A1106-AppR-SD1
Supporting document 1Safety assessment report – Application A1106 (at Approval)Food derived from Herbicide-tolerant & Insect-protected Corn Line 4114Summary and conclusionsBackgroundA genetically modified (GM) corn line with OECD Unique Identifier DP-004114-3, hereafter referred to as line 4114, has been developed by Pioneer Hi-Bred International Inc. The corn has been modified to be tolerant to the herbicide glufosinate ammonium and protected against lepidopteran insect pests, particularly European corn borer (Ostrinia nubilalis) and coleopteran insect pests, particularly western corn rootworm (Diabrotica virgifera virgifera).Tolerance to glufosinate ammonium is achieved through expression of the enzyme phosphinothricin acetyltransferase (PAT) encoded by the pat gene derived from the common soil bacterium Streptomyces viridochromogenes. Protection against lepidopteran insect pests is conferred by the cry1F gene, which is a synthetic and truncated version of a gene from Bacillus thuringiensis var. aizawai. Protection against coleopteran insect pests is conferred by two genes, cry34Ab1 and cry35Ab1 both from B. thuringiensis strain PS149B1.In conducting a safety assessment of food derived from line 4114, a number of criteria have been addressed including: a characterisation of the transferred gene sequences, their origin, function and stability in the corn genome; the changes at the level of DNA, and protein in the whole food; compositional analyses; and evaluation of intended and unintended changes. This safety assessment report addresses only food safety and nutritional issues. It therefore does not address: environmental risks related to the environmental release of GM plants used in food productionthe safety of animal feed, or animals fed with feed, derived from GM plantsthe safety of food derived from the non-GM (conventional) plant.History of UseIn terms of production, corn is the world’s dominant cereal crop, ahead of wheat and rice and is grown in over 160 countries. It has a long history of safe use in the food supply. Sweet corn is consumed directly while corn-derived products are routinely used in a large number and diverse range of foods (e.g. cornflour, starch products, breakfast cereals and high fructose corn syrup). Corn is also widely used as a feed for domestic livestock.Molecular characterisationLine 4114 was generated through Agrobacterium-mediated transformation and contains four expression cassettes: Comprehensive molecular analyses of line 4114 indicate there is a single insertion site comprising a single, complete copy of each of the cry1F, cry34Ab1, cry35Ab1, and pat genes. The introduced genetic elements are stably inherited from one generation to the next. There are no antibiotic resistance marker genes present in the line and no plasmid backbone has been incorporated into the transgenic locus.Characterisation and safety assessment of new substancesNewly expressed proteinsCorn line 4114 is a molecular stack expressing four novel proteins, Cry1F, Cry34Ab1, Cry35Ab1 and PAT. These proteins have previously been assessed in two corn lines DAS-01507-1 (line 1507) and DAS-59122-7 (line 59122) where the coding regions and regulatory elements of the various gene cassettes are the same as in line 4114. A breeding stack created from these two lines is also commercially availableFor Cry34Ab1, Cry35Ab1 and PAT, mean levels were lowest in the pollen (9.2, 0.34 ?g/g dry weight and <LOQ respectively). The highest levels of these three proteins were in the leaf samples at either the R1 or R4 stages. In contrast, the level of Cry1F was lowest in leaf tissue at R6 (the stage at which grain is harvested) and highest in pollen (35 ?g/g dry weight). In the grain, levels of Cry1F, Cry34Ab1, Cry35Ab1 and PAT were 3.3, 24, 1.1 and <LOQ ?g/g dry weight respectively.Western blot analyses confirmed that the four novel proteins in line 4114 have the expected molecular weights and immunoreactivity and are equivalent to the corresponding proteins produced in the breeding stack.Herbicide MetabolitesThe herbicide residues resulting from the application of glufosinate to lines carrying the pat or bar genes have been assessed in previous applications. There are no concerns that the spraying of line 4114 with glufosinate would result in the production of any novel metabolites that have not been previously positional analysesDetailed compositional analyses were done to establish the nutritional adequacy of grain from line 4114 and to characterise any unintended compositional changes. Analyses were done of proximates, fibre, minerals, amino acids, fatty acids, vitamins, secondary metabolites and anti-nutrients. The levels were compared to levels in a) an appropriate non-GM hybrid line, PHNAR x PHTFE, b) a tolerance interval compiled from results taken for eight non-GM hybrid lines grown in similar field trials in