ammonium tolerante planten uit deze test tevens
    resistent bleken te zijn tegen de Europese maïsboorder in een bio-assay.
         Uit planten, afkomstig van de eerste terugkruising van de originele transformant met
    een zuivere lijn werd een generatie nakomelingen voortgebracht door zelfbevruchting.
    Deze planten werden gebruikt voor DNA-analyse door middel van Southern blotting.
    Ook planten ontstaan door vijf opeenvolgende terugkruisingen van de originele transfor-
    mant met een zuivere lijn werden op deze wijze geanalyseerd. Bij deze experimenten
    werden steeds dezelfde hybridiserende DNA-fragmenten gevonden.
         Uit deze resultaten blijkt dat het nieuwe DNA stabiel wordt overgedragen naar vol-
    gende generaties. Dit feit wordt ook bevestigd door de aantoonbare aanwezigheid van
    het CRY1F-eiwit in plantenmateriaal afkomstig van maïslijn 1507 uit een aantal veld-
    proeven (deze resultaten zijn beschreven in de volgende paragraaf).
20  Insectenresistente en herbicide-tolerante maïs (1507)
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<pre>3.6 VI Specificiteit van de expressie van het nieuwe genetisch materiaal
    In het seizoen 1998-1999 werden veldproeven uitgevoerd met 1507-maïs op vier plaat-
    sen in Chili. Om de expressie van de nieuwe genen te onderzoeken werden monsters
    genomen van verschillende plantendelen in verschillende stadia van de groei (Iow97,
    Iow03). Dit betreft blad in het V9-stadium, stuifmeel, zaadpluis en stengel in het R1-sta-
    dium, zaden na volledige rijping (R6 stadium) en hele planten in het R4-stadium en na
    verdroging op het veld. Op elke locatie werden verschillende monsters genomen van elk
    van deze weefseltypen. Daarin werden vervolgens de hoeveelheden van CRY1F- en
    PAT-eiwit als fractie van de totale hoeveelheid oplosbaar eiwit bepaald via een ELISA-
    methode met specifieke antisera tegen deze eiwitten. De aanvrager vermeldt per weef-
    seltype gemiddelde waarden, minima en maxima, waarbij de waarden voor de vier loca-
    ties zijn samengenomen. Op een vergelijkbare manier werden analyses uitgevoerd op
    materiaal uit veldproeven in 1999 op drie plaatsen in Frankrijk en drie plaatsen in Italië.
    Behalve bovenstaande weefseltypen werden hier ook nog hele planten in het V9- en het
    R1-stadium onderzocht. Tevens werden van hele planten in het R4-stadium en van maïs-
    korrels ook monsters onderzocht na behandeling van de planten met glufosinaat-ammo-
    nium. De aanvrager verstrekte later ook gegevens van een derde serie veldproeven,
    uitgevoerd in 2000 op drie locaties in Frankrijk, twee in Italië en één in Bulgarije. Hier-
    bij werden ook alle hiervoor genoemde weefseltypen onderzocht, nu echter in alle
    gevallen zowel met als zonder behandeling met glufosinaat-ammonium. De resultaten
    van de laatstgenoemde experimenten werden ook omgerekend naar hoeveelheden van
    de nieuwe eiwitten per eenheid drooggewicht van het onderzochte weefsel. In alle weef-
    seltypen van 1507-maïs kon de aanwezigheid van CRY1F-eiwit worden vastgesteld, ter-
    wijl dit niet aantoonbaar was in niet-transgene planten. Bij de veldproeven in Europa in
    2000 werd het gehalte CRY1F eiwit in maïskorrels vastgesteld op 2.2 ng per mg droog-
    gewicht voor de onbehandelde maïslijn 1507 en 2.5 ng per mg drooggewicht voor maïs-
    lijn 1507 na behandeling met glufosinaat-ammonium. Voor het PAT-eiwit konden
    daarentegen geen waarden worden vastgesteld, doordat het gehalte in de meeste geval-
    len onder de detectiegrens lag.
         Extracten van blad, stuifmeel, maïskorrels en hele planten van de veldproeven in
    Chili werden ook gebruikt voor Western blotting met antisera tegen het CRY1F- en
    PAT-eiwit. In alle weefseltypen was het CRY1F-eiwit detecteerbaar. Het PAT-eiwit
    kon alleen worden aangetoond in bladweefsel.
    Interpretatie en evaluatie van voorgelegde gegevens                                         21
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<pre>3.7 VII Overdracht van genetisch materiaal vanuit het GGO
    De aanvrager stelt dat van maïs afgeleide voedselproducten DNA kunnen bevatten. Het
    gehalte aan DNA is afhankelijk van het gevolgde productieproces. De commissie stelt
    dat mensen dagelijks grote hoeveelheden plantaardig en dierlijk DNA innemen. Het is
    voorstelbaar dat delen van dit DNA in de vorm van intacte DNA-fragmenten in de dar-
    men terechtkomen en daar aan de residente microflora worden overgedragen. Als dit al
    gebeurt, zullen in de praktijk deze genen niet of nauwelijks tot expressie komen, omdat
    er geen goede promoter aan gekoppeld is. Als deze genen toch tot expressie zouden
    komen, bieden ze in veruit de meeste gevallen de betreffende bacteriën geen competitief
    voordeel, en de gastheer geen nadeel. Er kan eigenlijk alleen een probleem optreden in
    het geval van overdracht van markergenen voor resistentie tegen antibiotica, waarbij dan
    ook nog de darmflora in de consument onder selectiedruk moet staan door het gebruik
    van het betreffende antibioticum. In maïslijn 1507 is geen gen aanwezig dat een antibio-
    ticumresistentie tot gevolg heeft. Daarom deelt de commissie de zienswijze van de aan-
    vrager dat geen nadelige gevolgen zijn te verwachten van overdracht van genetisch
    materiaal uit 1507-maïs, zo dit al zou plaatsvinden.
3.8 IX Verwachte opname en gebruiksfrequentie van het NV
    Hoewel de maïsplant en hele maïskorrels vooral als diervoeding worden gebruikt, wor-
    den van de maïskorrel afgeleide producten maïsolie en maïszetmeel in grote hoeveelhe-
    den verwerkt in producten voor menselijke consumptie. Ook uit maïszetmeel
    geproduceerde siropen en ethanol worden op grote schaal in de voedingsmiddelen-
    industrie gebruikt. Over het gebruik van maïs voor de productie van voedingsmiddelen
    is veel informatie beschikbaar. De nieuwe eigenschappen van maïslijn 1507 zijn van
    agronomische betekenis en zouden op grote schaal toepasbaar zijn. Hoewel 1507-maïs
    daarom in eerste instantie slechts in plaats van andere maïs zal worden gebruikt, kan niet
    geheel worden uitgesloten dat het in productie nemen van de nieuwe maïslijn kan leiden
    tot een betere concurrentiepositie als gevolg van een meer efficiënte productie van het
    betreffende gewas. In dat geval zou de consumptie door de mens van producten die van
    maïs zijn afgeleid (in het bijzonder olie en zetmeel) kunnen toenemen ten opzichte van
    overeenkomstige ingrediënten, afkomstig van andere gewassen. Volgens de commissie
    bestaan daartegen uit het oogpunt van voedingskunde echter geen bezwaren.
22  Insectenresistente en herbicide-tolerante maïs (1507)
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<pre>3.9  X Informatie op basis van eerdere blootstelling van de mens aan het NV of
     zijn bron
     Conventionele maïs heeft een lange geschiedenis van veilig gebruik binnen en buiten
     Europa. Over de gemodificeerde maïslijn 1507 zijn geen gebruiksgegevens beschikbaar.
3.10 XI Informatie over de voedingswaarde van het NV
     De aanvrager heeft voedingskundige analyses laten uitvoeren op de maïs van genetisch
     gemodificeerde en controleplanten. In de aanvraag werden resultaten gepresenteerd van
     veldproeven, uitgevoerd in Chili (1998-1999), Frankrijk (1999) en Italië (1999). De
     commissie constateerde echter dat aanvankelijk geen afzonderlijke waarden per locatie
     werden vermeld. Uitgangspunt voor de commissie is dat de analyse van de samenstel-
     ling van een gemodificeerd gewas gebaseerd moet zijn op veldproeven, die zijn uitge-
     voerd op meerdere geografische locaties, representatief voor de commerciële productie
     van het gewas. Om eventuele verschillen waar te kunnen nemen tussen de gemodifi-
     ceerde lijn en de best vergelijkbare controlelijn is het tevens van belang dat de waarne-
     mingen per locatie worden geanalyseerd. In reactie op dit commentaar heeft de
     aanvrager de resultaten van deze veldproeven opnieuw geanalyseerd, nu per locatie.
     Tevens verstrekte de aanvrager aanvullende resultaten van veldproeven, uitgevoerd in
     Frankrijk, Italië en Bulgarije in 2000. Op verzoek van de commissie beargumenteerde
     de aanvrager dat deze locaties inderdaad representatief zijn voor commerciële toepas-
     sing van dit gewas.
          Bij al deze experimenten werd het gehalte aan vet, eiwit, koolhydraten, vezels (ADF
     en NDF) en as bepaald in planten en maïskorrels. In de maïskorrels is tevens het gehalte
     bepaald aan calcium, fosfor, koper, ijzer, magnesium, mangaan, kalium, natrium, zink,
     vitamine B1, vitamine B2, foliumzuur, tocoferolen, palmitinezuur, stearinezuur, olie-
     zuur, linolzuur en linoleenzuur. Ook is de aminozuursamenstelling bepaald en het
     gehalte van de secundaire plantenstoffen inositol, raffinose, p-coumaarzuur, furfural en
     ferulinezuur en de antinutriënten fytinezuur en trypsine inhibitor. Behoudens kleine aan-
     passingen, die door de aanvrager zijn beargumenteerd, komt deze lijst van geanaly-
     seerde componenten overeen met aanbevelingen gedaan door de OESO (OECD02).
          In Chili (seizoen 1998-1999) is op vier verschillende locaties 1507-maïs gekweekt
     naast controleplanten met een vergelijkbare genetische achtergrond. De 1507-maïs werd
     behandeld met glufosinaat-ammonium, waarna gevoelige planten werden verwijderd.
     Het proefveld op elke locatie was ingedeeld in zes blokken, die als herhalingen van het
     experiment gezien kunnen worden. Voor de analyse van de samenstelling zijn per loca-
     tie uit elk blok monsters genomen van de bovengrondse delen van drie planten en van
     Interpretatie en evaluatie van voorgelegde gegevens                                       23
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<pre>   maïskorrels uit drie maïskolven. Voor elke geanalyseerde component vermeldt de aan-
   vrager de gemiddelde waarde voor maïslijn 1507 en voor de controle maïs, evenals een
   maat voor de spreiding. Er werden een aantal statistisch significante verschillen gevon-
   den, die merendeels willekeurig verdeeld lijken te zijn over de verschillende proefvel-
   den. Wel valt op dat op alle locaties een hoger gehalte linoleenzuur werd gevonden voor
   1507-maïs dan voor de controleplanten. Verder werden voor één locatie afwijkende
   waarden gevonden voor negen aminozuren. Alle vastgestelde gehaltes blijven evenwel
   binnen bekende literatuurwaarden.
       Het onderzoek in Frankrijk (1999) betreft monsters van 1507-maïs en controleplan-
   ten uit zes blokken op één locatie. In Italië (1999) werden monsters genomen op drie
   locaties, elk in drievoud, waarbij ook 1507-maïs behandeld met glufosinaat-ammonium
   werd onderzocht. Voor de drie proefvelden in Italië werden afwijkende waarden gevon-
   den bij een groot aantal van de metingen voor verschillende aminozuren in maïslijn
   1507 ten opzichte van de controleplanten. De overige gevonden verschillen lijken wille-
   keurig verdeeld. Alle vastgestelde gehaltes blijven binnen bekende literatuurwaarden.
       De aanvrager verstrekte aanvullende informatie over veldproeven, die in 2000 zijn
   uitgevoerd op drie locaties in Frankrijk, twee in Italië en één in Bulgarije. Het proefveld
   op elke locatie was ingedeeld in drie blokken. Elk blok bevatte 1507-maïs, met glufosi-
   naat-ammonium behandelde 1507-maïs en controle-maïs. Hieruit werden monsters
   genomen bestaande uit drie hele planten (R4 stadium) en zaden uit vijf maïskolven van
   afzonderlijke planten (R6 stadium). Er werden enkele statistisch significante verschillen
   waargenomen. Er is geen duidelijk patroon in de verdeling hiervan over de locaties en
   de waargenomen verschillen bleven binnen de natuurlijke variatie die uit de literatuur
   bekend is. Op één locatie in Frankrijk werden evenwel verschillen tussen de transgene
   maïs en de controle waargenomen voor vrijwel alle afzonderlijke aminozuren. Voor de
   controle werden hier consequent iets lagere waarden gevonden dan voor maïslijn 1507,
   terwijl dat op andere locaties niet het geval was.
       Ook beschrijft de aanvrager een voederproef met slachtkuikens, waarbij de groei
   van de (uitsluitend mannelijke) dieren werd onderzocht. Gedurende de onderzoekspe-
   riode van 42 dagen consumeerde één groep dieren maïskorrels van lijn 1507 en één
   groep maïskorrels van een controlelijn met vergelijkbare genetische achtergrond. Daar-
   naast werden dieren in vier controlegroepen gevoederd met maïskorrels van commer-
   ciële lijnen. Het voer bestond in alle gevallen gedurende de eerste twintig dagen voor 54
   procent uit maïskorrels en in de daarop volgende periode voor 57 procent. Iedere groep
   bestond uit 35 dieren. Er werden geen significante verschillen gevonden in sterfte, toe-
   name in lichaamsgewicht of voedselconversie.
       Samenvattend werd er een groot aantal verschillen waargenomen in aminozuurge-
   haltes van maïskorrels van de gemodificeerde lijn en controleplanten voor één locatie in
   Chili (1998-1999), drie locaties in Italië (1999) en één locatie in Frankrijk (2000). Zulke
24 Insectenresistente en herbicide-tolerante maïs (1507)
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<pre>     verschillen zijn echter niet waargenomen voor de andere veldproeven. In alle gevallen
     blijven de gehaltes binnen de natuurlijke variatie die uit de literatuur bekend is. Ook de
     afwijkende waarden voor linoleenzuur die zijn gevonden bij de vier veldproeven in
     Chili blijven binnen dit bereik. De aanvrager stelt dat de waargenomen verschillen toe te
     schrijven zijn aan natuurlijke variabiliteit. De commissie is van mening dat zelfs indien
     deze verschillen zijn toe te schrijven aan de genetische modificatie van maïslijn 1507,
     dit voedingskundig niet bezwaarlijk is, gezien de geringe omvang van die verschillen,
     binnen de grenzen van bekende natuurlijke variatie. De commissie onderschrijft daarom
     de conclusie van de aanvrager dat 1507-maïs voedingskundig niet verschilt van conven-
     tionele maïs. Dit wordt ook door de beschreven voederproef bevestigd.
3.11 XII Microbiologische informatie over het NV
     Het is niet te verwachten dat er op de nieuwe maïs of daarvan afgeleide producten
     andere micro-organismen of microbiële metabolieten voorkomen.
3.12 XIII Toxicologische informatie over het NV
     De aanvrager beschrijft toxicologisch onderzoek van de beide nieuwe eiwitten CRY1F
     en PAT. Omdat het CRY1F-eiwit slechts in geringe hoeveelheid in de transgene maïs-
     planten voorkomt, moest het eiwit voor een dierproef in bacteriën worden geproduceerd.
     Om een efficiënte productie te realiseren werd een fusie-eiwit tot expressie gebracht in
     de bacterie Pseudomonas fluorescens, bestaande uit het N-terminale deel van het
     CRY1F-eiwit en het C-terminale deel van het verwante CRY1A(b)-eiwit. Door enzyma-
     tische splitsing werd hieruit een eiwit geïsoleerd dat overeenkomt met aminozuur 28 tot
     612 van het natuurlijke CRY1F-eiwit. Het CRY1F-eiwit in maïslijn 1507 komt overeen
     met aminozuur 1 tot 605, met een substitutie op positie 604. Uit onderzoek door de aan-
     vrager blijkt dat deze beide eiwitten vergelijkbaar zijn voor wat betreft biologische acti-
     viteit in gevoelige insecten, reactiviteit met specifieke antisera bij Western blotting,
     afwezigheid van post-translationele glycosylering en grootte van de gevormde peptides
     na digestie met trypsine. Het in bacteriën geproduceerde CRY1F-eiwit bleek zijn biolo-
     gische activiteit te verliezen na incubatie gedurende 30 minuten bij 75ºC.
          Eventuele acute toxiciteit van het bacteriële CRY1F-eiwit werd onderzocht bij
     CD-1 muizen. Vijf mannelijke en vijf vrouwelijke muizen kregen een dosis van 576 mg
     CRY1F-eiwit per kg lichaamsgewicht oraal toegediend, verdeeld over twee gelijke por-
     ties met een tussenpoos van één uur. Hiervoor werd 5050 mg per kg lichaamsgewicht
     gebruikt van een preparaat dat 11,4 procent CRY1F-eiwit bevat. Alle dieren bleven in
     leven en er werden geen klinische afwijkingen waargenomen gedurende de daaropvol-
     Interpretatie en evaluatie van voorgelegde gegevens                                         25
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<pre>   gende veertien dagen. Bij sectie na deze periode werden geen macroscopisch zichtbare
   afwijkingen waargenomen.
        Op verzoek van de commissie leverde de aanvrager een verdere onderbouwing van
   de veiligheid van het CRY1F-eiwit. Daarin wordt beargumenteerd dat de bekende toxi-
   sche werking van de CRY-eiwitten beperkt is tot bepaalde insectensoorten met speciale
   receptoren voor dit eiwit in de darmwand. De aanvrager wijst er op dat verschillende
   Bacillus thuringiensis variëteiten reeds tientallen jaren veilig als biopesticiden worden
   gebruikt. Dit geldt ook voor de ondersoort aizawai, waaruit het CRY1F-eiwit afkomstig
   is. Verder heeft de aanvrager de aminozuurvolgorde van het CRY1F-eiwit vergeleken
   met een database van bekende eiwitten. Daarbij werden overeenkomsten gevonden met
   181 sequenties. Naast sequenties van CRY-eiwitten betreft dit slechts drie andere eiwit-
   ten, die geen van alle toxines zijn. Verder is de veiligheid van nauw verwante CRY-
   eiwitten, zoals het CRY1A(b)-eiwit, in het verleden experimenteel bevestigd. Genetisch
   gemodificeerde gewassen waarin onder meer het CRY1A(b)-eiwit tot expressie is
   gebracht, zijn de afgelopen jaren op grote schaal geproduceerd. Er zijn geen nadelige
   effecten gerapporteerd van consumptie van hiervan afgeleide voedingsmiddelen.
        Bij een ander onderzoek werd het PAT-eiwit toegediend aan CD-1 muizen. Vijf
   mannelijke en vijf vrouwelijke muizen kregen een dosis van circa 5000 mg PAT-eiwit
   per kg lichaamsgewicht oraal toegediend, verdeeld over twee porties met een tussenpoos
   van één uur. Hiervoor werd 6000 mg per kg lichaamsgewicht gebruikt van een preparaat
   dat in bacteriën werd geproduceerd en dat 84 procent PAT-eiwit bevat. Alle dieren ble-
   ven in leven en er werden geen klinische afwijkingen waargenomen gedurende de daar-
   opvolgende veertien dagen. Bij sectie na deze periode werden geen macroscopisch
   zichtbare afwijkingen waargenomen. Tevens werd een onderzoek naar eventuele orale
   toxiciteit van het PAT-eiwit bij herhaalde blootstelling uitgevoerd met Wistar ratten
   door toevoeging gedurende veertien dagen van dit eiwit in concentraties van 0, 5.000 en
   50.000 ppm aan een dieet met laag eiwitgehalte. Het hiervoor gebruikte PAT-eiwit was
   geproduceerd in E.coli en had een zuiverheid van 98 procent. De totale hoeveelheid toe-
   gevoegd eiwit was in deze drie groepen gelijk door toevoeging van respectievelijk
   50.000, 45.000 en 0 ppm soja-eiwit. Elke groep bestond uit vijf mannelijke en vijf vrou-
   welijke ratten. Uit de voedselopname per groep werd een gemiddelde inneming van het
   PAT-eiwit berekend van 0, 712 en 7619 mg per kg lichaamsgewicht per dag voor de
   mannelijke dieren en van 0, 703 en 7965 mg per kg lichaamsgewicht per dag voor de
   vrouwelijke dieren. Een controlegroep kreeg een standaard dieet. Alle dieren bleven in
   leven en er werden geen klinische verschijnselen waargenomen gedurende de daarop-
   volgende veertien dagen. Na deze periode werd bloed en urine afgenomen en werden de
   dieren gedood. Een groot aantal algemene en klinisch chemische bepalingen werden uit-
   gevoerd voor bloed en urine. Voor alle dieren werden orgaangewichten bepaald. Een
   groot aantal weefsels en organen werden microscopisch onderzocht van de dieren uit de
26 Insectenresistente en herbicide-tolerante maïs (1507)
</pre>