different seasons, and c) levels recorded in the literature. Only five of the 56 analytes that were considered in a statistical analysis deviated in level from the control in a statistically significant manner. However, the mean levels of all five of these analytes fell within both the tolerance interval and the historical range from the literature. It is further noted that the differences between the line 4114 and control means for each of the five analytes were less than the variation found within the control. It can therefore be concluded that grain from line 4114 is compositionally equivalent to grain from conventional corn varieties.ConclusionNo potential public health and safety concerns have been identified in the assessment of herbicide-tolerant and insect-protected corn line 4114. On the basis of the data provided in the present Application, and other available information, food derived from line 4114 is considered to be as safe for human consumption as food derived from conventional corn varieties.Table of Contents TOC \o "1-2" \h \z \u Summary and conclusions PAGEREF _Toc430248230 \h iBackground PAGEREF _Toc430248231 \h iHistory of Use PAGEREF _Toc430248232 \h iMolecular characterisation PAGEREF _Toc430248233 \h iiCharacterisation and safety assessment of new substances PAGEREF _Toc430248234 \h iiCompositional analyses PAGEREF _Toc430248235 \h iiConclusion PAGEREF _Toc430248236 \h iiiList of Figures PAGEREF _Toc430248237 \h 2List of Tables PAGEREF _Toc430248238 \h 2List of Abbreviations PAGEREF _Toc430248239 \h 31Introduction PAGEREF _Toc430248240 \h 42History of Use PAGEREF _Toc430248241 \h 52.1Host organism PAGEREF _Toc430248242 \h 52.2Donor organisms PAGEREF _Toc430248243 \h 63Molecular characterisation PAGEREF _Toc430248244 \h 73.1Method used in the genetic modification PAGEREF _Toc430248245 \h 83.2Function and regulation of introduced genes PAGEREF _Toc430248246 \h 93.3Breeding of corn line DP-004114-3 PAGEREF _Toc430248247 \h 123.4Characterisation of the genetic modification in the plant PAGEREF _Toc430248248 \h 133.5Stability of the genetic changes in line 4114 PAGEREF _Toc430248249 \h 153.6Antibiotic resistance marker genes PAGEREF _Toc430248250 \h 163.7Conclusion PAGEREF _Toc430248251 \h 164Characterisation and safety assessment of new substances PAGEREF _Toc430248252 \h 164.1Newly expressed proteins PAGEREF _Toc430248253 \h 164.2Herbicide metabolites PAGEREF _Toc430248254 \h 255Compositional analysis PAGEREF _Toc430248255 \h 255.1Key components PAGEREF _Toc430248256 \h 265.2Study design and conduct for key components PAGEREF _Toc430248257 \h 265.3Analyses of key components in grain PAGEREF _Toc430248258 \h 275.4Conclusion from compositional analysis PAGEREF _Toc430248259 \h 336Nutritional impact PAGEREF _Toc430248260 \h 33References PAGEREF _Toc430248261 \h 34List of Figures TOC \h \z \c "Figure" Figure 1: The corn wet milling process (diagram taken from CRA (2006)) PAGEREF _Toc414460100 \h 6Figure 2: Genes and regulatory elements contained in plasmid PHP27118 PAGEREF _Toc414460101 \h 8Figure 3: Representation of the genetic elements in the T-DNA insert of plasmid PHP27118 PAGEREF _Toc414460102 \h 9Figure 4: Breeding diagram for corn line DP-004114-3 PAGEREF _Toc414460103 \h 12Figure 5: Amino acid sequence of the Cry1F protein PAGEREF _Toc414460104 \h 17Figure 6: Amino acid sequences of the Cry34Ab1 and Cry35Ab1 proteins PAGEREF _Toc414460105 \h 18Figure 7: Amino acid sequence of the PAT protein PAGEREF _Toc414460106 \h 19List of Tables TOC \h \z \c "Table" Table 1: Description of the genetic elements contained in the T-DNA of PHP27118 PAGEREF _Toc417973322 \h 9Table 2: Line 4114 generations used for various analyses PAGEREF _Toc417973323 \h 13Table 3: Segregation of the DP-004114-3 T-DNA sequences over five generations PAGEREF _Toc417973324 \h 15Table 4:Cry1F, Cry34Ab1, Cry35Ab1 and PAT protein content in line 4114 parts at different growth stages (averaged across 5 sites) PAGEREF _Toc417973325 \h 19Table 5: Summary of line 4114 protein concentrations as a ratio of line 1507, line 59122 and 1507 x 59122 PAGEREF _Toc417973326 \h 21Table 6: Summary of consideration of Cry1F, Cry34Ab1, Cry35Ab1 and PAT in previous FSANZ safety assessments PAGEREF _Toc417973327 \h 23Table 7: Mean (range) percentage dry weight (%dw) of proximates and fibre in grain from glufosinate-treated line 4114 and the hybrid (PHNAR x PHTFE) control. PAGEREF _Toc417973328 \h 28Table 8: Mean (range) percentage composition, relative to total fat, of major fatty acids in grain from glufosinate-treated line 4114 and the hybrid (PHNAR x PHTFE) control. PAGEREF _Toc417973329 \h 29Table 9: Mean (range) %dw of amino acids in grain from glufosinate-treated line 4114 and the hybrid (PHNAR x PHTFE) control. PAGEREF _Toc417973330 \h 30Table 10: Mean (range) levels of minerals in the grain of glufosinate-treated line 4114 and the hybrid (PHNAR x PHTFE) control. PAGEREF _Toc417973331 \h 30Table 11: Mean (range) weight (mg/k g dw) of vitamins in grain from glufosinate-treated line 4114 and the hybrid (PHNAR x PHTFE) control. PAGEREF _Toc417973332 \h 31Table 12: Mean (range) of anti-nutrients in grain from glufosinate-treated line 4114 and the hybrid (PHNAR x PHTFE) control. PAGEREF _Toc417973333 \h 32Table 13: Mean %dwt (range) of three secondary metabolites in grain from glufosinate-treated line 4114 and the hybrid (PHNAR x PHTFE) control. PAGEREF _Toc417973334 \h 32Table 14: Summary of analyte levels found in grain of glufosinate-treated line 4114 that are significantly (P < 0.05) different from those found in grain of the hybrid (PHNAR x PHTFE) control. PAGEREF _Toc417973335 \h 32List of AbbreviationsADFacid detergent fibreBLASTPBasic Local Alignment Search Tool: Proteinbpbase pairsBtBacillus thuringiensisCrycrystalDIGdigoxigeninDNAdeoxyribonucleic acidT-DNAtransferred DNAdwdry weightELISAenzyme linked immunosorbent assayFAOFood and Agriculture Organization of the United NationsFARRPFood Allergy Research and Resource ProgramFASTAFast Alignment Search Tool - AllFDRFalse discovery rateFSANZFood Standards Australia New ZealandGMgenetically modifiedkDakilo DaltonLBLeft Border of T-DNALOQLimit of quantitationMRLmaximum residue limitNDFneutral detergent fibreOECDOrganisation for Economic Co-operation and DevelopmentOGTROffice of the Gene Technology RegulatorORFopen reading framePATphosphinothricin acetyltransferasePCRpolymerase chain reactionP-valueprobability valueRBRight Border of T-DNASASStatistical Analysis SoftwareSDS-PAGEsodium dodecyl sulfate polyacrylamide gel electrophoresisU.S.United States of AmericaWHOWorld Health Organization1IntroductionPioneer Hi-Bred Australia Ltd on behalf of Pioneer Hi-Bred International Inc has submitted, an application to FSANZ to vary Standard 1.5.2 – Food produced using Gene Technology – in the Australia New Zealand Food Standards Code (the Code) to include food from a new genetically modified (GM) corn line with OECD Unique Identifier DP-004114-3 (also referred to as line 4114). The corn has been modified to be tolerant to the herbicide glufosinate ammonium (glufosinate) and protected against lepidopteran insect pests, particularly European corn borer (Ostrinia nubilalis) and coleopteran insect pests, particularly western corn rootworm (Diabrotica virgifera virgifera) ADDIN REFMGR.CITE <Refman><Cite><Author>Diehn</Author><Year>2011</Year><RecNum>1707</RecNum><IDText>Maize event DP-004114-3 and methods for detection thereof</IDText><MDL Ref_Type="Patent"><Ref_Type>Patent</Ref_Type><Ref_ID>1707</Ref_ID><Title_Primary>Maize event DP-004114-3 and methods for detection thereof</Title_Primary><Authors_Primary>Diehn,S.</Authors_Primary><Authors_Primary>Lu,A.L.</Authors_Primary><Authors_Primary>Nowatzki,T.M.</Authors_Primary><Authors_Primary>Nubel,D.S.</Authors_Primary><Authors_Primary>Pascual,M.A.</Authors_Primary><Authors_Primary>Register III,J.C.</Authors_Primary><Authors_Primary>Scelonge,C.J.</Authors_Primary><Authors_Primary>Young,J.K.</Authors_Primary><Authors_Primary>Crowgey,E.L.</Authors_Primary><Date_Primary>2011</Date_Primary><Keywords>maize</Keywords><Keywords>methods</Keywords><Keywords>Dna</Keywords><Keywords>Plants</Keywords><Reprint>Not in File</Reprint><Issue>US 2011/0154523 A1</Issue><Web_URL><u>;\References\GM References_in RefMan\Diehn et al_2011_Maize event DP004114-3 patent.pdf</u></Web_URL_Link2><ZZ_WorkformID>22</ZZ_WorkformID></MDL></Cite></Refman>(Diehn et al. 2011).Tolerance to glufosinate is achieved through expression of the enzyme phosphinothricin acetyltransferase (PAT) encoded by the pat gene derived from the common soil bacterium Streptomyces viridochromogenes. This protein has been considered in 19 previous FSANZ approvals and globally is represented in six major crop species and over 30 approved GM single plant events. Protection against lepidopteran insect pests is conferred by the cry1F gene, which is a synthetic version of a gene from Bacillus thuringiensis var. aizawai, and encodes a truncated version of an insecticidal protein, Cry1F. Protection against coleopteran insect pests is conferred by two genes, cry34Ab1 and cry35Ab1 both from B. thuringiensis strain PS149B1 and encoding the insecticidal proteins Cry34Ab1 and Cry35Ab1. These proteins have both been considered previously by FSANZ Line 4114 is a molecular stack that, in terms of traits, is the equivalent of a breeding stack (known commercially as Herculex? XTRA) obtained by crossing two corn lines, DAS-01507-1 (line 1507) and DAS-59122-7 (line 59122). Food from both of these lines has been approved by FSANZ in applications A446 ADDIN REFMGR.CITE <Refman><Cite><Author>FSANZ</Author><Year>2003</Year><RecNum>354</RecNum><IDText>Application A446: Food derived from Insect-Protected and Glufosinate Ammonium-Tolerant Corn Line 1507</IDText><MDL