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<br><br>====================================================================== Pagina 27 ======================================================================

<pre>hoge dosisgroep, de controlegroep waarbij alleen soja-eiwit aan het voer werd toege-
voegd en de controlegroep, die een standaard dieet consumeerde. Er werden geen rele-
vante afwijkingen gevonden, die zijn gerelateerd aan de blootstelling aan het PAT-eiwit.
     Naast mogelijke acute of subacute toxiciteit bij orale blootstelling is ook mogelijke
allergeniteit van de nieuwe eiwitten van belang voor de beoordeling van de voedselvei-
ligheid van 1507-maïs. Om aannemelijk te maken dat allergische reacties ten gevolge
van de nieuwe eiwitten onwaarschijnlijk zijn, levert de aanvrager gegevens over de bron
van de nieuwe eiwitten, eventuele overeenkomsten met bekende allergenen en de mate
van resistentie tegen afbraak door proteolytische enzymen (COD02). De aanvrager stelt
dat geen allergeniteit is beschreven van de algemeen voorkomende bodembacteriën
waaruit de nieuwe genen zijn geïsoleerd, Bacillus thuringiensis en Streptomyces
viridochromogenes. Een vergelijking van de aminozuurvolgorde van het CRY1F- en het
PAT-eiwit met bekende allergenen uit een algemene eiwitdatabase leverde geen over-
eenkomsten op, als werd gezocht naar een venster van acht opeenvolgende identieke
aminozuren. Bij een later onderzoek naar het CRY1F-eiwit heeft de aanvrager gebruik
gemaakt van een specifieke database van 2033 sequenties van allergene eiwitten. Dit
onderzoek is uitgevoerd volgens de aanbevelingen van FAO/WHO (FAO01). Er werden
geen overeenkomstige stukken van zeven of acht identieke opeenvolgende aminozuren
geïdentificeerd. Wel werden overeenkomstige sequenties van zes opeenvolgende ami-
nozuren gevonden met drie eiwitten uit de gebruikte database. De aanvrager acht dit
gegeven echter niet relevant, omdat bij een venster van zes of zeven aminozuren vaak
vals-positieve resultaten worden gevonden, zoals al vermeld werd in paragraaf 3.4. Het
CRY1F-eiwit werd ook met dezelfde bekende allergenen vergeleken door toepassing
van het programma FASTA bij een venster van 80 aminozuren. Daarbij werden geen
overeenkomsten gevonden van meer dan 35 procent, het percentage dat door de FAO/
WHO als grens voor mogelijke significantie werd gesteld. Verder werd het CRY1F-
eiwit binnen één minuut afgebroken in gesimuleerd maagsap bij een verhouding van één
molecuul pepsine op 188 moleculen CRY1F. In een later onderzoek werd aangetoond
dat het CRY1F- eiwit zelfs binnen vijftien seconden werd afgebroken in gesimuleerd
maagsap met ongeveer 0,3 procent pepsine. Het PAT-eiwit werd binnen vijf seconden
afgebroken in gesimuleerd maagsap met dezelfde concentratie pepsine. Een dergelijke
snelle afbraak van nieuwe eiwitten bij in vitro digestie experimenten met pepsine, wordt
in het algemeen gezien als een argument tegen eventuele allergeniteit.
     Het uitgangspunt bij de genoemde onderzoeken is dat het verschil tussen maïslijn
1507 en conventionele maïs uitsluitend de aanwezigheid is van de geïntroduceerde
genen, de genproducten daarvan en de daardoor veroorzaakte nieuwe eigenschappen.
De commissie is echter van mening dat de moleculair-biologische karakterisering van
de nieuwe maïslijn de aanwezigheid van onbedoelde additionele veranderingen in het
genoom van de maïs niet geheel kan uitsluiten, en verzocht de aanvrager daarom om
Interpretatie en evaluatie van voorgelegde gegevens                                        27
</pre>

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<pre>   naast het onderzoek van de nieuwe eiwitten ook een toxiciteitsonderzoek uit te voeren
   met de gehele maïskorrel, om aldus een extra garantie voor de veiligheid te verkrijgen
   voor humane, duurzame consumptie van 1507-maïs. In antwoord hierop heeft de aan-
   vrager vervolgens de resultaten overlegd van een subchronisch toxiciteitsonderzoek, uit-
   gevoerd met ratten (stam Crl:CD(SD)IGS BR). Gedurende 90 dagen kregen testgroepen
   33 procent of 11 procent w/w 1507-maïs in het voer toegediend. Controlegroepen kre-
   gen 33 procent of 11 procent w/w van een vergelijkbare, niet-gemodificeerde maïs te
   eten. In de 11 procent-groepen werd het voer aangevuld met 22 procent w/w van een
   commerciële niet-gemodificeerde maïsvariant. Een andere controlegroep kreeg in het
   voer 33 procent w/w van deze niet-gemodificeerde maïs toegediend. De test- en contro-
   legroepen bestonden ieder uit twaalf mannelijke en twaalf vrouwelijke ratten. Lichaams-
   gewicht en voedselconsumptie werden gedurende de eerste week dagelijks bepaald en
   daarna wekelijks. Klinische observaties werden twee maal daags gedaan op direct zicht-
   bare verschijnselen van toxiciteit en wekelijks door een algemeen onderzoek. Aan het
   begin en het einde van de onderzoeksperiode werd oogonderzoek en gedragskundig
   onderzoek uitgevoerd. Na afloop van de onderzoeksperiode werden bloed- en urinemon-
   sters genomen en werden de dieren gedood. Een aantal algemene en klinisch chemische
   parameters werden bepaald voor bloed en urine. Het gewicht van een aantal organen
   werd bepaald. Tevens werden een groot aantal weefsels en organen van de hoge dosis-
   groepen voor 1507-maïs en voor de vergelijkbare, niet-gemodificeerde maïs microsco-
   pisch onderzocht. Voor zover significante verschillen werden waargenomen, waren
   deze niet toe te schrijven aan de toediening van 1507-maïs. Op basis van dit proefdierex-
   periment en gegevens van FAO/WHO over inneming van maïs en maïsproducten bere-
   kent de aanvrager een margin of exposure van 12 voor het CRY1F-eiwit voor Europese
   consumenten ten opzichte van de hoogst geteste dosis. Daarbij is uitgegaan van het
   maximale gemeten gehalte aan CRY1F in maïskorrels en gebruik van uitsluitend 1507-
   maïs voor de productie van alle geconsumeerde maïsproducten. Tevens wordt veronder-
   steld dat er geen verlies van CRY1F optreedt door de bewerking tot voedingsmiddelen.
   Zeker de laatste twee aannames zijn zeer conservatief te noemen, dat wil zeggen, het is
   aannemelijk dat de werkelijke consumptie van dit eiwit uit maïslijn 1507 aanmerkelijk
   lager zal zijn.
       De commissie is van mening dat de aanvrager op een juiste wijze onderzoek heeft
   verricht naar de eigenschappen van de nieuwe eiwitten CRY1F en PAT. Daarbij zijn
   geen aanwijzingen gevonden voor toxiciteit of allergeniteit. Uit de resultaten van het
   door de aanvrager uitgevoerde subchronische toxiciteitsonderzoek bij ratten blijkt
   bovendien dat bij consumptie van 1507-maïs geen nadelige effecten zijn te verwachten
   ten gevolge van eventuele onbekende veranderingen in het genoom van de plant.
28 Insectenresistente en herbicide-tolerante maïs (1507)
</pre>

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<pre>      Literatuur
COD02 Report of the third session of the CODEX ad hoc intergovernmental task force on foods derived from
      biotechnology. Proposed draft annex on the assessment of possible allergenicity of the draft guideline for
      the conduct of food safety assessment of foods derived from recombinant-DNA plants. Rome: FAO, 2002.
EG97  Verordening (EG) nr. 258/97 van het Europees parlement en de Raad van 27 januari 1997 betreffende
      nieuwe voedingsmiddelen en nieuwe voedselingrediënten. Publikatieblad van de Europese
      Gemeenschappen 1997; L43: 1-6.
EG97a Aanbeveling (EG) nr. 97/618/EC van de Commissie van 29 juli 1997 betreffende de wetenschappelijke
      aspecten en de presentatie van de informatie die nodig is om aanvragen voor het in de handel brengen van
      nieuwe voedingsmiddelen en nieuwe voedselingrediënten te ondersteunen alsmede het opstellen van de
      verslagen van de eerste beoordeling uit hoofde van Verordening (EG) nr. 258/97 van het Europees
      Parlement en de Raad. Publikatieblad van de Europese Gemeenschappen 1997; L253: 1-36.
EG98  Verordening (EG) Nr. 1139/98 van de Raad van 26 mei 1998 betreffende de verplichte opneming in de
      etikettering van bepaalde met genetisch gemodificeerde organismen geproduceerde levensmiddelen van
      andere gegevens dan die waarin Richtlijn 79/112/EEG voorziet. Publikatieblad van de Europese
      Gemeenschappen 1998; L159: 4-7.
EG00  Verordening (EG) nr. 49/2000 van de Commissie van 10 januari 2000 houdende wijziging van Verordening
      (EG) nr. 1139/98 van de Raad betreffende de verplichte opneming in de etikettering van bepaalde met
      genetisch gemodificeerde organismen geproduceerde levensmiddelen van andere gegevens dan die waarin
      Richtlijn 79/112/EEG voorziet. Publikatieblad van de Europese Gemeenschappen 2000; L6: 13-14.
EG01  Richtlijn 2001/18/EG van het Europees Parlement en de Raad van 12 maart 2001 inzake de doelbewuste
      introductie van genetisch gemodificeerde organismen in het milieu en tot intrekking van Richtlijn 90/220/
      EEG van de Raad. Publikatieblad van de Europese Gemeenschappen 2001; L106: 1-38.
      Literatuur                                                                                                 29
</pre>

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<br><br>====================================================================== Pagina 30 ======================================================================

<pre>EG03   Verordening (EG) nr. 1829/2003 van het Europees Parlement en de Raad van 22 september 2003 inzake
       genetisch gemodificcerde levensmiddelen en diervoerders. Publikatieblad van de Europese
       Gemeenschappen 2003; L268: 1-23.
EG03a  Verordening (EG) nr. 1830/2003) van het Europees Parlement en de Raad van 22 september 2003 inzake
       genetisch gemodificeerde organismen en de traceerbaarheid van met genetisch gemodificeerde organismen
       geproduceerde levensmiddelen en diervoerders en tot wijziging van Richtlijn 2001/18/EG. Publikatieblad
       van de Europese Gemeenschappen 2003; L268: 24-28.
FAO96  Biotechnology and Food Safety. Report of a joint FAO/WHO Consultation. Rome: FAO, 1996.
FAO01  Evaluation of allergenicity of genetically modified foods. Report of a joint FAO/WHO expert consultation
       on allergenicity of foods derived from biotechnology. Rome: FAO, 2001.
GR92   Commissie Toxicologische aspecten van biotechnologisch bereide producten. Productveiligheid bij nieuwe
       biotechnologie (publicatienummer 1992/03). Den Haag: Gezondheidsraad, 1992.
Hil02  Hileman RE, Silvanovich A, Goodman RE, Rice EA, Holleschak G, Astwood JD et al. Bioinformatic
       methods for allergenicity assessment using a comprehensive allergen database. Int Arch Allergy Immunol
       2002; 128(4): 280-291.
Iow97  Iowa State University of Science and Technology. How a corn plant develops. Special report no. 48. Ames,
       Iowa: reprinted 1997.
Iow03  Iowa State University of Science and Technology. How a corn plant develops. Special report no. 48.
       Website http://maize.agron.iastate.edu/corngrows.html, geraadpleegd op 21 oktober 2003.
Kle02  Kleter GA, Peijnenburg AA. Screening of transgenic proteins expressed in transgenic food crops for the
       presence of short amino acid sequences identical to potential, IgE - binding linear epitopes of allergens.
       BMC Struct Biol 2002; 2(1): 8.
Koh03  Kohli A, Twyman RM, Abranches R, Wegel E, Stoger E, Christou P. Transgene integration, organization
       and interaction in plants. Plant Mol Biol 2003;52(2):247-58.
OECD93 Safety evaluation of foods derived by modern biotechnology. Concepts and principles. Paris: OECD, 1993.
OECD96 OECD Workshop on Food Safety Evaluation. Paris: OECD, 1996.
OECD98 Report of the OECD workshop on the toxicological and nutritional testing of novel foods. Paris: OECD,
       1998 (draft).
OECD99 Consensus document on general information concerning the genes and their enzymes that confer tolerance
       to phosphinothricin herbicide. Paris: OECD, 1999.
OECD00 Report of the task force for the safety of novel foods. Paris: OECD, 2000.
OECD02 Consensus document on compositional considerations for new varieties of maize (Zea mays): Key food and
       feed nutrients, anti-nutrients and secondary plant metabolites. Paris: OECD, 2002.
Pio01  Pioneer Hi-Bred en Mycogen Seeds. Aanvraag voor toelating op de markt van voedingsmiddelen en
       voedselingrediënten afkomstig van maïslijn 1507 in het kader van de EU-verordening 258/97. Brussel:
       Pioneer Overseas Corporation, 2001.
Pio02  Pioneer Hi-Bred en Mycogen Seeds. Beantwoording van vragen van de Commissie Veiligheidsbeoordeling
       Nieuwe Voedingsmiddelen van 28/06/2001. Brussel: Pioneer Overseas Corporation, 2002.
30     Insectenresistente en herbicide-tolerante maïs (1507)
</pre>