Ref_Type="Report"><Ref_Type>Report</Ref_Type><Ref_ID>354</Ref_ID><Title_Primary>Application A446: Food derived from Insect-Protected and Glufosinate Ammonium-Tolerant Corn Line 1507</Title_Primary><Authors_Primary>FSANZ</Authors_Primary><Date_Primary>2003</Date_Primary><Reprint>Not in File</Reprint><Publisher>Report prepared by Food Standards Australia New Zealand</Publisher><Web_URL><u>;(FSANZ 2003) and A543 ADDIN REFMGR.CITE <Refman><Cite><Author>FSANZ</Author><Year>2005</Year><RecNum>355</RecNum><IDText>Application A543: Food derived from Insect-Protected, Glufosinate Ammonium-Tolerant Corn Line DAS-59122-7</IDText><MDL Ref_Type="Report"><Ref_Type>Report</Ref_Type><Ref_ID>355</Ref_ID><Title_Primary>Application A543: Food derived from Insect-Protected, Glufosinate Ammonium-Tolerant Corn Line DAS-59122-7</Title_Primary><Authors_Primary>FSANZ</Authors_Primary><Date_Primary>2005</Date_Primary><Reprint>Not in File</Reprint><Publisher>Report prepared by Food Standards Australia New Zealand</Publisher><Web_URL><u>;(FSANZ 2005) respectively and hence, food from the breeding stack is also approved to enter the Australian and New Zealand food supplies. The breeding stack was grown on approximately 10,000,000 ha in the U.S. in 2013 (Pioneer Hi-Bred proprietary data). Food from line 4114 requires a separate approval since it represents a unique molecular event although the expressed traits are the same as those already assessed by FSANZ. The purpose in developing the line 4114 molecular stack was to have all of the inserted genes on a single transformation construct integrated at a single genetic locus in the corn genome. Line 4114 therefore has an advantage over the breeding stack because having three linked traits at a single locus will simplify future breeding efforts. The Applicant has stated that line 4114 is not intended to be a stand-alone product and will be crossed by conventional breeding with other approved GM corn lines as well as conventional lines.The Applicant indicates that any lines containing the DP-004114-3 event will be grown primarily in North America, and approval for cultivation in Australia or New Zealand is not currently being sought. Therefore, if approved, food derived from this line may enter the Australian and New Zealand food supply as imported food products.2History of Use2.1Host organismThe host organism is a conventional Pioneer proprietary inbred corn (Zea mays) line PHWWE ADDIN REFMGR.CITE <Refman><Cite><Author>Wang</Author><Year>2008</Year><RecNum>1708</RecNum><IDText>Highly transformable elite inbred line - PHWWE</IDText><MDL Ref_Type="Patent"><Ref_Type>Patent</Ref_Type><Ref_ID>1708</Ref_ID><Title_Primary>Highly transformable elite inbred line - PHWWE</Title_Primary><Authors_Primary>Wang,N.</Authors_Primary><Authors_Primary>Heetland,L.L.</Authors_Primary><Authors_Primary>Solawetz,W.E.</Authors_Primary><Authors_Primary>Zhao,Z.-Y.</Authors_Primary><Date_Primary>2008</Date_Primary><Reprint>Not in File</Reprint><Issue>US 2008/0072344 A1</Issue><Web_URL_Link2>;\References\GM References_in RefMan\Wang et al_2008_patent for PHWWE corn.pdf</u></Web_URL_Link2><ZZ_WorkformID>22</ZZ_WorkformID></MDL></Cite></Refman>(Wang et al. 2008). It is a homozygous line with an elite genotype that also has high transformability and good response in tissue culture. The line was produced by initially crossing a Hi-II hybrid corn line with pollen from the elite line PHO9B and then going through a series of backcrossing to PHO9B and self-pollination while selecting for the desired characteristics. Mature corn (Zea mays) plants contain both female and male flowers and usually reproduce sexually by wind-pollination. This provides for both self-pollination and natural out-crossing between plants, both of which are undesirable since the random nature of the crossing leads to lower yields ADDIN REFMGR.CITE <Refman><Cite><Author>CFIA</Author><Year>1994</Year><RecNum>58</RecNum><IDText>The Biology of Zea mays L. (Corn/Maize)</IDText><MDL Ref_Type="Report"><Ref_Type>Report</Ref_Type><Ref_ID>58</Ref_ID><Title_Primary>The Biology of <i>Zea mays</i> L. (Corn/Maize)</Title_Primary><Authors_Primary>CFIA</Authors_Primary><Date_Primary>1994</Date_Primary><Keywords>Zea mays</Keywords><Reprint>Not in File</Reprint><Volume>BIO1994-11</Volume><Pub_Place>Ottawa</Pub_Place><Publisher>Canadian Food Inspection Agency</Publisher><Web_URL><u>;(CFIA 1994). The commercial production of corn now utilises controlled cross-pollination of two inbred lines (using conventional techniques) to combine desired genetic traits and produce hybrid varieties known to be superior to open-pollinated varieties in terms of their agronomic characteristics. This inbred-hybrid concept and resulting yield response is the basis of the modern corn seed industry and hybrid corn varieties are used in most developed countries for consistency of performance and production.In terms of production, corn is the world’s dominant cereal crop, ahead of wheat and rice and is grown in over 160 countries ADDIN REFMGR.CITE <Refman><Cite><Author>FAOSTAT</Author><Year>2014</Year><RecNum>549</RecNum><IDText>Online database of the Food and Agriculture Organization of the United Nations</IDText><MDL Ref_Type="Online Source"><Ref_Type>Online Source</Ref_Type><Ref_ID>549</Ref_ID><Title_Primary>Online database of the Food and Agriculture Organization of the United Nations</Title_Primary><Authors_Primary>FAOSTAT</Authors_Primary><Date_Primary>2014</Date_Primary><Reprint>Not in File</Reprint><Web_URL><u>;(FAOSTAT 2014). In 2012, worldwide production of corn was over 872 million tonnes, with the United States and China being the major producers (~273 and 208 million tonnes, respectively) ADDIN REFMGR.CITE <Refman><Cite><Author>FAOSTAT</Author><Year>2014</Year><RecNum>549</RecNum><IDText>Online database of the Food and Agriculture Organization of the United Nations</IDText><MDL Ref_Type="Online Source"><Ref_Type>Online Source</Ref_Type><Ref_ID>549</Ref_ID><Title_Primary>Online database of the Food and Agriculture Organization of the United Nations</Title_Primary><Authors_Primary>FAOSTAT</Authors_Primary><Date_Primary>2014</Date_Primary><Reprint>Not in File</Reprint><Web_URL><u>;(FAOSTAT 2014). Corn is not a major crop in Australia or New Zealand and in 2012, production was approximately 450,000 and 211,00 tonnes respectively ADDIN REFMGR.CITE <Refman><Cite><Author>FAOSTAT</Author><Year>2014</Year><RecNum>549</RecNum><IDText>Online database of the Food and Agriculture Organization of the United Nations</IDText><MDL Ref_Type="Online Source"><Ref_Type>Online Source</Ref_Type><Ref_ID>549</Ref_ID><Title_Primary>Online database of the Food and Agriculture Organization of the United Nations</Title_Primary><Authors_Primary>FAOSTAT</Authors_Primary><Date_Primary>2014</Date_Primary><Reprint>Not in File</Reprint><Web_URL><u>;(FAOSTAT 2014). Domestic production is supplemented by the import of corn grain and corn-based products, the latter of which are used, for example, in breakfast cereals, baking products, extruded confectionery and food coatings. In 2011, Australia and New Zealand imported, respectively, 856 and 5,800 tonnes of corn grain, 10,600 and 306 tonnes of frozen sweet corn and 8,427 and 900 tonnes of otherwise-processed sweet corn ADDIN REFMGR.CITE <Refman><Cite><Author>FAOSTAT</Author><Year>2014</Year><RecNum>549</RecNum><IDText>Online database of the Food and Agriculture Organization of the United Nations</IDText><MDL Ref_Type="Online Source"><Ref_Type>Online Source</Ref_Type><Ref_ID>549</Ref_ID><Title_Primary>Online database of the Food and Agriculture Organization of the United Nations</Title_Primary><Authors_Primary>FAOSTAT</Authors_Primary><Date_Primary>2014</Date_Primary><Reprint>Not in File</Reprint><Web_URL><u>;(FAOSTAT 2014). Corn product imports to Australia and New Zealand included 4,734 and 2,100 tonnes of corn flour and 1,520 and 13 tonnes of corn oil respectively ADDIN REFMGR.CITE <Refman><Cite><Author>FAOSTAT</Author><Year>2014</Year><RecNum>549</RecNum><IDText>Online database of the Food and Agriculture Organization of the United Nations</IDText><MDL Ref_Type="Online Source"><Ref_Type>Online Source</Ref_Type><Ref_ID>549</Ref_ID><Title_Primary>Online database of the Food and Agriculture Organization of the United Nations</Title_Primary><Authors_Primary>FAOSTAT</Authors_Primary><Date_Primary>2014</Date_Primary><Reprint>Not in File</Reprint><Web_URL><u>;(FAOSTAT 2014). Corn is a major source of crystalline fructose and high fructose corn syrup, both of which are processed from cornstarch. Approximately 3,000 tonnes of crystalline fructose, but negligible high fructose corn syrup, were imported into Australia in 2011 ADDIN REFMGR.CITE <Refman><Cite><Author>Green Pool</Author><Year>2012</Year><RecNum>1670</RecNum><IDText>Sugar consumption in Australia. A statistical update</IDText><MDL Ref_Type="Report"><Ref_Type>Report</Ref_Type><Ref_ID>1670</Ref_ID><Title_Primary>Sugar consumption in Australia. A statistical update</Title_Primary><Authors_Primary>Green Pool</Authors_Primary><Date_Primary>2012</Date_Primary><Reprint>Not in File</Reprint><Pub_Place>Brisbane</Pub_Place><Publisher>Green Pool Commodity Specialists</Publisher><Web_URL><u>:\References\GM References_in RefMan<u>\Green Pool_2012_sugar consumption in Aus.pdf</u></Web_URL_Link2><ZZ_WorkformID>24</ZZ_WorkformID></MDL></Cite></Refman>(Green Pool 2012); neither Australia nor New Zealand currently produce fructose (either crystalline or as high fructose corn syrup). The majority of grain and forage derived from corn is used as animal feed, however corn also has a long history of safe use as food for human consumption. There are five main types of corn grown for food:Flour – Zea mays var. amylaceaFlint – Z. mays var. indurataDent – Z. mays