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<br><br>====================================================================== Pagina 31 ======================================================================

<pre>Pio03  Pioneer Hi-Bred en Mycogen Seeds. Aanvullende beantwoording van vragen van de Commissie
       Veiligheidsbeoordeling Nieuwe Voedingsmiddelen van 28/06/2001. Brussel: Pioneer Overseas
       Corporation, 2003.
Pio03a Pioneer Hi-Bred en Mycogen Seeds. Beantwoording van vragen van de Commissie Veiligheidsbeoordeling
       Nieuwe Voedingsmiddelen van 25/03/2003. Brussel: Pioneer Overseas Corporation, 2003.
Pio03b Pioneer Hi-Bred en Mycogen Seeds. Beantwoording van vragen van de Commissie Veiligheidsbeoordeling
       Nieuwe Voedingsmiddelen van 11/06/2003. Brussel: Pioneer Overseas Corporation, 2003.
SCF99  Scientific Committee on Food. Opinion concerning the scientific basis for determining whether food
       products, derived from genetically modified maize, could be included in a list of food products which do not
       require labelling because they do not contain (detectable) traces of DNA or protein. Brussels: Scientific
       Committee on Food of the EU, 1999.
SSC99  Scientific Steering Committee. Opinion of the Scientific Steering Committee on microbial resistance,
       Brussels: Scientific Steering Committee of the EU, 1999.
Sha88  Shaw, RH. Climatic requirement. P. 3-29 in: Corn and corn improvement, Sprague, GF and Dudley, JW
       (eds.). Madison, Wisconsin: American Society of Agronomy, Inc., Crop Science Society of America, Inc.,
       1988.
Whi95  White, PJ and Pollack, LM. Corn as a food source in the United States: Part II. Processes, Products,
       composition and nutritive values. Cereal foods world 1995; 40: 756-762.
WHO91  WHO. Strategies for assessing the safety of foods procuced by biotechnology. Report of a joint FAO/ WHO
       consultation. Geneva: WHO, 1991.
WHO00  Safety aspects of genetically modified foods of plant origin. Report of a joint FAO/WHO expert
       consultation on foods derived from biotechnology. Geneva: WHO, 2000.
       Literatuur                                                                                                   31
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<pre>32 Insectenresistente en herbicide-tolerante maïs (1507)</pre>

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<pre>A De adviesaanvraag
B De commissie
C EU-procedure
D Samenvatting van het dossier
E Schematische weergave van de genetische modificatie
  Bijlagen
                                                      33
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<pre>34 Insectenresistente en herbicide-tolerante maïs (1507)</pre>

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<pre>Bijlage A
        De adviesaanvraag
        Op 18 augustus 1999 schreef de Minister van Volksgezondheid, Welzijn en Sport aan de
        Voorzitter van de Gezondheidsraad (brief kenmerk GZB/VVB 993428):
        Sinds mei 1997 is in de Europese Unie de Verordening (EG) 258/97 van kracht inzake nieuwe voedings-
        middelen en nieuwe voedselingrediënten. Daarmee werd de veiligheidsbeoordeling onderdeel van een com-
        munautaire procedure.
             Met u is reeds de mogelijkheid besproken de beoordeling door de Gezondheidsraad te laten uitvoeren.
        Ik verzoek u dan ook mede namens de Staatssecretaris van Landbouw, Natuurbeheer en Visserij, in deze
        eerste fase van uitvoering van de Europese Verordening (EG) 258/97 gedurende een aantal jaren, de veilig-
        heidsbeoordeling gestalte te geven. Voor het onderbrengen bij de Gezondheidsraad pleit het experimentele
        karakter dat de beoordeling de eerste jaren zal hebben. Dit experimentele karakter komt voort uit het feit dat
        het een nieuw soort beoordeling betreft van deels nieuwe categorieën van voedingsmiddelen of voedse-
        lingrediënten. Het is namelijk een veiligheidsbeoordeling vóór het op de markt brengen van met name voe-
        dingsmiddelen van een genetisch gemodificeerde oorsprong en zogenaamd functional foods (nutriceutica).
        Daarnaast ga ik ervan uit dat de onafhankelijke wetenschappelijke advisering door de Gezondheidsraad het
        vertrouwen van de Europese Commissie en de andere lidstaten in het Nederlandse oordeel nog versterkt.
             Mijn beleid is erop gericht een zo groot mogelijke openheid en transparantie te realiseren van de
        gevolgde procedure en de beoordeling om de consument vertrouwen te geven in de veiligheid van de
        De adviesaanvraag                                                                                              35
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<pre>   nieuwe voedingsmiddelen. Ik verzoek de Gezondheidsraad hieraan bij te dragen door bijvoorbeeld inzage te
   geven in de dossiers waarvoor een aanvraag wordt ingediend, waarbij uiteraard bedrijfsvertrouwelijke gege-
   vens worden beschermd en door de criteria, waarop de veiligheid zal worden beoordeeld, te publiceren.
   De Minister van Volksgezondheid, Welzijn en Sport,
   w.g. dr E. Borst-Eilers
36 Insectenresistente en herbicide-tolerante maïs
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<pre>Bijlage B
        De commissie
        •  Prof. dr LM Schoonhoven, voorzitter
           emeritus hoogleraar entomologie; Wageningen Universiteit en Researchcentrum
        •  Prof. dr CAFM Bruijnzeel-Koomen
           hoogleraar dermatologie/allergologie; Academisch Ziekenhuis Utrecht
        •  Ir EJ Kok
           toxicoloog; RIKILT-DLO Wageningen
        •  Dr CF van Kreijl
           moleculair-bioloog; RIVM Bilthoven
        •  Prof. dr P van der Laan
           hoogleraar statistiek; Technische Universiteit Eindhoven
        •  Dr FM Nagengast
           gastro-enteroloog; Academisch Ziekenhuis Nijmegen
        •  Dr ir JMA van Raaij
           voedingsfysioloog; Wageningen Universiteit and Researchcentrum
        •  Prof. dr ir G Schaafsma
           hoogleraar voeding; TNO Voeding, Zeist
        •  Prof. dr EG Schouten
           hoogleraar epidemiologie; Wageningen Universiteit and Researchcentrum
        •  Dr GJA Speijers
           toxicoloog; RIVM Bilthoven
        •  Prof. dr WJ Stiekema
           hoogleraar bioinformatica; Wageningen Universiteit en Researchcentrum
        De commissie                                                                   37
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<pre>   •   Ir R Top, adviseur
       Ministerie van VWS; Den Haag
   •   Prof. dr WM de Vos
       hoogleraar microbiologie; Wageningen Universiteit en Researchcentrum
   •   Dr ir F van der Wilk, adviseur
       COGEM, Bilthoven
   •   Dr RA Woutersen
       toxicoloog; TNO Voeding, Zeist
   •   Dr CMA van Rossum, secretaris
       Gezondheidsraad, Den Haag
   Administratieve ondersteuning: drs CL Vuijst; Gezondheidsraad, Den Haag
   Layout: J van Kan; Gezondheidsraad, Den Haag
38 Insectenresistente en herbicide-tolerante maïs
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<pre>Bijlage C
        EU-procedure
        Als een fabrikant een nieuw voedingsmiddel op de markt brengt, dient de veiligheid
        voor de consument gewaarborgd te zijn. In 1997 werd de Europese verordening van
        kracht waarin de procedure is geregeld voor de goedkeuring voor marktintroductie van
        een nieuw voedingsmiddel (EG97). Bij deze procedure zijn verschillende actoren
        betrokken. De aanvrager moet beoordelen of het product werkelijk 'nieuw' is, dat wil
        zeggen dat het nog niet eerder in de Europese Unie in substantiële mate voor menselijke
        voeding is gebruikt en ook niet wezenlijk gelijkwaardig is aan een bestaand product.
        (Voor een wezenlijk gelijkwaardig product kan worden volstaan met een kennisgeving
        van de marktintroductie.) Ook moet het niet gaan om een levensmiddelenadditief, aroma
        of extractiemiddel, omdat deze producten op een andere wijze worden beoordeeld. Voor
        een nieuw voedingsmiddel in de zin van de Europese verordening moet de aanvrager
        een veiligheidsdossier overleggen volgens aanbevelingen van de Europese Commissie
        (EG97a). Deze aanbevelingen zijn gebaseerd op rapporten van verschillende instanties
        die zich met het onderwerp nieuwe voedingsmiddelen bezighouden, te weten de OECD
        (OECD93, OECD96) en de WHO/FAO (FAO96, WHO91). Ook de Gezondheidsraad
        heeft zich al eerder over dit onderwerp gebogen (GR92). Sinds het verschijnen van de
        aanbevelingen van de EU wordt in internationaal verband gewerkt aan explicitering en
        aanpassing aan de stand van de wetenschap (FAO01, OECD98, OECD00, SCF99,
        SSC99, WHO00). In de nabije toekomst zullen levensmiddelen geproduceerd uit
        genetisch gemodificeerde organismen niet meer volgens deze procedure worden beoor-
        deeld, maar overeenkomstig een nieuwe Europese verordening (EU03).
        EU-procedure                                                                            39
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<pre>       De fabrikant levert het volgens de richtlijnen samengestelde dossier in bij het land
   waar het product het eerst op de markt zal komen. Daarop komt de nationale veiligheids-
   beoordelingsautoriteit in actie. In Nederland is dat de Minister van Volksgezondheid,
   Welzijn en Sport. Zij heeft de Gezondheidsraad verzocht haar van advies te dienen. De
   Voorzitter van de Gezondheidsraad heeft hiertoe de commissie Veiligheidsbeoordeling
   nieuwe voedingsmiddelen (commissie VNV) ingesteld.
       De commissie beoordeelt op basis van de huidige stand van de wetenschap of de
   door de fabrikant geleverde gegevens juist en volledig zijn en of zij het eens is met diens
   conclusies. Zij maakt een verslag van haar bevindingen — ook volgens de Europese
   aanbevelingen (EG97a, deel III) — en biedt dat de minister aan. De minister formuleert
   het Nederlandse oordeel over een voedingsmiddel en brengt dat in bij het Europese
   overleg in het Permanent Comité voor de voedselketen en de diergezondheid. Alle Euro-
   pese lidstaten worden uitgenodigd hun oordeel (de zogeheten tweede beoordeling) te
   geven over het dossier en over de eerste beoordeling alvorens genoemd Comité een
   eindoordeel velt. Als een dossier veel vragen oproept, gaat er een adviesvraag van de
   Europese Commissie naar het Wetenschappelijk Comité voor de menselijke voeding.
   Komt men dan nog niet tot overeenstemming dan beslist de Europese Ministerraad.
40 Insectenresistente en herbicide-tolerante maïs
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<pre>Bijlage D
        Samenvatting van het dossier
        Samenvatting van het dossier 41
</pre>

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<br><br>====================================================================== Pagina 42 ======================================================================