var. indentataSweet – Z. mays var. saccharata & Z. mays var. rugosaPop – Z. mays var. evertaDent corn is the most commonly grown for grain and silage and is the predominant type grown in the U.S. ADDIN REFMGR.CITE <Refman><Cite><Author>OGTR</Author><Year>2008</Year><RecNum>803</RecNum><IDText>The biology of Zea mays L. ssp mays (maize or corn)</IDText><MDL Ref_Type="Report"><Ref_Type>Report</Ref_Type><Ref_ID>803</Ref_ID><Title_Primary>The biology of <i>Zea mays</i> L. ssp <i>mays</i> (maize or corn)</Title_Primary><Authors_Primary>OGTR</Authors_Primary><Date_Primary>2008</Date_Primary><Keywords>Zea mays</Keywords><Reprint>Not in File</Reprint><Publisher>Prepared by the Office of the Gene Technology Regulator, Australia</Publisher><Web_URL><u>:\References\GM References_in RefMan<u>\OGTR_2008_corn biology.pdf</u></Web_URL_Link2><ZZ_WorkformID>24</ZZ_WorkformID></MDL></Cite></Refman>(OGTR 2008). Line 4114 is a yellow dent corn but could be crossed with other types. Two main grain processing routes are followed for dent corn ADDIN REFMGR.CITE <Refman><Cite><Author>White</Author><Year>1995</Year><RecNum>57</RecNum><IDText>Corn as a food source in the United States: Part II. Processes, products, composition, and nutritive values</IDText><MDL Ref_Type="Book Chapter"><Ref_Type>Book Chapter</Ref_Type><Ref_ID>57</Ref_ID><Title_Primary>Corn as a food source in the United States: Part II. Processes, products, composition, and nutritive values</Title_Primary><Authors_Primary>White,P.J.</Authors_Primary><Authors_Primary>Pollak,L.M.</Authors_Primary><Date_Primary>1995</Date_Primary><Reprint>Not in File</Reprint><Start_Page>756</Start_Page><End_Page>762</End_Page><Title_Secondary>Cereal Foods World 40</Title_Secondary><ZZ_WorkformID>3</ZZ_WorkformID></MDL></Cite></Refman>(White and Pollak 1995):Dry milling that gives rise to food by-products such as flour and hominy grits.Wet milling ADDIN REFMGR.CITE <Refman><Cite><Author>CRA</Author><Year>2006</Year><RecNum>866</RecNum><IDText>Corn oil</IDText><MDL Ref_Type="Book, Whole"><Ref_Type>Book, Whole</Ref_Type><Ref_ID>866</Ref_ID><Title_Primary>Corn oil</Title_Primary><Authors_Primary>CRA</Authors_Primary><Date_Primary>2006</Date_Primary><Reprint>In File</Reprint><Volume>5</Volume><Pub_Place>Washington D.C.</Pub_Place><Publisher>Corn Refiners Association</Publisher><Web_URL><u>:\References\GM References_in RefMan<u>\CRA_2006_corn oil.pdf</u></Web_URL_Link2><ZZ_WorkformID>2</ZZ_WorkformID></MDL></Cite></Refman>(CRA 2006), that involves steeping the grain, coarse and fine grinding, centrifugation and evaporating the steep, to yield food by-products such as starch (for cornstarch, corn syrup and individual sweeteners such as dextrose and fructose) and germ (for oil) – see REF _Ref277762301 \h \* MERGEFORMAT Figure 1. Corn products are used widely in processed foods.Figure SEQ Figure \* ARABIC 1: The corn wet milling process (diagram taken from CRA ADDIN REFMGR.CITE <Refman><Cite ExcludeAuth="1"><Author>CRA</Author><Year>2006</Year><RecNum>866</RecNum><IDText>Corn oil</IDText><MDL Ref_Type="Book, Whole"><Ref_Type>Book, Whole</Ref_Type><Ref_ID>866</Ref_ID><Title_Primary>Corn oil</Title_Primary><Authors_Primary>CRA</Authors_Primary><Date_Primary>2006</Date_Primary><Reprint>In File</Reprint><Volume>5</Volume><Pub_Place>Washington D.C.</Pub_Place><Publisher>Corn Refiners Association</Publisher><Web_URL><u>:\References\GM References_in RefMan<u>\CRA_2006_corn oil.pdf</u></Web_URL_Link2><ZZ_WorkformID>2</ZZ_WorkformID></MDL></Cite></Refman>(2006))2.2Donor organisms2.2.1Bacillus thuringiensisMany different subspecies of Bacillus thuringiensis (Bt) have been isolated from dead or dying insects, mostly from the orders Coleoptera, Diptera and Lepidoptera, but many subspecies have also been found in the soil, aquatic environments and other habitats ADDIN REFMGR.CITE <Refman><Cite><Author>WHO</Author><Year>1999</Year><RecNum>161</RecNum><IDText>Microbial pest control agent Bacillus thuringiensis.</IDText><MDL Ref_Type="Serial (Book,Monograph)"><Ref_Type>Serial (Book,Monograph)</Ref_Type><Ref_ID>161</Ref_ID><Title_Primary>Microbial pest control agent <i>Bacillus thuringiensis</i>.</Title_Primary><Authors_Primary>WHO</Authors_Primary><Date_Primary>1999</Date_Primary><Keywords>Bacillus thuringiensis</Keywords><Reprint>In File</Reprint><Issue>217</Issue><Pub_Place>Geneva</Pub_Place><Publisher>World Health Organization</Publisher><Title_Series>Environmental Health Criteria</Title_Series><ZZ_WorkformID>25</ZZ_WorkformID></MDL></Cite></Refman>(WHO 1999). The source of the cry3Bb1 gene used in line 4114 is the Bt variety aizawai.Studies on mammals, particularly laboratory animals, demonstrate that B. thuringiensis is mostly non-pathogenic and non-toxic. B. thuringiensis has been demonstrated to be highly specific in its insecticidal activity and has demonstrated little, if any, direct toxicity to non-target insects ADDIN REFMGR.CITE <Refman><Cite><Author>NPTN</Author><Year>2000</Year><RecNum>361</RecNum><IDText>Bacillus thuringiensis (Technical Fact Sheet)</IDText><Prefix>see </Prefix><MDL Ref_Type="Pamphlet"><Ref_Type>Pamphlet</Ref_Type><Ref_ID>361</Ref_ID><Title_Primary><i>Bacillus thuringiensis</i> (Technical Fact Sheet)</Title_Primary><Authors_Primary>NPTN</Authors_Primary><Date_Primary>2000</Date_Primary><Keywords>Bacillus</Keywords><Keywords>Bacillus thuringiensis</Keywords><Reprint>In File</Reprint><Publisher>National Pesticide Telecommunications Network, Oregon State University & U.S. Environmental Protection Agency</Publisher><Web_URL><u>:\References\GM References_in RefMan<u>\NPTN_2000_Bt fact sheet.pdf</u></Web_URL_Link2><ZZ_WorkformID>21</ZZ_WorkformID></MDL></Cite><Cite><Author>OECD</Author><Year>2007</Year><RecNum>362</RecNum><IDText>Consensus document on safety information on transgenic plants expressing Bacillus thuringiensis - derived insect control proteins</IDText><Suffix> and references therein</Suffix><MDL Ref_Type="Report"><Ref_Type>Report</Ref_Type><Ref_ID>362</Ref_ID><Title_Primary>Consensus document on safety information on transgenic plants expressing <i>Bacillus thuringiensis</i> - derived insect control proteins</Title_Primary><Authors_Primary>OECD</Authors_Primary><Date_Primary>2007</Date_Primary><Keywords>Safety</Keywords><Keywords>Plants</Keywords><Keywords>Bacillus</Keywords><Keywords>Bacillus thuringiensis</Keywords><Keywords>Insect Control</Keywords><Keywords>Proteins</Keywords><Reprint>In File</Reprint><Volume>ENV/JM/MONO(2007)14</Volume><Publisher>Organisation for Economic Co-operation and Development</Publisher><Web_URL><u>(2007)14&docLanguage=En</u></Web_URL><Web_URL_Link2>file://Y:\References\GM References_in RefMan<u>\OECD_2007_Bt safety.pdf</u></Web_URL_Link2><ZZ_WorkformID>24</ZZ_WorkformID></MDL></Cite></Refman>(see NPTN 2000; OECD 2007 and references therein). Infection in humans is unusual although there have been at least two clinical reports, one in the wounds of a soldier ADDIN REFMGR.CITE <Refman><Cite><Author>Hernandez</Author><Year>1998</Year><RecNum>34</RecNum><IDText>Bacillus thuringiensis subsp. konkukian (serotype H34) superinfection: case report and experimental evidence of pathogenicity in immunosuppressed mice</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>34</Ref_ID><Title_Primary><i>Bacillus thuringiensis</i> subsp. <i>konkukian</i> (serotype H34) superinfection: case report and experimental evidence of pathogenicity in immunosuppressed mice</Title_Primary><Authors_Primary>Hernandez,E.</Authors_Primary><Authors_Primary>Ramisse,F.</Authors_Primary><Authors_Primary>Ducoureau,J.-P.</Authors_Primary><Authors_Primary>Cruel,T.</Authors_Primary><Authors_Primary>Cavallo,J.-D.</Authors_Primary><Date_Primary>1998</Date_Primary><Keywords>Bacillus</Keywords><Keywords>Bacillus thuringiensis</Keywords><Keywords>Mice</Keywords><Reprint>In File</Reprint><Start_Page>2138</Start_Page><End_Page>2139</End_Page><Periodical>Journal of Clinical Microbiology</Periodical><Volume>36</Volume><Web_URL_Link2>file://Y:\References\GM References_in RefMan<u>\Hernandez et al_1998_Bt infection.pdf</u></Web_URL_Link2><ZZ_JournalFull><f name="System">Journal of Clinical Microbiology</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(Hernandez et al. 1998) and one in burn wounds ADDIN REFMGR.CITE <Refman><Cite><Author>Damgaard</Author><Year>1997</Year><RecNum>35</RecNum><IDText>Characterization of Bacillus thuringiensis isolated from infections in burn wounds</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>35</Ref_ID><Title_Primary>Characterization of <i>Bacillus thuringiensis</i> isolated from infections in burn wounds</Title_Primary><Authors_Primary>Damgaard,P.H.</Authors_Primary><Authors_Primary>Granum,P.E.</Authors_Primary><Authors_Primary>Bresciani,J.</Authors_Primary><Authors_Primary>Torregrossa,M.V.</Authors_Primary><Authors_Primary>Eilenberg,J.</Authors_Primary><Authors_Primary>Valentino,L.</Authors_Primary><Date_Primary>1997</Date_Primary><Keywords>Bacillus</Keywords><Keywords>Bacillus thuringiensis</Keywords><Keywords>Aedes</Keywords><Keywords>Enterotoxins</Keywords><Reprint>In File</Reprint><Start_Page>47</Start_Page><End_Page>53</End_Page><Periodical>FEMS Immunology & Medical Microbiology</Periodical><Volume>18</Volume><ZZ_JournalFull><f name="System">FEMS Immunology & Medical Microbiology</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(Damgaard et al. 1997), and in both cases impaired immunosuppression was implicated in the cause of the infection. B. thuringiensis has also been rarely associated with gastroenteritis ADDIN REFMGR.CITE <Refman><Cite><Author>Jackson</Author><Year>1995</Year><RecNum>36</RecNum><IDText>Bacillus cereus and Bacillus thuringiensis isolated in a gastroeneritis outbreak investigation</IDText><Prefix>see eg </Prefix><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>36</Ref_ID><Title_Primary><i>Bacillus cereus</i> and <i>Bacillus thuringiensis</i> isolated in a gastroeneritis outbreak investigation</Title_Primary><Authors_Primary>Jackson,S.G.