<pre>                                     6. SUMMARY
In accordance with Article 6.1 of the Regulation (EC) No 2258/97, this is a summary
of the application for placing on the market of novel foods and novel food ingredients
jointly submitted to the competent authority and the food assessment body of The
Netherlands by Pioneer Hi-Bred International Inc. and Mycogen Seeds c/o Dow
AgroSciences LLC. Pioneer Hi-Bred International Inc., as represented by Pioneer
Overseas Corporation, Avenue Tedesco 7, B-1160 Brussels, Belgium, is taking the
lead for this submission.
 Description of the product
The product consists of foods from maize grain produced from genetically modified
B.t. Cry1F maize line 1507 and progeny derived from conventional breeding between
1507 maize with any traditionally bred non-GM maize. The term "1507 maize" refers
to grain derived from inbreds and hybrids of maize line 1507 including crosses with
traditionally bred maize.
The 1507 maize has been genetically modified (GM) to express CRY1F protein for
resistance to certain lepidopteran insect pests, such as the European corn borer (ECB),
and to express phosphinothricin-N-acetyltransferase (PAT) protein for tolerance to
glufosinate-ammonium herbicide.
Categorisation of the product
Based on the recommendations made by the EC Scientific Committee on Food (SCF)
for the scientific classification of the novel food (NF) and for the assessment of
wholesomeness, 1507 maize belongs to Class 3, Subclass 3.1, under GM plants and
their products (Commission Recommendation 97/618/EC of 29th July 1997). Under
NF Class 3, the GM plants can be consumed directly as unprocessed foods or after
having been processed into foods and food ingredients.
Identification and consultation of the essential information requirements
In the case of 1507 maize and based on Table II of the SCF recommendations, the
structured schemes identified to provide guidance for consultation of the essential
information requirements for the NF and the assessment of wholesomeness comprise
Schemes I to VII and IX to XIII.
Scheme I: Specification of the novel food (NF)
Maize (Zea mays L.) has well characterised specification belonging to the Gramineae
family, the genus Zea and the species Z. mays (2n = 20). The evidence presented in
this application confirms that foods derived from 1507 maize can be considered to be
substantially equivalent to food products derived from traditionally-bred non-GM
maize with no nutritional or toxicological changes. Traditionally-bred maize does not
contain any toxic or anti-nutritional factors that need to be controlled by a
specification and the characteristics, compositional analyses and safety evaluation of
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<pre>the genetic modification in 1507 maize does not entail a separate specification of the
NF.
A PCR detection method to confirm molecular identity and to ensure that the NF
marketed is similar to that evaluated is being developed. The detection method will
be submitted to the regulatory authority before the 1507 maize is placed on the EU
market for food purposes.
Scheme II: Effect of the production process applied to the NF
Foods from 1507 maize will be used and processed as any other commercial maize.
The production process applied to maize are well known and have a long history of
safe use. The safety evaluation contained in this application for foods derived from
1507 maize provides further verification that no specific conditions of use and
processing are required for 1507 maize, and therefore there should be no effects on the
existing production processes as applied to the NF.
Scheme HI: History of the organism used as the source of the NF
The NF, referred to as 1507 maize, is obtained from a maize plant (Zea mays L.)
which has a long history of safe use. The genetic modification of 1507 maize was
carried out by using the particle acceleration method to introduce into maize cells a
purified linear DNA fragment containing the cry1F and pat gene coding sequences
and the necessary regulatory components for expression of CRY1F and PAT proteins
in maize. No other gene coding sequences have been inserted during the genetic
modification of 1507 maize.
The genetic sequences obtained from the donor organisms and inserted in 1507 maize
do not have any pathogenic characteristics. First of all maize as the recipient plant,
and donor of the ubiZM1(2) promoter, is not a pathogenic organism and has a long
history of domestication and safe use as an agricultural food crop.
Secondly, Bacillus thuringiensis, donor of the crylF sequence, has a history of
decades of safe use as a pesticide (EPA, 1996). The subspecies aizawai is
commercially used to control wax moth larvae and various caterpillars, especially the
diamondback moth caterpillar (Cornell University, 1996). Agrobacterium
tumefaciens is the source of the ORF25PolyA terminator for the cry1F gene. No
sequences involved in plant pathogenicity are encoded by this transcription
terminator.
 Thirdly, Streptomyces viridochromogenes, donor of the pat gene, is a common soil
 bacterium that produces the tripeptide L-phosphinothricyl-L-alanyl-alanine (L-PPT),
 which was developed as a non-selective herbicide by Hoechst Ag and there is a
 history of safe use of the pat gene in GM crops (OECD, 1999; see Annex 5). The
 cauliflower mosaic virus, donor of the CaMV 35S promoter and terminator sequences,
 is a DNA caulimovirus with a host range restricted primarily to cruciferous plants
 (ICTV Database, 1998).
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<pre> Scheme IV: Effect of the genetic modification on the properties of the host
 organism
The 1507 maize has been modified to express CRY1F protein for resistance to certain
lepidopteran insect pests, such as the European com borer (ECB), and to express
phosphinothricin-N-acetyltransferase (PAT) protein for tolerance to glufosinate-
ammonium herbicide. The particle acceleration method was used to introduce into
maize cells a purified linear DNA fragment (PHI8999A; 6235 bp) containing the
cry1F and pat coding sequences and the necessary regulatory components only. The
nptII gene, conferring resistance to kanamycin, was not part of the purified linear
DNA fragment used in the transformation and it is not present in 1507 maize, as
confirmed by the molecular analyses carried out with Southern blots.
The insert, PHI8999A, consists of a linear DNA fragment containing we plant
optimized and truncated crylF gene and the plant optimized pat gene together with
the regulatory sequences necessary for their expression in maize plants. The insert
was obtained by digesting the plasmid PHP8999 (9504 bp) with the restriction
enzyme PmeI. This produced the 6235 bp insert, which was subsequently purified by
agarose gel electrophoresis and used in the transformation of 1507 maize. The 3269
bp fragment containing the nptII gene was discarded and was not used in the
transformation.
The amino acid sequence of the core CRY IF protein expressed in 1507 maize is, with
the exception of a single amino acid substitution, leucine at position 604, identical to
amino acids 1-605 of the native CRY1F protein which is 1174 amino acids long. This
change in the coding sequence was made to introduce aXhoI restriction site for fusion
of sequences encoding the C-terminal domain of the protein that forms the full length
protein. The amino acid sequence of the recombinant CRY1F protein (MR872)
produced in Pseudomonas fluorescens which has been used in toxicological studies
consists of a chimeric CRY1F/CRYlA(b) protein with equivalent biochemical
characteristics and biological activity to the maize expressed core CRY1F protein
(Evans, 1998).
The activity of the PAT protein is specific to catalysing the conversion of L-PPT to N-
acetyl-L-PPT. This is an inactive form which does not bind to glutamine synthetase
(De Block et al. 1987). The expression of the PAT protein in 1507 maize allows the
detoxification of ammonia to continue and confers tolerance to the herbicide
glufosinate-ammonium. The activity of the PAT protein has been described in detail
by the OECD (1999): "Consensus document on general information concerning the
genes and their enzymes that confer tolerance to phosphinothricin herbicide" (Annex
5).
 Detailed molecular characterization, based on Southern blot analyses, confirms that
 1507 maize contains a full-length copy of the DNA insert integrated into the maize
 genome and an additional copy of the cry1F gene (Glatt, 2000; Annex 6).
Scheme V: Genetic stability of the GMO used as NF source
 The 1507 maize has remained stable as inbreds and hybrids for at least six
 generations, through crossing and backcrossing to elite inbreds and selfing, as
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<pre>confirmed by early and late segregation data (BC2F1 and F1 generations,
respectively). This evidence, together with the molecular characterization by
Southern blotting of the 1507 maize (Scheme IV) and analyses of the expression of
CRY1F and PAT proteins (Scheme VI), confirms stable integration of the cry1F and
pat genes in the genome of 1507 maize. These analyses support the conclusion that
the cry1F and pat genes are inherited as Mendelian dominant genes and that the
additional copy of the cry1F gene is genetically linked to the insert containing the
cry1F and pat genes.
Scheme VI: Specificity of expression of novel genetic material
Analyses of expression of the novel genetic material (cry1F and pat genes) has been
carried out at ten locations over two growing seasons: four locations in Chile in 1998-
1999, and six locations in France and Italy in 1999. The results obtained from tissue
samples from 1507 maize have confirmed specificity of CRY1F protein expression in
samples of leaf (V9 stage), pollen, silk, stalk, whole plant (both at V9 and R4 stages),
grain and senescent whole plant tissues. The results have also confirmed specificity
of PAT protein expression in samples of leaf (V9 stage) and whole plant (V9 stage)
tissues. The ELISA results show that CRY1F is expressed in all plant material tested,
with significantly lower concentrations in grain than in vegetative tissue.
Additionally, Western blot analyses have confirmed that CRY1F and PAT proteins
expressed in 1507 maize are of the same molecular weight and immunoreactivity as
the microbially-derived proteins. Other than the expected bands for CRY1F and PAT
proteins, the Western blots did not show any other bands to indicate either partial or
fusion proteins in the 1507 maize tissues.
 Comparison of the agronomic performance in terms of grain yield, moisture,
 accumulated growing degree days to reach 50% pollen shed, accumulated growing
 degree days to reach 50% silking, grain density, plant height, ear height, early stand
 count establishment, visual rating of emergence vigour from spike to one-leaf stage,
 visual rating of vigour at three- to five-leaf stage, stalk lodging, root lodging, dropped
 ears per plot and top integrity between 1507 maize and non-GM control maize with
 comparable genetic backgrounds, further confirms that 1507 maize is comparable to
 non-GM control maize. With the exception of the intended resistance to certain
 lepidopteran insect pests and tolerance to glufosinate-ammonium herbicide, as derived
 from the genetic modification, no unexpected phenotypic differences have been
 observed in 1507 maize. The agronomic data also provides further evidence of the
 specificity of expression of the novel genetic material in 1507 maize
 Scheme VII: Transfer of genetic material from GMO
  Transfer of genetic material originating from 1507 maize to bacteria is a negligible
  concern as there is no known mechanism for, or definitive demonstration of, DNA
  transfer from plants to microbes under natural conditions. In any case, and even if
  horizontal gene transfer were to take place, transfer of the cry1F or pat gene from 1507
  maize does not represent a risk to human health or animal health.
  Scheme VIII: Ability of the GMM to survive in and colonize the human gut
  This is not applicable as the NF is not a GM microorganism (GMM).
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<pre> Scheme IX: Anticipated intake/extent of use of the NF
Majority of grain and forage derived from maize is used for animal feed and about 8%
of EU production is processed for human food products. The evidence provided in
the application shows that the inserted genetic material and the newly expressed
CRY1F and PAT proteins in 1507 maize are safe to human health. Furthermore, the
properties of 1507 maize regarding its nutritional value and compositional
characteristics are comparable and substantially equivalent to those of traditionally-
bred non-GM maize. Based on this evidence, the anticipated use of 1507 maize
derived foods will be no different from that of traditionally bred maize, and any
substitution of traditionally bred maize by 1507 maize will not be of any nutritional
significance.
Scheme X: Information from previous human exposure to the NF or its source
Food products derived from maize have been produced, prepared and consumed by
humans for centuries throughout the world. Based on the evidence presented in this
application, we conclude that exposure to food products derived from 1507 maize will
not give rise to nutritional, microbiological, lexicological and/or allergenicity
problems and that it is as safe as exposure to any other food products derived from
traditionally-bred non-GM maize.
Scheme XI: Nutritional information on the NF
Range of data from compositional analyses of maize grain are provided in the
application for the nutritional assessment of 1507 maize. Field studies representative
of pertinent conditions and growth stages of the commercial production of maize were
conducted on 1507 maize and non-GM control maize. Data from ten different
locations over two growing seasons have been analysed: four locations in Chile
(1998-1999) and six in the EU (France and Italy, 1999). At the locations in Italy, the
trials also included treatments to compare 1507 maize sprayed with glufosinate-
ammonium, 1507 maize unsprayed with glufosinate-ammonium, and non-GM control
maize. At all locations, the non-GM control maize was of comparable genetic
background to 1507 maize.
The compositional data determined the levels of protein, fiber (ADF, NDF),
carbohydrates, fat, ash, five different fatty acids, five different minerals, eighteen
different amino acids, four different vitamins, five different secondary metabolites and
two potential anti-nutrients. The analyses on the composition clearly demonstrated
that grain from 1507 maize, unsprayed or sprayed with glufosinate-ammonium, is
comparable to grain from non-GM maize and to the published range of composition
values in the literature. These analyses support the conclusion that 1507 maize is
substantially equivalent to traditionally-bred maize.
 In addition, nutritional equivalence between 1507 maize and traditionally-bred maize
 has been shown in a poultry feeding study where broiler chickens were fed over a 42-
 day period with diets containing 1507 maize grain or yellow dent grain from non-GM
 maize. The mortality, body weight gain and feed conversion of the chickens were
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<pre>compared and no statistically significant differences were observed between chickens
fed a diet containing grain from 1507 maize or grain from non-GM maize.
Scheme XII: Microbiological information on the NF
The NF does not contain any microorganisms and therefore no microbiological
information is provided.
SchemeXIII: Toxicological information on theNF
Toxicitv
Maize has a long history of use as food in the EU and constitutes a traditional
counterpart to 1507 maize that can be used as a baseline to facilitate the toxicological
assessment. Maize is not considered to have harmful toxicants and the genetic
modification in 1507 maize does not introduce any new toxicants harmful to humans
or animals.
The CRY1F protein has specific toxicity against certain lepidopteran insect pests
(target organisms), and there is no evidence for CRY proteins originating from
Bacillus thuringiensis to have any harmful effects on the health of humans and
animals (EPA, 1995a; McClintock et al., 1995; EPA, 1996). Furthermore, the
potential toxicity of the CRY1F protein to humans and animals was specifically
examined in an acute oral toxicology study where CRY1F protein was evaluated for
acute toxicity in mice (Kuhn, 1998). The relatively high dose tested did not give rise
to any toxicity and therefore the acute LD50 for CRY1F protein could not be
determined other than estimated to be higher than 576 mg CRY1F per kg body
weight.
The PAT protein has already been found safe to human health during the assessment
of glufosinate-ammonium tolerant maize (OECD, 1999; Annex 5). The pat gene was
originally obtained from Streptomyces viridochromogenes strain Tü494 which has no
known toxic or pathogenic potential. The PAT protein is enzymatically active but it
has high substrate specificity to the active ingredient of glufosinate-ammonium (L-
PPT). A toxicity study consisting of feeding rats with the PAT protein has been
carried out (Pfister et al., 1996; Health Canada, 1997). Results from the range of
doses tested (up to 50000 mg/kg body weight) showed no adverse effects on the
growth or histopathology of the animals. The PAT protein has also been tested in an
additional acute toxicity study in mice (Brooks, 2000). As before, the relatively high
dose tested did not give rise to any toxicity and therefore the acute LD50 of PAT
protein could not be determined other than estimated to be higher than 5000 mg PAT
per kg body weight.
 A 42-day long poultry feeding study has also been carried out with grain from 1507
 maize and the non-GM control maize with comparable genetics. The results show
 that mortality, body weight gain and feed conversion of the chickens fed with 1507
 maize was similar to chickens fed a standard diet containing yellow dent maize.
  Allergenicity
  The most important factor to consider in assessing allergenic potential is whether the
  source of the gene being introduced into plants is known to be allergenic (FDA,
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<pre> 1992). Neither Bacillus thuringiensis (the source of the cry1F gene) nor Streptomyces
 viridochromogenes (the source of the pat gene) have a history of causing allergy.
The biochemical profile of the CRY IF and PAT proteins also provides a basis for
allergenic assessment when compared with known protein allergens. A database
search was compiled by Meyer (1999) using the Wisconsin Genetics Computer Group
(GCG) sequence analysis computer program with the keyword "allergen" to search
standard DNA and protein sequence databases. Comparison of the 15 most
homologous database sequences confirmed that the CRY1F and PAT proteins do not
share any significant amino acid sequence homology with known protein allergens.
Furthermore, protein allergens are typically stable to the peptic and tryptic digestion,
and to the acid conditions of the human digestive system, which allows them to reach
and pass through the intestinal mucosa to elicit an allergenic response. Both CRY1F
and PAT proteins are readily degradable in simulated digestive juice, minimising any
potential for these proteins to be absorbed by the intestinal mucosa when consumed.
The CRY1F protein was nearly completely proteolysed in simulated gastric
conditions within one minute at a molar ratio of 188:1 (CRY1F:pepsin) (Evans,
1998). In addition, the immunoblot detection technique has demonstrated that
CRY1F is not glycosylated, which is an additional indicator of the absence of
allergenic potential in the CRY1F protein. Furthermore, CRY1F looses
immunoreactivity after heat processing and it has a history of safe use in microbial
pesticides (Evans, 1998). In addition, a study on the heat lability of CRY1F protein at
various temperatures showed that CRY1F looses biological activity after exposure at
75°C or greater for 30 minutes (Herman, 2000). The PAT protein degraded to non-
detectable levels within 5 seconds after introduction to simulated gastric fluid
containing pepsin (Glatt, 1999; OECD, 1999; Annex 5).
Therefore, the cry1F and pat genes introduced into 1507 maize do not encode for
known allergens, and neither the CRY1F nor the PAT proteins share immunologically
significant amino acid sequences with known allergens. This together with the heat
lability and rapid breakdown of these proteins under digestive conditions, confirms
that the CRY1F and PAT proteins should not pose any allergenic risk.
 Evaluation and conclusion
 The safety evaluation of food products derived from B.t. Cry1F maize line 1507,
 referred to as 1507 maize, presented throughout this application has been carried out
 following thorough consideration of a series of key parameters that show
 wholesomeness and substantial equivalence between 1507 maize and traditionally-
 bred non-GM maize: Southern blot analyses and molecular characterisation,
 agronomic performance, Mendelian inheritance, protein expression analyses,
 compositional analyses, nutritional equivalence and lexicological information have
 demonstrated that food products from 1507 maize are as safe and nutritious as other
 maize products already on the EU market.
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<pre>Bijlage          E
                 Schematische weergave van de
                 genetische modificatie
     Schematische weergave van de genetische modificatie in 1507 maïs
     9                     = primaire insertie
     4, 7a/b/c, 8, 10, 13  = gedeeltelijke duplicaties van de primaire insertie
     5, 6, 11, 12, 14      = overeenkomst met chloroplast DNA
     2,3                   = overeenkomst met bekend maïs genomisch DNA
     1, 15                 = onbekend, waarschijnlijk maïs genomisch DNA
                                                 6 7b 7c                                                        14
              1       2      3          4    5    7a     8                                     10  11   12    13          15
         5’ flankerende sequentie                                                                         3’ flankerende sequentie
                                                                      primaire insertie
                                                                                9
                                                                                                     T 35S
                    P ubiZM1(2)                                cry1F                 T ORF25 P 35S    pat
                                                                                                      7b         herkomst van
                                                                                                   7a            gedeeltelijke
             8                                             7c         4                10          13            duplicaties
           0               1000              2000               3000              4000        5000            6000
                                                                                                            lengte in basenparen
                 Schematische weergave van de genetische modificatie                                                               49
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<pre>50 Insectenresistente en herbicide-tolerante maïs</pre>

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<pre>Insect-resistant and herbicide-tolerant
maize (1507)
Assessment of safety for the consumer, in accordance with European
Regulation 258/97 concerning novel foods and novel food ingredients
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<pre></pre>

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<pre>Letter to the Dutch Minister of Health,
Welfare and Sport
On November 4, 2003, professor dr JGAJ Hautvast, Vice-President of the Health Coun-
cil of the Netherlands wrote as follows to the Minister of Health, Welfare and Sport:
Herewith I present you an advisory report that is prepared in response to your request for
advice regarding the safety of novel foods and novel food ingredients, also made on
behalf of the Minister of Agriculture, Nature and Food Quality. This advice is a so called
initial assessment in the context of European Regulation (EC) 258/97, concerning
Insect-resistant and herbicide-tolerant maize (1507). The assessment was carried out by
the Committee on the Safety Assessment of Novel Foods of the Health Council of the
Netherlands.
     This advisory report is also presented to the Minister of Agriculture, Nature and
Food Quality.
Signed
professor dr JGAJ Hautvast
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<pre></pre>

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<pre>Insect-resistant and herbicide-tolerant
maize (1507)
Assessment of safety for the consumer, in accordance with European
Regulation 258/97 concerning novel foods and novel food ingredients
Health Council of the Netherlands:
Committee on the Safety Assessment of Novel Foods
to:
the Minister of Health, Welfare and Sport
the Minister of Agriculture, Nature and Food Quality
No. 2003/04VNV, The Hague, November 4, 2003
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<pre>The Health Council of the Netherlands, established in 1902, is an independent scientific
advisory body. Its remit is “to advise the government and Parliament on the current level
of knowledge with respect to public health issues...” (Section 21, Health Act).
     The Health Council receives most requests for advice from the Ministers of Health,
Welfare & Sport, Housing, Spatial Planning & the Environment, Social Affairs &
Employment, and Agriculture, Nature and Food Quality. The Council can publish advi-
sory reports on its own initiative. It usually does this in order to ask attention for devel-
opments or trends that are thought to be relevant to government policy.
     Most Health Council reports are prepared by multidisciplinary committees of Dutch
or, sometimes, foreign experts, appointed in a personal capacity. The reports are avail-
able to the public.
Preferred citation:
Health Council of the Netherlands: Committee on the Safety Assessment of Novel
Foods. Insect-resistant and herbicide-tolerant maize (1507). The Hague: Health Council
of the Netherlands, 2003; publication no. 2003/04VNV.
all rights reserved
A public version of this advisory report can be found on the website of the Health
Council of the Netherlands: www.healthcouncil.nl.
</pre>

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<pre>    Contents
    Executive summary, conclusions and recommendations 59
1   Introduction 61
2   Completeness and accuracy of the dossier 63
2.1 Administrative data 63
2.2 General description of the food 64
2.3 Classification of the food for assessment 64
2.4 Information about the food 64
2.5 Brief summary by the applicant 65
2.6 Other assessments 65
2.7 Labelling proposal by the applicant 65
3   Interpretation and evaluation of the data presented 67
3.1 I Specification of the novel food (NF) 67
3.2 II Effects of the production process applied to the NF 67
3.3 III History of the organism used as the source of the NF 68
3.4 IV Effect of the genetic modification on the properties of the host organism 68
3.5 V Genetic stability of the genetically modified organism (GMO) used as NF source 70
3.6 VI Specificity of the expression of the novel genetic material 70
3.7 VII Transfer of genetic material from the GMO 71
3.8 IX Anticipated intake and extent of use of the NF 72
    Contents                                                                            57
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<pre>3.9  X Information from previous human exposure to the NF or its source 72
3.10 XI Nutritional information on the NF 72
3.11 XII Microbiological information on the NF 74
3.12 XIII Toxicological information on the NF 75
     Literature 79
     Annexes 83
A    Request for advice 85
B    The Committee 87
C    EU-procedure 89
D    Executive summary of the dossier 91
E    Schematic representation of the genetic modification 99
58   Insect-resistant and herbicide-tolerant maize (1507)
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<pre>Executive summary, conclusions and
recommendations
Two companies, Pioneer Hi-Bred and Mycogen Seeds, jointly presented a safety dossier
for the genetically modified maize line 1507. This dossier contains molecular biological,
nutritional and toxicological information. The reference is a conventional maize line
with a history of safe use within the European Union.
     The modified maize line differs from a conventional line, due to the presence of the
cry1F and pat genes and their expression products, the CRY1F and PAT proteins. The
inserted genes, which are of bacterial origin, have been modified for optimal expression
in the plant. This renders the maize plant resistant to damage by certain insect pests and
tolerant to treatment with the herbicide glufosinate ammonium in the field. Molecular
biological analysis of the new crop revealed that a single, complete copy of each of these
genes is present at a single location in the DNA of 1507 maize. There are no indications
that the new proteins are toxic or allergenic for humans in the concentrations at which
they occur.
     The intended change is associated with unintended rearrangements of the DNA
flanking the insertion, which have also been analysed in detail. There are no indications
that the changes in the maize plant genome result in the unintended production of other
new proteins.
     The modified maize line’s composition has been compared with that of a near
isogenic, non-modified maize line by means of a chemical analysis of a large number of
components: micronutrients, macronutrients, antinutrients and secondary metabolites.
To this end, field trials were conducted at several locations, the results of which were
processed for each location separately. Observed variations in the components investi-
Executive summary, conclusions and recommendations                                         59
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<pre>   gated remained within the figures cited in the references and have no health-related con-
   sequences.
        Furthermore, in a subchronic toxicity study on animals, no adverse effects of the
   modification in 1507 maize were observed.
        The Committee is of the opinion that the information in the dossier provides suffi-
   cient basis for a safety assessment. The dossier contains a correct interpretation of the
   data submitted. Based on current scientific knowledge, the Committee’s opinion is that
   the consumption of 1507 maize, as well as foods and food ingredients derived from this,
   is just as safe for humans as the consumption of non-genetically modified maize and
   maize products.
60 Insect-resistant and herbicide-tolerant maize (1507)
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<pre>Chapter 1
        Introduction
        On 28 February 2001 the Minister of Health, Welfare and Sport requested the advice of
        the Committee on the Safety Assessment of Novel Foods (VNV), hereinafter referred to
        as ‘the Committee’, concerning the safety for consumers of foods and food ingredients
        produced from a new maize variant. This maize, which is referred to as line 1507, origi-
        nates from a plant (Zea mays L.) which, as a result of genetic modification, produces
        two new proteins, the CRY1F and PAT proteins. The CRY1F protein protects the plant
        against damage by certain insects, such as the European Corn Borer (Ostrinia nubilalis).
        The PAT protein renders the plant tolerant to the herbicide glufosinate ammonium. As a
        result, it is possible to control weeds in the field during the growth phase of these plants
        by treatment with the herbicide, without damaging the maize plants. The maize variant
        was developed by Pioneer Hi-Bred and Mycogen Seeds, who also submitted the applica-
        tion (Pio01). The application contains a safety assessment with a number of accompany-
        ing research reports. In June 2001, the Committee requested the applicant to supplement
        the dossier with additional data regarding the molecular characterisation, and the com-
        positional analysis of the new maize line. The Committee also requested the applicant to
        submit the results of additional animal experiments and to complete the dossier by send-
        ing all of the data cited in the references. In February 2002, the applicant supplied some
        of the new information requested (Pio02), followed by additional data in February 2003
        (Pio03). In March 2003, the Committee responded to this by requesting further clarifica-
        tion concerning the molecular biological analysis, the study into the modified maize’s
        composition and the toxicological study into the new proteins. The applicant supplied a
        part of the new information requested in May 2003 (Pio03a). In July 2003, additional
        Introduction                                                                                 61
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<pre>   information was supplied, after the Committee had further specified its previous ques-
   tion (Pio03b). The Committee devoted several meetings to discussing the dossier and
   completed its assessment in November 2003. This advisory report contains the Com-
   mittee’s findings.
62 Insect-resistant and herbicide-tolerant maize (1507)
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<pre>Chapter 2
        Completeness and accuracy of the
        dossier
2.1     Administrative data
        The names and addresses of the applicants, hereafter referred to as ‘Pioneer/Mycogen’
        or ‘the applicant’, are as follows:
        Pioneer Hi-bred International Inc.
        400 Locust Street, Suite 800
        Des Moines, IA 50309, USA
        Mycogen Seeds
        c/o Dow Agroscience LLC
        9330 Zionsville Road
        Indianapolis, IN 46268-1054, USA
        For the purpose of this application, these companies are represented by:
        Pioneer Overseas Company,
        Avenue Tedesco 7
        B-1160 Brussels, Belgium.
        Completeness and accuracy of the dossier                                              63
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<pre>2.2 General description of the food
    The application concerns the marketing and trading of maize line 1507 on the European
    market for direct consumption and for further processing into foods and food ingredi-
    ents. The application also covers crosses of maize line 1507 with non-genetically modi-
    fied maize lines.
2.3 Classification of the food for assessment
    The dossier contains arguments for classification in class 3.1, one of the six main classes
    and sub classes of novel foods as referred to in table 1, section 1 of Recommendation 97/
    618 of the European Commission (EC97a). This concerns a genetically modified plant,
    the conventional counterpart of which has a history of safe use in the European Union.
    The Committee concurs with this classification.
2.4 Information about the food
    The applicant specified the information that is essential for a safety assessment of novel
    food consumption in accordance with the themes prescribed in Recommendation 97/618
    of the European Commission (EC97a):
    I.    Specification of the novel food (NF)
    II. Effects of the production process applied to the NF
    III. History of the organism used as the source of the NF
    IV. Effect of the genetic modification on the properties of the host organism
    V. Genetic stability of the genetically modified organism (GMO) used as NF source
    VI. Specificity of the expression of the novel genetic material
    VII. Transfer of genetic material from the GMO
    IX. Anticipated intake and extent of use of the NF
    X. Information from previous human exposure to the NF or its source
    XI. Nutritional information on the NF
    XII. Microbiological information on the NF
    XIII. Toxicological information on the NF.
    For every subject, the applicant follows each step in the flow charts and refers to the
    appendices or the references for the data used.
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<pre>2.5 Brief summary by the applicant
    The dossier contains a brief summary that was sent to the EU member states in accord-
    ance with article 6, section 2 of the European Regulation (EC) 258/97 (EC97).
2.6 Other assessments
    In the context of Directive 2001/18/EC (EG01) on the deliberate release of genetically
    modified organisms into the environment, molecular biological aspects of this novel
    food will be assessed in the Netherlands by the Committee on Genetic Modification
    (COGEM) at the request of the Ministry of Spatial Planning, Housing and the Environ-
    ment.
         In the Netherlands, animal feed safety assessments of genetically modified crops are
    carried out by the National Institute for Quality Control of Agricultural Products
    (RIKILT).
         In the Netherlands, permission to treat this maize in the field with glufosinate
    ammonium rests with the Board for the Authorisation of Pesticides (CTB).
2.7 Labelling proposal by the applicant
    The dossier contains a labelling proposal. Recently, European Regulation (EC) No.
    1830/2003 was published, concerning the traceability and labelling of genetically modi-
    fied organisms and traceability of derived food and feed products (EC03a). After the
    entry into force of this regulation, not all articles shall apply with effect instantly. Until
    the new regulation comes into force, labelling should satisfy the requirements in EC
    Regulations 258/97 (EC97), 1139/98 (EC98), and 49/2000 (EC00). However, in the
    Netherlands, a labelling proposal is discussed in the Regular Consultation on the Com-
    modity Act and is not further assessed by this Committee.
    Completeness and accuracy of the dossier                                                       65
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<pre>66 Insect-resistant and herbicide-tolerant maize (1507)</pre>

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<pre>Chapter 3
        Interpretation and evaluation of the data
        presented
3.1     I Specification of the novel food (NF)
        This application concerns a maize line, into which two new genes have been introduced.
        One of these genes is the pat gene, which codes for the protein phosphinothricin-N-
        acetyltransferase (PAT). This is an enzyme which occurs naturally in the soil bacterium
        Streptomyces viridochromogenes. It causes the modified maize plant to be tolerant to the
        herbicide glufosinate ammonium. The second introduced gene is cry1F. This codes for a
        protein which corresponds to the biologically active part of the CRY1F protein from the
        soil bacterium Bacillus thuringiensis, subspecies aizawai. Despite this distinction, the
        new protein in the transgenic plant is also designated as CRY1F. The production of this
        protein in the modified maize plant protects the plant from damage by certain insects,
        such as the larvae of the European Corn Borer.
3.2     II Effects of the production process applied to the NF
        Whole maize kernels are used as animal feed on a large scale, but only on a small scale
        for direct human consumption (in the case of sweet corn). However, flour, starch and oil
        obtained from maize are important base materials in the production of foods. For a large
        proportion of the maize starch, this involves conversion into syrups or ethanol. The
        modification in maize line 1507 is of agronomic importance and does not influence the
        production processes used to process maize into foods and food ingredients. The appli-
        Interpretation and evaluation of the data presented                                      67
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<pre>    cant refers to the literature for a description of the production processes normally used
    for this purpose (Whi95).
3.3 III History of the organism used as the source of the NF
    The source of the novel food plant is a conventionally cultivated maize variety, Zea
    mays Hi-II (respectively the genus, the species and the variety). Two genes of bacterial
    origin were inserted into the maize genome using genetic modification. These genes
    have been modified for optimal expression in the plant. The maize plant has a long his-
    tory of safe use for foods and food ingredients. It is cultivated in many areas throughout
    the world (Sha88).
3.4 IV Effect of the genetic modification on the properties
    of the host organism
    This application concerns a maize line into which two new genes, cry1F and pat, were
    introduced by the transfer of a single, linear DNA fragment. This was accomplished by
    using a particle acceleration method to transfer the novel DNA, precipitated onto micro-
    scopic tungsten particles, into embryonic maize cells. Transgenic maize plants were
    regenerated from plant cells whose genome incorporated the new DNA. These were first
    selected for tolerance to the herbicide glufosinate ammonium, then for the presence of
    the CRY1F protein. Maize line 1507 was created by crossing one of these plants to an
    inbred maize line. The intended insertion of new DNA into this plant was accompanied
    by rearrangements in the flanking DNA.
         The cry1F gene originates from the bacterium Bacillus thuringiensis, subspecies
    aizawai. In order to achieve optimal expression in plant cells, a truncated version of this
    gene was used for the transformation. Its nucleotide sequence had also been optimised
    for plant codon usage. The CRY1F protein in the transgenic plant corresponds to the
    first 605 amino acids of the bacterial protein, with a single amino acid substitution at
    position 604. This section contains the biologically active part of the protein, that specif-
    ically kills certain insect pests by damaging these animals’ intestinal cells. Expression of
    the cry1F gene in the transgenic plant is controlled by the ubiZM1(2) promoter from
    maize and the ORF25PolyA terminator from the bacterium Agrobacterium tumefaciens.
         The pat gene, which originates from the soil bacterium Streptomyces viridochromo-
    genes, codes for the PAT protein. This protein catalyses the conversion of L-phosphino-
    thricin (L-PPT), the active ingredient of the herbicide glufosinate ammonium, to N-
    acetyl-L-PPT. L-PPT binds to the enzyme glutamine synthetase and kills susceptible
    plants by impeding the essential detoxification of ammonia. Since N-acetyl-L-PPT does
    not bind to this enzyme, expression of the PAT protein renders transgenic plants tolerant
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<pre>to the herbicide (OECD99). The pat gene in the modified maize has been optimised for
plant codon usage, to achieve optimal expression. Expression of the pat gene in the
transgenic plant is under the control of the 35S promoter and the 35S terminator from
cauliflower mosaic virus (CaMV).
     The applicant analysed DNA from two different generations of maize line 1507 by
means of Southern blotting (discussed in detail in section 3.5). These experiments
revealed the presence of a single complete copy of the new DNA, the primary insertion.
They also revealed an extra copy of part of the cry1F gene. This technique was also used
to confirm that the DNA of maize line 1507 contains no other parts of the original plas-
mid, from which the DNA used for the transformation was isolated.
     The Committee felt that a more detailed analysis of the genetic modification was
required. It therefore asked the applicant to perform additional DNA sequence analysis,
including the DNA immediately upstream and immediately downstream of the intended
insertion. The primary insertion was indeed shown to consist of a single, virtually com-
plete copy of the DNA of interest. The sequence of approximately 2500 base pairs
upstream of the primary insertion and approximately 1600 base pairs downstream of the
primary insertion revealed the presence of seven extra fragments of the new DNA. It
also identified five fragments that may have originated from chloroplast DNA (see sche-
matic representation in Annex E). Such rearrangements have often been observed, espe-
cially following transformations achieved through the use of the ballistic technique
(Koh03).
     At the Committee’s request, the applicant characterised the open reading frames
which originate from the presence of extra fragments of the new DNA. If neighbouring
DNA sequences were to make transcription possible, then these open reading frames
could potentially be translated into fusion proteins. None of the Northern blot, Western
blot and RT-PCR experiments carried out by the applicant gave any indication that such
fusion proteins had actually formed. On the basis of the DNA sequence, 24 open reading
frames were identified spanning the junction sites of fragments of new DNA. All con-
ceivable translation products of these open reading frames were investigated for possi-
ble similarities with known toxic or allergenic proteins. Following a broad comparison
of the amino acid sequences with a general protein database, no similarities to known
toxic proteins were found. Comparisons were also carried out using an allergenic protein
database created by the applicant in accordance with FAO/WHO recommendations
(FAO01). Searches were carried out for similarities with these known allergens using
each 80-amino acid window, and for the occurrence of identical sequences of six, seven
or eight contiguous amino acids. None of the possible proteins showed more than a 35%
similarity with a known allergen over a length of 80 amino acids. Nor were any identical
sequences of seven or eight contiguous amino acids found. However, six instances were
found of sequences of six identical amino acids in comparisons with known allergens.
Interpretation and evaluation of the data presented                                      69
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<pre>    The applicant states that random similarities without clinical relevance will often be
    found when comparing fragments consisting of just six or seven amino acids. For this
    reason, the applicant sees no indication of possible allergenicity. The Committee feels
    that this position is acceptable and that it is supported by recent publications (Hil02,
    Kle02).
         In summary, the Committee feels that the molecular biological analysis was very
    thorough, and that it found no effects of the genetic modification other than the produc-
    tion of both of the intended new proteins, CRY1F and PAT.
3.5 V Genetic stability of the genetically modified organism (GMO)
    used as NF source
    The inheritance of glufosinate ammonium tolerance in the progeny of the original trans-
    formant was studied in three successive generations of backcrossing to an inbred line.
    The expected 1:1 segregation of this characteristic was confirmed. This generation of
    plants was then backcrossed once again to an inbred line, followed by self-pollination.
    Progeny plants were treated to glufosinate ammonium, thereby selecting only tolerant
    plants. These were then crossed with a non-transgenic inbred line. The expected 2:1 seg-
    regation of tolerant and susceptible plants was confirmed in the resultant progeny. The
    applicant reports the results of a bio-assay, which showed that the glufosinate-ammo-
    nium tolerant plants from this experiment were also resistant to the European Corn
    Borer.
         Plants derived from the first backcross of the original transformant to an inbred line
    were self-pollinated to produce a new generation of progeny. These plants were sub-
    jected to DNA analysis, by means of Southern blotting. The same analytical technique
    was also applied to plants derived from five successive generations of backcrossing of
    the original transformant to an inbred line. All of these experiments detected the same
    hybridising DNA fragments.
         These results show that there is a stable transfer of the new DNA to subsequent gen-
    erations. This fact is confirmed by the results of several field trials (described in the next
    section) which show that the CRY1F protein is present in plant material derived from
    maize line 1507.
3.6 VI Specificity of the expression of the novel genetic material
    During the 1998-1999 season, field trials of 1507 maize were carried out at four loca-
    tions in Chile. In order to examine the expression of the new genes, samples were taken
    from different plants at different stages of growth (Iow97, Iow03). These samples were
    leaves at the V9 stage, pollen, silk and stalks at the R1 stage, mature kernels (R6 stage)
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<pre>    and whole plants at the R4 stage, and after drying out in the field. Multiple samples of
    each of these tissue types were taken at each location. Next, the amounts of CRY1F pro-
    tein and PAT protein were measured as fractions of the total soluble protein content by
    means of an ELISA method, which used specific antisera to these proteins. The appli-
    cant provides average, minimum and maximum values for each tissue type. The data
    from the four locations was combined for this purpose. Material from field trials carried
    out in 1999, at three locations in France and three in Italy, was subjected to comparable
    analyses. Aside from the above-mentioned tissue types, whole plants at the V9 and R1
    stages were also investigated. In addition, samples from maize kernels and whole plants
    at the R4 stage were also investigated following treatment with glufosinate ammonium.
    Later, the applicant also submitted data from a third series of field trials that had been
    carried out in the year 2000, at three locations in France, two in Italy and one in Bul-
    garia. In these trials, all of the above-mentioned tissue types were investigated. Here,
    however, both material from plants that had been treated with glufosinate ammonium
    and material from untreated plants was examined. The results of the latter experiments
    were also converted into amounts of the new proteins per unit of dry weight of the tissue
    examined. CRY1F protein was found to be present in all tissue types from 1507 maize,
    whereas it was not detected in non-transgenic plants. In the year 2000 European field tri-
    als, kernels from untreated plants of maize line 1507 were found to contain 2.2 ng of
    CRY1F protein per mg dry weight. Kernels from plants of maize line 1507 that had been
    treated with glufosinate ammonium contained 2.5 ng of CRY1F protein per mg dry
    weight. Conversely, no values for the PAT protein could be determined, since the levels
    were generally below the limit of detection.
         Extracts from leaves, pollen, maize kernels and whole plants from the field trials in
    Chile were also used for Western blotting using antisera to the CRY1F and PAT proteins.
    The CRY1F protein was present at detectable levels in all tissue types. Only leaf tissue
    contained detectable levels of the PAT protein.
3.7 VII Transfer of genetic material from the GMO
    The applicant states that food products derived from maize can contain DNA. The
    amount of DNA present depends on the production process used. The Committee states
    that people ingest large amounts of vegetable and animal DNA every day. It is conceiva-
    ble that parts of this DNA, in the form of intact DNA fragments, could enter the intes-
    tines and be transferred to the resident microflora. Should this occur, there will be little,
    if any, expression of these genes in practice, as they are not linked to a suitable promoter.
    If these genes were nevertheless to be expressed, they would, in the vast majority of
    cases, not provide the bacteria concerned with a competitive advantage and the host
    would not be disadvantaged. In fact, the only problem that might occur involves the
    Interpretation and evaluation of the data presented                                           71
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<pre>     transfer of marker genes for antibiotic resistance, and then only if consumers’ intestinal
     flora are exposed to selective pressure following the use of the antibiotic in question.
     Maize line 1507 does not contain any genes that confer antibiotic resistance. Therefore,
     the Committee shares the applicant’s viewpoint that no adverse effects are to be
     expected from the transfer of genetic material from 1507 maize, if such a transfer were
     to occur.
3.8  IX Anticipated intake and extent of use of the NF
     Although the maize plant and whole maize kernels are mostly used as animal feed,
     maize oil and maize starch derived from the maize kernels are used in large quantities in
     products for human consumption. Syrups and ethanol produced from maize starch are
     also used on a large scale in the food industry. A great deal of information is available
     about the use of maize in the production of foods. The new properties of maize line 1507
     are of agronomic importance and could be used on a large scale. Although 1507 maize
     will therefore initially just replace other maize, the possibility cannot be ruled out that
     the cultivation of the new maize line could lead to a better competitive position owing to
     a more efficient production of the crop concerned. In that case, the human consumption
     of products derived from maize (in particular oil and starch) could increase compared to
     equivalent ingredients derived from other crops. The Committee feels that there are no
     objections to this from a nutritional viewpoint.
3.9  X Information from previous human exposure to the NF or its source
     Conventional maize has a long history of safe use both within Europe’s borders and
     beyond. No data are available concerning the previous use of modified maize line 1507.
3.10 XI Nutritional information on the NF
     The applicant has had nutritional analyses performed on maize from genetically modi-
     fied plants and control plants. In the application, the results were presented of field trials
     that had been carried out in Chile (1998-1999), France (1999) and Italy (1999). How-
     ever, the Committee notes that the values were initially pooled, rather than being
     reported on a location-by-location basis. The Committee's general opinion is that any
     analysis of the composition of a modified crop must be based on field trials carried out
     in several geographical locations that are representative of the crop’s commercial pro-
     duction. In order to detect any differences between the modified line and the best com-
     parable control line it is also important that observations be analysed on a location-by-
     location basis. In response to these comments, the applicant has re-analysed the results
72   Insect-resistant and herbicide-tolerant maize (1507)
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<pre>of these field trials, this time on a location-by-location basis The applicant also submit-
ted additional results from field trials that had been carried out in the year 2000, in
France, Italy and Bulgaria. At the Committee’s request, the applicant made a case to
support the view that these locations were indeed representative of the commercial use
of this crop.
     These experiments all measured the levels of fat, protein, carbohydrates, fibre (ADF
and NDF) and ash in maize kernels and maize plants. The maize kernels were also ana-
lysed to determine the levels of calcium, phosphorous, copper, iron, magnesium, manga-
nese, potassium, sodium, zinc, vitamin B1, vitamin B2, folic acid, tocopherols, palmitic
acid, stearic acid, oleic acid, linoleic acid and linolenic acid. The amino-acid composi-
tion was also determined, as were the levels of the secondary metabolites inositol, raffi-
nose, p-coumaric acid, furfural and ferulic acid. Measurements were also made of the
levels of the antinutrients phytic acid and trypsin inhibitor. Aside from minor modifica-
tions, for which the applicant has made a case, this list of analysed components complies
with the recommendations made by the OECD (OECD02).
     During the 1998-1999 season, 1507 maize was cultivated at four different locations
in Chile, alongside control plants with a comparable genetic background. The 1507
maize was treated with glufosinate ammonium, after which susceptible plants were
removed. The trial field at each location was divided up into six blocks, which can be
seen as repetitions of the experiment. For the purpose of compositional analysis, sam-
ples of the above-ground parts of three plants and of maize kernels from three ears of
maize were taken from each block at each location. For each analysed component, the
applicant reports the average value for maize line 1507 and for the control maize, as well
as a measure of spread. Several statistically significant differences were found, most of
which appeared to be randomly distributed between the various trial fields. Remarkably,
at every location 1507 maize was found to contain higher levels of linolenic acid than
the control plants. In addition, divergent values for nine amino acids were found at one
particular location. Nonetheless, all of the measured levels fall within value ranges that
have been published in the literature.
     The study carried out in France (1999) involved samples of 1507 maize and control
plants from six blocks at a single location. In Italy (1999), samples were taken at three
locations, each in triplicate. Samples of 1507 maize that had been treated with glufosi-
nate ammonium were also investigated. A large number of measurements of material
from the three trial fields in Italy produced divergent values for various amino acids.
The remaining differences seemed to be randomly distributed. All of the measured lev-
els fall within value ranges that have been published in the literature.
     The applicant supplied new information about field trials that had been carried out
in the year 2000, at three locations in France, two in Italy and one in Bulgaria. The trial
fields at each location were divided up into three blocks. Each block contained 1507
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<pre>     maize, together with 1507 maize that had been treated with glufosinate ammonium and
     with control maize. Samples taken from these blocks consisted of three entire plants (R4
     stage) and seeds from five ears of maize taken from different plants (R6 stage). Several
     statistically significant differences were observed. There was no clear pattern in their
     distribution across the various locations. In addition, the observed differences remained
     within the range of natural variation reported in the literature. At one location in France,
     differences were also found between the transgenic maize and the control, in terms of
     virtually all individual amino acids. Here, consistently lower values were found for the
     control than for maize line 1507, although this was not the case at other locations.
          The applicant also describes an animal feeding experiment with broiler chicks,
     where the growth of male chicks was investigated. Throughout the study period of 42
     days, one group of animals were fed maize kernels from line 1507 while another group
     were fed maize kernels from a control line with a comparable genetic background. In
     addition, animals in four control groups were fed maize kernels from commercial lines.
     In all cases, 54% of the feed provided during the first 20 days consisted of maize ker-
     nels. During the subsequent period, this figure was 57%. Each group consisted of 35 ani-
     mals. No significant differences were found in terms of mortality, body weight gain or
     food conversion.
          In summary, a large number of differences were observed in terms of amino-acid
     levels in maize kernels from the modified lines and from control plants at a single loca-
     tion in Chile (1998-1999), three locations in Italy (1999) and a single location in France
     (2000). No such differences were observed in the other field trials. In all cases the levels
     remained within the range of natural variation reported in the literature. The divergent
     values for linolenic acid found during the four field trials in Chile also fall within this
     range. The applicant states that the observed differences can be attributed to natural var-
     iation. The Committee takes the view that even if these differences could be ascribed to
     the genetic modification of maize line 1507, there can be no nutritional objections, given
     the limited extent of the differences, within the limits of known natural variation. The
     Committee therefore endorses the applicant’s conclusion that 1507 maize does not differ
     nutritionally from conventional maize. This was also confirmed by the feeding experi-
     ment described above.
3.11 XII Microbiological information on the NF
     Neither the new maize, nor products derived from it, are expected to contain unusual
     microorganisms or microbial metabolites.
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<pre>3.12 XIII Toxicological information on the NF
     The applicant describes toxicological studies into both new proteins, CRY1F and PAT.
     For the purposes of an animal study, it was necessary to produce the CRY1F protein in
     bacteria, since the protein only occurs in limited quantities in transgenic maize plants. In
     order to achieve efficient production, a fusion protein was expressed in the bacterium
     Pseudomonas fluorescens, consisting of the N-terminal region of the CRY1F protein
     and the C-terminal region of the related CRY1A(b) protein. This was then subjected to
     enzymatic splitting, after which a protein was isolated that corresponded to the region of
     the natural CRY1F protein from amino acid 28 to amino acid 612. The CRY1F protein
     in maize line 1507 corresponds to the region from amino acid 1 to amino acid 605, with
     a substitution at position 604. Research carried out by the applicant has shown that these
     proteins are comparable in terms of their biological activity in susceptible insects, their
     reactivity with specific antisera in Western blotting, the absence of post-translational
     glycosylation and the size of the peptides remaining after digestion with trypsin. The
     CRY1F protein produced in bacteria was shown to lose its biological activity after being
     incubated at a temperature of 75ºC for 30 minutes.
          The bacterial CRY1F protein was investigated for possible acute toxicity, using
     CD-1 mice. Five male mice and five female mice received a dose of 576 mg of CRY1F
     protein per kg of body weight. The protein was administered orally, as two equal por-
     tions separated by an interval of one hour. For this purpose a dose of 5050 mg per kg
     body weight was used, from a preparation containing 11.4 % of CRY1F protein. None of
     the animals died nor were any clinical anomalies observed throughout the subsequent
     14-day period. Autopsies conducted after this period revealed no macroscopically visi-
     ble anomalies.
          At the Committee’s request, the applicant provided further material in support of the
     safety of the CRY1F protein. This made the case that the known toxic action of the CRY
     proteins is limited to certain insect species, which have specific receptors for that protein
     in the cells of the intestinal wall. The applicant points out that various Bacillus thuring-
     iensis varieties have been safely used as biopesticides for several decades. This also
     applies to the subspecies aizawai, from which the CRY1F protein was derived. In addi-
     tion, the applicant has compared the amino-acid sequence of the CRY1F protein with a
     database of known proteins. The search showed that there were similarities with 181
     sequences. Besides sequences from CRY proteins, there were only three other proteins
     in this list, none of which were toxins. Furthermore, past experiments have confirmed
     the safety of closely related CRY proteins, such as the CRY1A(b) protein. Genetically
     modified crops in which proteins such as CRY1A(b) are expressed, have been produced
     Interpretation and evaluation of the data presented                                           75
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<pre>   on a large scale in recent years. No adverse effects have been reported in relation to the
   consumption of foods derived from these crops.
        In another study, PAT protein was administered to CD-1 mice. Five male mice and
   five female mice received a dose of approximately 5000 mg of PAT protein per kg of
   body weight. The protein was administered orally, as two equal portions separated by an
   interval of one hour. For this purpose a dose of 6000 mg per kg body weight was used,
   from a preparation produced in bacteria, which contained 84% of PAT protein. None of
   the animals died nor were any clinical anomalies observed throughout the subsequent
   14-day period. Autopsies conducted after this period revealed no macroscopically visi-
   ble anomalies. A study was also carried out into the possible oral toxicity of the PAT
   protein following repeated exposure. This protein was incorporated into a low-protein
   diet for Wistar rats over a period of 14 days, at concentrations of 0 ppm, 5000 ppm and
   50000 ppm. The PAT protein used for this purpose was produced in E.coli and had a
   purity of 98%. The total amount of added protein in these three groups was adjusted to a
   uniform level by the addition of 50000 ppm, 45000 ppm and 0 ppm soya protein respec-
   tively. Each group contained five male and five female rats. The food uptake per group
   was used to calculate an average PAT intake. The values were 0, 712 and 7619 mg per
   kg body weight per day for the male animals and 0, 703 and 7965 mg per kg body
   weight per day for the female animals. A control group was fed a standard diet. None of
   the animals died nor were any clinical symptoms observed throughout the subsequent
   14-day period. At the end of this period, blood and urine samples were taken and the
   animals were sacrificed. Numerous general and clinical chemistry tests were performed
   on the blood and urine samples. Organ weights were recorded for all animals. Numerous
   tissue and organ samples were microscopically examined for animals in the high-dose
   group, the control group in which only soya protein had been added to the diet and the
   control group which had been fed on a standard diet. The findings showed no relevant
   anomalies related to exposure to the PAT protein.
        In addition to possible acute or sub-acute oral toxicity, possible allergenicity of the
   new proteins is another important issue in the food safety assessment of 1507 maize. In
   order to make the case that the new proteins are unlikely to give rise to allergic reac-
   tions, the applicant has supplied data about the source of the new proteins, possible sim-
   ilarities to known allergens and the degree of resistance to breakdown by proteolytic
   enzymes (COD02). The applicant states that no cases of allergenicity have been
   reported in relation to the widely occurring soil bacteria (Bacillus thuringiensis and
   Streptomyces viridochromogenes) from which the new genes were isolated. A compari-
   son of the amino acid sequences of CRY1F protein and PAT protein with known aller-
   gens in a general protein database found no similarities, when a search was made for a
   window of eight contiguous, identical amino acids. In a subsequent investigation of the
   CRY1F protein, the applicant used a specific database of 2033 sequences of allergenic
76 Insect-resistant and herbicide-tolerant maize (1507)
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<pre>proteins. This study was carried out in accordance with FAO/WHO recommendations
(FAO01). No matches of seven or eight identical, contiguous amino acids were identi-
fied. However, corresponding sequences of six contiguous amino acids were found in
three proteins from the database used. Nonetheless, the applicant does not consider this
fact to be relevant, since using a window of six or seven amino acids often produces
false positive results, as stated in section 3.4. The CRY1F protein was also compared to
the same known allergens, using the FASTA program and an 80-amino acid window.
This produced no results with a similarity in excess of 35%, the percentage set by the
FAO/WHO as the limit for possible significance. Furthermore, the CRY1F protein was
digested in simulated gastric fluid within one minute, at a ratio of one molecule of pep-
sin to 188 molecules of CRY1F. A subsequent study showed that the CRY1F protein
was digested within as little as 15 seconds in simulated gastric fluid containing approx.
0.3% pepsin. The PAT protein was digested within five seconds in simulated gastric
fluid containing the same concentration of pepsin. When new proteins are digested so
rapidly during in vitro digestion experiments with pepsin, it is generally seen as an argu-
ment against possible allergenicity.
     The above-mentioned studies are based on the assumption that the only difference
between maize line 1507 and conventional maize is the presence of the introduced
genes, their gene products and the resultant new properties. However, the Committee
feels that molecular biological characterisation of the new maize line cannot entirely
eliminate the possibility that unintended additional changes might have occurred in the
maize genome. Accordingly, it requested the applicant to supplement the studies of the
new proteins with a toxicity study using whole maize kernels, to provide an extra guar-
antee of 1507 maize’s safety in terms of long-term human consumption. In response to
this, the applicant then submitted the results of a sub-chronic toxicity study in rats (strain
Crl:CD(SD)IGS BR). The test groups received 33% or 11% w/w 1507 maize in their
feed over a 90-day period. Control groups received 33% or 11% w/w of comparable,
non-modified maize in their feed. In the 11% groups, the feed was supplemented with
22% w/w of a commercial non-modified maize variant. Another control group received
33% w/w of this non-modified maize in their feed. The test and control groups contained
12 male and 12 female rats. Body weight and food consumption were measured each
day during the first week, and once a week thereafter. Clinical observations were made
twice a day for visible symptoms of toxicity and weekly by means of a general examina-
tion. Eye examinations and behavioural experiments were conducted at the start and end
of the study period. Once the study had been completed, blood and urine samples were
taken and the animals were sacrificed. Numerous general and clinical chemistry param-
eters of the blood and urine samples were measured. The weight of a number of organs
was determined. A large number of tissue and organ samples from the animals in the
high dose groups for 1507 maize and for comparable, non-modified maize were also
Interpretation and evaluation of the data presented                                            77
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<pre>   investigated microscopically. Any significant differences found were not attributable to
   the administration of 1507 maize. On the basis of this experimental animal study and of
   data from FAO/WHO concerning the intake of maize and maize products, the applicant
   has calculated that, in relation to the highest dose tested, the CRY1F protein’s margin of
   exposure for European consumers has a value of 12. This was based on the maximum
   measured content of CRY1F in maize kernels, and the sole use of 1507 maize for the
   production of all maize products consumed. In addition, it was assumed that no CRY1F
   would be lost during the processing of maize into foods. The latter two assumptions in
   particular can be said to be extremely conservative, therefore the actual consumption of
   this protein from maize line 1507 is likely to be substantially lower.
        The Committee feels that the applicant’s study of the properties of the new proteins,
   CRY1F and PAT, was appropriately designed and correctly carried out. The results gave
   no indication of either toxicity or allergenicity. In addition, the results of the subchronic
   toxicity study in rats carried out by the applicant show that no adverse effects from the
   consumption of 1507 maize can be expected as a consequence of possible unknown
   changes in the genome of the plant.
78 Insect-resistant and herbicide-tolerant maize (1507)
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<pre>      Literature
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      biotechnology. Proposed draft annex on the assessment of possible allergenicity of the draft guideline for
      the conduct of food safety assessment of foods derived from recombinant-DNA plants. Rome: FAO, 2002.
EC97  Regulation (EC) 258/97 of the European Parliament and of the Council of 27 January 1997 concerning
      novel foods and novel food ingredients. Official Journal of the European Communities 1997; L43: 1-6.
EC97a 97/618/EC: Commission Recommendation of 29 July 1997 concerning the scientific aspects and the
      presentation of information necessary to support applications for the placing on the market of novel foods
      and novel food ingredients and the preparation of initial assessment reports under Regulation (EC) No 258/
      97 of the European Parliament of the Council. Official Journal of the European Communities 1997; L253:
      1-36.
EC98  Council Regulation (EC) No. 1139/98 of 26 May 1998 concerning the compulsory indication of the
      labelling of certain foodstuffs produced from genetically modified organisms of particulars other than those
      provided for in Directive 79/112/EEC.Official Journal of the European Communities 1998; L159: 4-7.
EC00  Commission Regulation (EC) No. 49/2000 of 10 January 2000 amending Council Regulation (EC) No.
      1139/98 concerning the compulsory indication on the labelling of certain foodstuffs produced from
      genetically modified organisms of particulars other than those provided for in Directive 79/112/EEC.
      Official Journal of the European Communities 2000; L6: 13-14.
EC01  Directive 2001/18/EG of the European Parliament and of the Council of 12 March 2001 on the deliberate
      release into the environment of genetically modified organisms and repealing Council Directive 90/220/
      EEC. Offical Journal of the European Communities 2001; L106: 1-38.
EC03  Regulation (EC) No 1830/2003 of the European Parliament and of the Council of 22 September 2003 on
      genetically modified food and feed. Offical Journal of the European Communities 2003; L268: 1-23.
      Literature                                                                                                   79
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<pre>EC03a  Regulation (EC) No 1830/2003 of the European Parliament and of the Council of 22 September 2003
       concerning the traceability and labelling of genetically modified organisms and the traceability of food and
       feed products produced from genetically modified organisms and amending Directive 2001/18/EG. Offical
       Journal of the European Communities 2003; L268: 24-28.
FAO96  Biotechnology and Food Safety. Report of a joint FAO/WHO Consultation. Rome: FAO, 1996.
FAO01  Evaluation of allergenicity of genetically modified foods. Report of a joint FAO/WHO expert consultation
       on allergenicity of foods derived from biotechnology. Rome: FAO 2001.
HCN92  Commissie Toxicologische aspecten van biotechnologisch bereide producten. Productveiligheid bij nieuwe
       biotechnologie (publication number 1992/03). The Hague: Health Council of the Netherlands, 1992.
Hil02  Hileman RE, Silvanovich A, Goodman RE, Rice EA, Holleschak G, Astwood JD et al. Bioinformatic
       methods for allergenicity assessment using a comprehensive allergen database. Int Arch Allergy Immunol
       2002; 128(4): 280-291.
Iow97  Iowa State University of Science and Technology. How a corn plant develops. Special report no. 48. Ames,
       Iowa: reprinted 1997.
Iow03  Iowa State University of Science and Technology. How a corn plant develops. Special report no. 48.
       Website http://maize.agron.iastate.edu/corngrows.html, consulted on 21 October, 2003.
Kle02  Kleter GA, Peijnenburg AA. Screening of transgenic proteins expressed in transgenic food crops for the
       presence of short amino acid sequences identical to potential, IgE - binding linear epitopes of allergens.
       BMC Struct Biol 2002; 2(1): 8.
Koh03  Kohli A, Twyman RM, Abranches R, Wegel E, Stoger E, Christou P. Transgene integration, organization
       and interaction in plants. Plant Mol Biol 2003; 52(2):247-58.
OECD93 Safety evaluation of foods derived by modern biotechnology. Concepts and principles. Paris: OECD, 1993.
OECD96 OECD Workshop on Food Safety Evaluation. Paris: OECD, 1996.
OECD98 Report of the OECD workshop on the toxicological and nutritional testing of novel foods. Paris: OECD,
       1998.
OECD99 Consensus document on general information concerning the genes and their enzymes that confer tolerance
       to phosphinothricin herbicide. Paris: OECD, 1999.
OECD00 Report of the task force for the safety of novel foods and feeds. Paris: OECD, 2000.
OECD02 Consensus document on compositional considerations for new varieties of maize (Zea mays): Key food and
       feed nutrients, anti-nutrients and secondary plant metabolites. Paris: OECD, 2002.
Pio01  Pioneer Hi-Bred en Mycogen Seeds. Application for placing on the market of novel foods and novel food
       ingredients containing genetically modified organisms in accordance with regulation (EC) No. 258/97: 1507
       maize. Brussels: Pioneer Overseas Corporation, 2001.
Pio02  Pioneer Hi-Bred en Mycogen Seeds. Response to the VNV-Committee’s questions of 28/06/2001. Brussels:
       Pioneer Overseas Corporation, 2002.
Pio03  Pioneer Hi-Bred en Mycogen Seeds. Additional response to the VNV-Committee’s questions of 28/06/2001.
       Brussels: Pioneer Overseas Corporation, 2003.
Pio03a Pioneer Hi-Bred en Mycogen Seeds. Response to the VNV-Committee’s questions of 25/03/2003. Brussels:
       Pioneer Overseas Corporation, 2003.
80     Insect-resistant and herbicide-tolerant maize (1507)
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<pre>Pio03b Pioneer Hi-Bred en Mycogen Seeds. Response to the VNV-Committee’s questions of 11/06/2003. Brussels:
       Pioneer Overseas Corporation, 2003.
SCF99  Opinion concerning the scientific basis for determining whether food products, derived from genetically
       modified maize, could be included in a list of food products which do not require labelling because they do
       not contain (detectable) traces of DNA or protein. Brussels: Scientific Committee on Food of the EU, 1999.
SSC99  Opinion of the Scientific Steering Committee on microbial resistance. Brussels: Scientific Steering
       Committee of the EU, 1999.
Sha88  Shaw, RH. Climatic requirement. P. 3-29 in: Corn and corn improvement, Sprague, GF and Dudley, JW
       (eds.). Madison, Wisconsin: American Society of Agronomy, Inc., Crop Science Society of America, Inc.,
       1988.
Whi95  White, PJ and Pollack, LM. Corn as a food source in the United States: Part II. Processes, Products,
       composition and nutritive values. Cereal foods world 1995; 40: 756-762.
WHO91  Strategies for assessing the safety of foods produced by biotechnology. Report of a joint FAO/WHO
       Consultation. Geneva: WHO, 1991.
WHO00  Safety aspects of genetically modified foods of plant origin. Report of a joint FAO/WHO expert
       consultation on foods derived from biotechnology. Geneva: WHO, 2000.
       Literature                                                                                                  81
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<pre>82 Insect-resistant and herbicide-tolerant maize (1507)</pre>

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<pre>A Request for advice
B The committee
C EU-procedure
D Executive summary of the dossier
E Schematic representation of the genetic modification
  Annexes
                                                       83
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<pre>84 Insect-resistant and herbicide-tolerant maize (1507)</pre>

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<pre>Annex A
      Request for advice
      On 18 August 1999, the Minister of Health, Welfare and Sport wrote as follows to the
      President of the Health Council of the Netherlands (under reference GZB/VVB
      993428):
      Since May 1997, Regulation (EC) 258/97 concerning novel foods and novel food ingredients has been in
      force in the European Union. Under the Regulation, the safety of novel foods has to be assessed as part of a
      community procedure.
            Following discussions regarding the possibility of the Health Council making such assessments, the
      State Secretary for Agriculture, Nature Management and Fisheries and I wish the Council to take responsi-
      bility for safety assessment for a period of several years during the fist phase of implementation of European
      Regulation (EC) 258/97. It is considered appropriate that the Health Council should initially take on this
      role because the assessment activities will be of an experimental nature, involving both a new form of
      assessment (i.e. pre-marketing assessment) and, in many cases, new categories of foodstuff (primarily food-
      stuffs with a genetically modified basis and functional foods or nutraceuticals). We also feel that if assess-
      ments are made by a body with the Council's independent scientific status, this will support the validity of
      the Netherlands' opinion in the eyes of the European Committee and other member states.
      Request for advice                                                                                             85
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<pre>         My wish is to make the procedure and the assessment as open and transparent as possible, so as to
   enhance consumer trust in the safety of novel foods. I would like the Health Council to support this objec-
   tive by, for example, allowing perusal of the application dossier (insofar as consistent with the need to pro-
   tect the confidentiality of commercially sensitive information) and publishing the criteria upon which safety
   assessments are made.
   The Minister of Health, Welfare and Sport,
   (signed) dr E. Borst-Eilers
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<pre>Annex B
      The Committee
      •  Prof. dr LM Schoonhoven, chairman
         emeritus professor of entomology; Wageningen University and Research centre
      •  Prof. dr CAFM Bruijnzeel-Koomen
         professor of dermatology/allergology; Academic Hospital Utrecht
      •  Ir EJ Kok
         toxicologist; National Institute for Quality Control of Agricultural Products,
         Wageningen
      •  Dr CF van Kreijl
         molecular biologist; National Institute of Public Health and the Environment,
         Bilthoven
      •  Prof. dr P van der Laan
         professor of statistics; Technical University Eindhoven
      •  Dr FM Nagengast
         gastro enterologist; Academic Hospital Nijmegen
      •  Dr ir JMA van Raaij
         food physiologist; Wageningen University and Research centre
      •  Prof. dr ir G Schaafsma
         professor of nutrition; TNO Nutrition and Food Research, Zeist
      •  Prof. dr EG Schouten
         professor of epidemiology; Wageningen University and Research centre
      •  Dr GJA Speijers
         toxicologist; National Institute of Public Health and the Environment, Bilthoven
      The Committee                                                                       87
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<pre>   •   Prof. Dr WJ Stiekema
       professor of bioinformatics; Wageningen University and Research centre
   •   Ir R Top, advisor
       Ministry of Health, Welfare and Sport; The Hague
   •   Prof. dr WM de Vos
       professor of microbiology; Wageningen University and Research centre
   •   Dr ir F van der Wilk, advisor
       Committee on Genetic Modification, Bilthoven
   •   Dr RA Woutersen
       toxicologist; TNO Nutrition and Food Research, Zeist
   •   Dr CMA van Rossum, scientific staff member
       Health Council of the Netherlands, The Hague
   Administrative assistance:
   CL Vuijst, MSc; Health Council of the Netherlands, The Hague
   Layout: J van Kan; Health Council of the Netherlands, The Hague
88 Insect-resistant and herbicide-tolerant maize (1507)
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<pre>Annex C
      EU-procedure
      When manufacturers bring novel foodstuffs onto the market, consumer safety has to be
      ensured. In 1997, a European Regulation (EC97) came into force, laying down the pro-
      cedure for approving the market introduction of novel foodstuffs. The procedure recog-
      nizes various actors. The applicant must decide whether a product is a novel foodstuff,
      i.e. a substance that has not previously been available for human consumption to any
      substantial extent within the European Union and is not substantially equivalent to any
      existing product. (If a foodstuff is substantially equivalent to any existing product, it is
      sufficient to inform the authorities of its market introduction). Food additives, aromas
      and extracts are excluded from the provisions of the directive, since they fall within the
      scope of an established assessment regime. Before marketing a novel foodstuff, the
      applicant must compile a safety dossier that complies with the Recommendations of the
      European Commission (EC97a). These Recommendations are based on reports by a
      number of bodies that have studied the issue of novel foodstuffs, in particular the OECD
      (OECD93, OECD96) and the WHO/FAO (FAO96, WHO91). The Health Council of the
      Netherlands has also considered the question earlier (HCN92). Since publication of the
      EU recommendations, international efforts have been made to clarify and adapt the lat-
      est scientific knowledge in the field (FAO01, OECD98, OECD00, SCF99, SSC99,
      WHO00). In the near future, foods derived from genetically modified organisms will no
      longer be assessed using this procedure, but according to a new European regulation
      (EC03).
           Having compiled a dossier in line with the guidelines, the manufacturer has to sub-
      mit it to the competent authority in the country where the product is to be marketed first.
      EU-procedure                                                                                 89
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<pre>   This dossier is assessed by the national safety assessment authority. In the Netherlands,
   this is the Minister of Health, Welfare and Sport, who is advised by the Health Council.
   The President of the Health Council has created a Committee on the Safety Assessment
   of Novel Foods (VNV Committee) to advise the minister on behalf of the Council.
        On the basis of the scientific state of the art, the committee has to decide whether the
   information provided by the manufacturer is accurate and complete and whether the
   manufacturer’s conclusions are sound. The committee then draws up a report on its find-
   ings for the minister; this report must also comply with the European Recommendation
   (EC97a, part III). After considering the report, the minister formulates the Netherlands’
   opinion regarding the foodstuff in question, which is discussed at European level in the
   Standing Committee on the Food Chain and Animal Health. All other European member
   states are invited to express a ‘second opinion’ regarding the dossier and the first opin-
   ion. The Standing Committee then arrives at a final judgement. If a dossier is particu-
   larly contentious, the European Commission calls upon the Scientific Committee on
   Food for advice. If consensus still cannot be reached, the issue is referred to the Euro-
   pean Council of Ministers.
90 Insect-resistant and herbicide-tolerant maize (1507)
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<pre>Annex D
      Executive summary of the dossier
      Executive summary of the dossier 91
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<pre>                                     6. SUMMARY
In accordance with Article 6.1 of the Regulation (EC) No 2258/97, this is a summary
of the application for placing on the market of novel foods and novel food ingredients
jointly submitted to the competent authority and the food assessment body of The
Netherlands by Pioneer Hi-Bred International Inc. and Mycogen Seeds c/o Dow
AgroSciences LLC. Pioneer Hi-Bred International Inc., as represented by Pioneer
Overseas Corporation, Avenue Tedesco 7, B-1160 Brussels, Belgium, is taking the
lead for this submission.
 Description of the product
The product consists of foods from maize grain produced from genetically modified
B.t. Cry1F maize line 1507 and progeny derived from conventional breeding between
1507 maize with any traditionally bred non-GM maize. The term "1507 maize" refers
to grain derived from inbreds and hybrids of maize line 1507 including crosses with
traditionally bred maize.
The 1507 maize has been genetically modified (GM) to express CRY1F protein for
resistance to certain lepidopteran insect pests, such as the European corn borer (ECB),
and to express phosphinothricin-N-acetyltransferase (PAT) protein for tolerance to
glufosinate-ammonium herbicide.
Categorisation of the product
Based on the recommendations made by the EC Scientific Committee on Food (SCF)
for the scientific classification of the novel food (NF) and for the assessment of
wholesomeness, 1507 maize belongs to Class 3, Subclass 3.1, under GM plants and
their products (Commission Recommendation 97/618/EC of 29th July 1997). Under
NF Class 3, the GM plants can be consumed directly as unprocessed foods or after
having been processed into foods and food ingredients.
Identification and consultation of the essential information requirements
In the case of 1507 maize and based on Table II of the SCF recommendations, the
structured schemes identified to provide guidance for consultation of the essential
information requirements for the NF and the assessment of wholesomeness comprise
Schemes I to VII and IX to XIII.
Scheme I: Specification of the novel food (NF)
Maize (Zea mays L.) has well characterised specification belonging to the Gramineae
family, the genus Zea and the species Z. mays (2n = 20). The evidence presented in
this application confirms that foods derived from 1507 maize can be considered to be
substantially equivalent to food products derived from traditionally-bred non-GM
maize with no nutritional or toxicological changes. Traditionally-bred maize does not
contain any toxic or anti-nutritional factors that need to be controlled by a
specification and the characteristics, compositional analyses and safety evaluation of
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<pre>the genetic modification in 1507 maize does not entail a separate specification of the
NF.
A PCR detection method to confirm molecular identity and to ensure that the NF
marketed is similar to that evaluated is being developed. The detection method will
be submitted to the regulatory authority before the 1507 maize is placed on the EU
market for food purposes.
Scheme II: Effect of the production process applied to the NF
Foods from 1507 maize will be used and processed as any other commercial maize.
The production process applied to maize are well known and have a long history of
safe use. The safety evaluation contained in this application for foods derived from
1507 maize provides further verification that no specific conditions of use and
processing are required for 1507 maize, and therefore there should be no effects on the
existing production processes as applied to the NF.
Scheme HI: History of the organism used as the source of the NF
The NF, referred to as 1507 maize, is obtained from a maize plant (Zea mays L.)
which has a long history of safe use. The genetic modification of 1507 maize was
carried out by using the particle acceleration method to introduce into maize cells a
purified linear DNA fragment containing the cry1F and pat gene coding sequences
and the necessary regulatory components for expression of CRY1F and PAT proteins
in maize. No other gene coding sequences have been inserted during the genetic
modification of 1507 maize.
The genetic sequences obtained from the donor organisms and inserted in 1507 maize
do not have any pathogenic characteristics. First of all maize as the recipient plant,
and donor of the ubiZM1(2) promoter, is not a pathogenic organism and has a long
history of domestication and safe use as an agricultural food crop.
Secondly, Bacillus thuringiensis, donor of the crylF sequence, has a history of
decades of safe use as a pesticide (EPA, 1996). The subspecies aizawai is
commercially used to control wax moth larvae and various caterpillars, especially the
diamondback moth caterpillar (Cornell University, 1996). Agrobacterium
tumefaciens is the source of the ORF25PolyA terminator for the cry1F gene. No
sequences involved in plant pathogenicity are encoded by this transcription
terminator.
 Thirdly, Streptomyces viridochromogenes, donor of the pat gene, is a common soil
 bacterium that produces the tripeptide L-phosphinothricyl-L-alanyl-alanine (L-PPT),
 which was developed as a non-selective herbicide by Hoechst Ag and there is a
 history of safe use of the pat gene in GM crops (OECD, 1999; see Annex 5). The
 cauliflower mosaic virus, donor of the CaMV 35S promoter and terminator sequences,
 is a DNA caulimovirus with a host range restricted primarily to cruciferous plants
 (ICTV Database, 1998).
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<pre> Scheme IV: Effect of the genetic modification on the properties of the host
 organism
The 1507 maize has been modified to express CRY1F protein for resistance to certain
lepidopteran insect pests, such as the European com borer (ECB), and to express
phosphinothricin-N-acetyltransferase (PAT) protein for tolerance to glufosinate-
ammonium herbicide. The particle acceleration method was used to introduce into
maize cells a purified linear DNA fragment (PHI8999A; 6235 bp) containing the
cry1F and pat coding sequences and the necessary regulatory components only. The
nptII gene, conferring resistance to kanamycin, was not part of the purified linear
DNA fragment used in the transformation and it is not present in 1507 maize, as
confirmed by the molecular analyses carried out with Southern blots.
The insert, PHI8999A, consists of a linear DNA fragment containing we plant
optimized and truncated crylF gene and the plant optimized pat gene together with
the regulatory sequences necessary for their expression in maize plants. The insert
was obtained by digesting the plasmid PHP8999 (9504 bp) with the restriction
enzyme PmeI. This produced the 6235 bp insert, which was subsequently purified by
agarose gel electrophoresis and used in the transformation of 1507 maize. The 3269
bp fragment containing the nptII gene was discarded and was not used in the
transformation.
The amino acid sequence of the core CRY IF protein expressed in 1507 maize is, with
the exception of a single amino acid substitution, leucine at position 604, identical to
amino acids 1-605 of the native CRY1F protein which is 1174 amino acids long. This
change in the coding sequence was made to introduce aXhoI restriction site for fusion
of sequences encoding the C-terminal domain of the protein that forms the full length
protein. The amino acid sequence of the recombinant CRY1F protein (MR872)
produced in Pseudomonas fluorescens which has been used in toxicological studies
consists of a chimeric CRY1F/CRYlA(b) protein with equivalent biochemical
characteristics and biological activity to the maize expressed core CRY1F protein
(Evans, 1998).
The activity of the PAT protein is specific to catalysing the conversion of L-PPT to N-
acetyl-L-PPT. This is an inactive form which does not bind to glutamine synthetase
(De Block et al. 1987). The expression of the PAT protein in 1507 maize allows the
detoxification of ammonia to continue and confers tolerance to the herbicide
glufosinate-ammonium. The activity of the PAT protein has been described in detail
by the OECD (1999): "Consensus document on general information concerning the
genes and their enzymes that confer tolerance to phosphinothricin herbicide" (Annex
5).
 Detailed molecular characterization, based on Southern blot analyses, confirms that
 1507 maize contains a full-length copy of the DNA insert integrated into the maize
 genome and an additional copy of the cry1F gene (Glatt, 2000; Annex 6).
Scheme V: Genetic stability of the GMO used as NF source
 The 1507 maize has remained stable as inbreds and hybrids for at least six
 generations, through crossing and backcrossing to elite inbreds and selfing, as
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<pre>confirmed by early and late segregation data (BC2F1 and F1 generations,
respectively). This evidence, together with the molecular characterization by
Southern blotting of the 1507 maize (Scheme IV) and analyses of the expression of
CRY1F and PAT proteins (Scheme VI), confirms stable integration of the cry1F and
pat genes in the genome of 1507 maize. These analyses support the conclusion that
the cry1F and pat genes are inherited as Mendelian dominant genes and that the
additional copy of the cry1F gene is genetically linked to the insert containing the
cry1F and pat genes.
Scheme VI: Specificity of expression of novel genetic material
Analyses of expression of the novel genetic material (cry1F and pat genes) has been
carried out at ten locations over two growing seasons: four locations in Chile in 1998-
1999, and six locations in France and Italy in 1999. The results obtained from tissue
samples from 1507 maize have confirmed specificity of CRY1F protein expression in
samples of leaf (V9 stage), pollen, silk, stalk, whole plant (both at V9 and R4 stages),
grain and senescent whole plant tissues. The results have also confirmed specificity
of PAT protein expression in samples of leaf (V9 stage) and whole plant (V9 stage)
tissues. The ELISA results show that CRY1F is expressed in all plant material tested,
with significantly lower concentrations in grain than in vegetative tissue.
Additionally, Western blot analyses have confirmed that CRY1F and PAT proteins
expressed in 1507 maize are of the same molecular weight and immunoreactivity as
the microbially-derived proteins. Other than the expected bands for CRY1F and PAT
proteins, the Western blots did not show any other bands to indicate either partial or
fusion proteins in the 1507 maize tissues.
 Comparison of the agronomic performance in terms of grain yield, moisture,
 accumulated growing degree days to reach 50% pollen shed, accumulated growing
 degree days to reach 50% silking, grain density, plant height, ear height, early stand
 count establishment, visual rating of emergence vigour from spike to one-leaf stage,
 visual rating of vigour at three- to five-leaf stage, stalk lodging, root lodging, dropped
 ears per plot and top integrity between 1507 maize and non-GM control maize with
 comparable genetic backgrounds, further confirms that 1507 maize is comparable to
 non-GM control maize. With the exception of the intended resistance to certain
 lepidopteran insect pests and tolerance to glufosinate-ammonium herbicide, as derived
 from the genetic modification, no unexpected phenotypic differences have been
 observed in 1507 maize. The agronomic data also provides further evidence of the
 specificity of expression of the novel genetic material in 1507 maize
 Scheme VII: Transfer of genetic material from GMO
  Transfer of genetic material originating from 1507 maize to bacteria is a negligible
  concern as there is no known mechanism for, or definitive demonstration of, DNA
  transfer from plants to microbes under natural conditions. In any case, and even if
  horizontal gene transfer were to take place, transfer of the cry1F or pat gene from 1507
  maize does not represent a risk to human health or animal health.
  Scheme VIII: Ability of the GMM to survive in and colonize the human gut
  This is not applicable as the NF is not a GM microorganism (GMM).
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<pre> Scheme IX: Anticipated intake/extent of use of the NF
Majority of grain and forage derived from maize is used for animal feed and about 8%
of EU production is processed for human food products. The evidence provided in
the application shows that the inserted genetic material and the newly expressed
CRY1F and PAT proteins in 1507 maize are safe to human health. Furthermore, the
properties of 1507 maize regarding its nutritional value and compositional
characteristics are comparable and substantially equivalent to those of traditionally-
bred non-GM maize. Based on this evidence, the anticipated use of 1507 maize
derived foods will be no different from that of traditionally bred maize, and any
substitution of traditionally bred maize by 1507 maize will not be of any nutritional
significance.
Scheme X: Information from previous human exposure to the NF or its source
Food products derived from maize have been produced, prepared and consumed by
humans for centuries throughout the world. Based on the evidence presented in this
application, we conclude that exposure to food products derived from 1507 maize will
not give rise to nutritional, microbiological, lexicological and/or allergenicity
problems and that it is as safe as exposure to any other food products derived from
traditionally-bred non-GM maize.
Scheme XI: Nutritional information on the NF
Range of data from compositional analyses of maize grain are provided in the
application for the nutritional assessment of 1507 maize. Field studies representative
of pertinent conditions and growth stages of the commercial production of maize were
conducted on 1507 maize and non-GM control maize. Data from ten different
locations over two growing seasons have been analysed: four locations in Chile
(1998-1999) and six in the EU (France and Italy, 1999). At the locations in Italy, the
trials also included treatments to compare 1507 maize sprayed with glufosinate-
ammonium, 1507 maize unsprayed with glufosinate-ammonium, and non-GM control
maize. At all locations, the non-GM control maize was of comparable genetic
background to 1507 maize.
The compositional data determined the levels of protein, fiber (ADF, NDF),
carbohydrates, fat, ash, five different fatty acids, five different minerals, eighteen
different amino acids, four different vitamins, five different secondary metabolites and
two potential anti-nutrients. The analyses on the composition clearly demonstrated
that grain from 1507 maize, unsprayed or sprayed with glufosinate-ammonium, is
comparable to grain from non-GM maize and to the published range of composition
values in the literature. These analyses support the conclusion that 1507 maize is
substantially equivalent to traditionally-bred maize.
 In addition, nutritional equivalence between 1507 maize and traditionally-bred maize
 has been shown in a poultry feeding study where broiler chickens were fed over a 42-
 day period with diets containing 1507 maize grain or yellow dent grain from non-GM
 maize. The mortality, body weight gain and feed conversion of the chickens were
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<pre>compared and no statistically significant differences were observed between chickens
fed a diet containing grain from 1507 maize or grain from non-GM maize.
Scheme XII: Microbiological information on the NF
The NF does not contain any microorganisms and therefore no microbiological
information is provided.
SchemeXIII: Toxicological information on theNF
Toxicitv
Maize has a long history of use as food in the EU and constitutes a traditional
counterpart to 1507 maize that can be used as a baseline to facilitate the toxicological
assessment. Maize is not considered to have harmful toxicants and the genetic
modification in 1507 maize does not introduce any new toxicants harmful to humans
or animals.
The CRY1F protein has specific toxicity against certain lepidopteran insect pests
(target organisms), and there is no evidence for CRY proteins originating from
Bacillus thuringiensis to have any harmful effects on the health of humans and
animals (EPA, 1995a; McClintock et al., 1995; EPA, 1996). Furthermore, the
potential toxicity of the CRY1F protein to humans and animals was specifically
examined in an acute oral toxicology study where CRY1F protein was evaluated for
acute toxicity in mice (Kuhn, 1998). The relatively high dose tested did not give rise
to any toxicity and therefore the acute LD50 for CRY1F protein could not be
determined other than estimated to be higher than 576 mg CRY1F per kg body
weight.
The PAT protein has already been found safe to human health during the assessment
of glufosinate-ammonium tolerant maize (OECD, 1999; Annex 5). The pat gene was
originally obtained from Streptomyces viridochromogenes strain Tü494 which has no
known toxic or pathogenic potential. The PAT protein is enzymatically active but it
has high substrate specificity to the active ingredient of glufosinate-ammonium (L-
PPT). A toxicity study consisting of feeding rats with the PAT protein has been
carried out (Pfister et al., 1996; Health Canada, 1997). Results from the range of
doses tested (up to 50000 mg/kg body weight) showed no adverse effects on the
growth or histopathology of the animals. The PAT protein has also been tested in an
additional acute toxicity study in mice (Brooks, 2000). As before, the relatively high
dose tested did not give rise to any toxicity and therefore the acute LD50 of PAT
protein could not be determined other than estimated to be higher than 5000 mg PAT
per kg body weight.
 A 42-day long poultry feeding study has also been carried out with grain from 1507
 maize and the non-GM control maize with comparable genetics. The results show
 that mortality, body weight gain and feed conversion of the chickens fed with 1507
 maize was similar to chickens fed a standard diet containing yellow dent maize.
  Allergenicity
  The most important factor to consider in assessing allergenic potential is whether the
  source of the gene being introduced into plants is known to be allergenic (FDA,
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<pre> 1992). Neither Bacillus thuringiensis (the source of the cry1F gene) nor Streptomyces
 viridochromogenes (the source of the pat gene) have a history of causing allergy.
The biochemical profile of the CRY IF and PAT proteins also provides a basis for
allergenic assessment when compared with known protein allergens. A database
search was compiled by Meyer (1999) using the Wisconsin Genetics Computer Group
(GCG) sequence analysis computer program with the keyword "allergen" to search
standard DNA and protein sequence databases. Comparison of the 15 most
homologous database sequences confirmed that the CRY1F and PAT proteins do not
share any significant amino acid sequence homology with known protein allergens.
Furthermore, protein allergens are typically stable to the peptic and tryptic digestion,
and to the acid conditions of the human digestive system, which allows them to reach
and pass through the intestinal mucosa to elicit an allergenic response. Both CRY1F
and PAT proteins are readily degradable in simulated digestive juice, minimising any
potential for these proteins to be absorbed by the intestinal mucosa when consumed.
The CRY1F protein was nearly completely proteolysed in simulated gastric
conditions within one minute at a molar ratio of 188:1 (CRY1F:pepsin) (Evans,
1998). In addition, the immunoblot detection technique has demonstrated that
CRY1F is not glycosylated, which is an additional indicator of the absence of
allergenic potential in the CRY1F protein. Furthermore, CRY1F looses
immunoreactivity after heat processing and it has a history of safe use in microbial
pesticides (Evans, 1998). In addition, a study on the heat lability of CRY1F protein at
various temperatures showed that CRY1F looses biological activity after exposure at
75°C or greater for 30 minutes (Herman, 2000). The PAT protein degraded to non-
detectable levels within 5 seconds after introduction to simulated gastric fluid
containing pepsin (Glatt, 1999; OECD, 1999; Annex 5).
Therefore, the cry1F and pat genes introduced into 1507 maize do not encode for
known allergens, and neither the CRY1F nor the PAT proteins share immunologically
significant amino acid sequences with known allergens. This together with the heat
lability and rapid breakdown of these proteins under digestive conditions, confirms
that the CRY1F and PAT proteins should not pose any allergenic risk.
 Evaluation and conclusion
 The safety evaluation of food products derived from B.t. Cry1F maize line 1507,
 referred to as 1507 maize, presented throughout this application has been carried out
 following thorough consideration of a series of key parameters that show
 wholesomeness and substantial equivalence between 1507 maize and traditionally-
 bred non-GM maize: Southern blot analyses and molecular characterisation,
 agronomic performance, Mendelian inheritance, protein expression analyses,
 compositional analyses, nutritional equivalence and lexicological information have
 demonstrated that food products from 1507 maize are as safe and nutritious as other
 maize products already on the EU market.
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<pre>Annex         E
              Schematic representation of the genetic
              modification
   Schematic representation of the genetic modification in 1507 maize
   9                    = primary insert
   4, 7a/b/c, 8, 10, 13 = partial duplications of the primary insert
   5, 6, 11, 12, 14     = homologous to chloroplast DNA
   2,3                  = homologous to known maize genomic DNA
   1, 15                = unknown, probably maize genomic DNA
                                               6 7b 7c                                                     14
            1       2      3        4      5    7a    8                                   10  11   12    13          15
       5’ flanking sequence                                                                          3’ flanking sequence
                                                                   primary insert
                                                                          9
                                                                                                T 35S
                  P ubiZM1(2)                              cry1F                T ORF25 P 35S    pat
                                                                                                 7b
                                                                                                            origin of partial
                                                                                              7a
                                                                                                            duplications
           8                                            7c         4              10          13
         0               1000              2000             3000             4000        5000            6000
                                                                                                       length in base pairs
              Schematic representation of the genetic modification                                                            99
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<pre>100 Insect-resistant and herbicide-tolerant maize (1507)</pre>

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<br><br>