</Authors_Primary><Authors_Primary>Goodbrand,R.B.</Authors_Primary><Authors_Primary>Ahmed,R.</Authors_Primary><Authors_Primary>Kasatiya,S.</Authors_Primary><Date_Primary>1995</Date_Primary><Keywords>Bacillus</Keywords><Keywords>Bacillus thuringiensis</Keywords><Reprint>In File</Reprint><Start_Page>103</Start_Page><End_Page>105</End_Page><Periodical>Letters in Applied Microbiology</Periodical><Volume>21</Volume><ZZ_JournalFull><f name="System">Letters in Applied Microbiology</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(see eg Jackson et al. 1995) but generally, B. thuringiensis present in drinking water or food has not been reported to cause adverse effects on human health PFJlZm1hbj48Q2l0ZT48QXV0aG9yPk5QVE48L0F1dGhvcj48WWVhcj4yMDAwPC9ZZWFyPjxSZWNO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ADDIN REFMGR.CITE PFJlZm1hbj48Q2l0ZT48QXV0aG9yPk5QVE48L0F1dGhvcj48WWVhcj4yMDAwPC9ZZWFyPjxSZWNO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ADDIN EN.CITE.DATA (WHO 1999; NPTN 2000; OECD 2007).The effect of B. thuringiensis products on human health and the environment was the subject of a critical review by the WHO International Programme on Chemical Safety ADDIN REFMGR.CITE <Refman><Cite><Author>WHO</Author><Year>1999</Year><RecNum>161</RecNum><IDText>Microbial pest control agent Bacillus thuringiensis.</IDText><MDL Ref_Type="Serial (Book,Monograph)"><Ref_Type>Serial (Book,Monograph)</Ref_Type><Ref_ID>161</Ref_ID><Title_Primary>Microbial pest control agent <i>Bacillus thuringiensis</i>.</Title_Primary><Authors_Primary>WHO</Authors_Primary><Date_Primary>1999</Date_Primary><Keywords>Bacillus thuringiensis</Keywords><Reprint>In File</Reprint><Issue>217</Issue><Pub_Place>Geneva</Pub_Place><Publisher>World Health Organization</Publisher><Title_Series>Environmental Health Criteria</Title_Series><ZZ_WorkformID>25</ZZ_WorkformID></MDL></Cite></Refman>(WHO 1999). The review concluded that ‘B. thuringiensis products are unlikely to pose any hazard to humans or other vertebrates or the great majority of non-target invertebrates provided that they are free from non-B. thuringiensis microorganisms and biologically active products other than the insecticidal proteins’. Approved GM crops incorporating Bt Cry proteins have been available for a number of years and have not raised any food safety concerns ADDIN REFMGR.CITE <Refman><Cite><Author>Koch</Author><Year>2015</Year><RecNum>1817</RecNum><IDText>The food and environmental safety of Bt crops</IDText><MDL Ref_Type="Journal (Full)"><Ref_Type>Journal (Full)</Ref_Type><Ref_ID>1817</Ref_ID><Title_Primary>The food and environmental safety of Bt crops</Title_Primary><Authors_Primary>Koch,M.S.</Authors_Primary><Authors_Primary>Ward,J.M.</Authors_Primary><Authors_Primary>Levine,S.L.</Authors_Primary><Authors_Primary>Baum,J.A.</Authors_Primary><Authors_Primary>Vicini,J.L.</Authors_Primary><Authors_Primary>Hammond,B.G.</Authors_Primary><Date_Primary>2015</Date_Primary><Keywords>Safety</Keywords><Keywords>Bt</Keywords><Keywords>Bacillus</Keywords><Keywords>Bacillus thuringiensis</Keywords><Keywords>Pesticides</Keywords><Keywords>Proteins</Keywords><Keywords>Genes</Keywords><Keywords>Biotechnology</Keywords><Keywords>Mammals</Keywords><Keywords>Safety assessment</Keywords><Keywords>unintended effects</Keywords><Keywords>adverse effects</Keywords><Keywords>Plants</Keywords><Keywords>Humans</Keywords><Keywords>Human</Keywords><Reprint>Not in File</Reprint><Periodical>Frontiers in Plant Science</Periodical><Volume>6:283</Volume><Misc_2><f name="TimesNewRomanPS-BoldItalicMT">doi:10.3389/fpls.2015.00283</f></Misc_2><Web_URL_Link2>;\References\GM References_in RefMan\Koch et al_2015_safety of Bt crops.pdf</u></Web_URL_Link2><ZZ_JournalFull><f name="System">Frontiers in Plant Science</f></ZZ_JournalFull><ZZ_WorkformID>32</ZZ_WorkformID></MDL></Cite></Refman>(Koch et al. 2015).Commercial Bt products are powders containing a mixture of dried spores and toxin crystals. Such products are approved for use on crops in Australia and New Zealand and in both countries there is an exemption from maximum residue limits (MRLs) when Bt is used as an insecticide.2.2.2Streptomyces viridochromogenesThe source of the pat gene is the bacterial species Streptomyces viridochromogenes, strain Tü494 ADDIN REFMGR.CITE <Refman><Cite><Author>Wohlleben</Author><Year>1988</Year><RecNum>351</RecNum><IDText>Nucleotide sequence of the phosphinothricin N-acetyltransferase gene from Streptomyces viridochromogenes Tü494 and its expression in Nicotiana tabacum</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>351</Ref_ID><Title_Primary>Nucleotide sequence of the phosphinothricin N-acetyltransferase gene from <i>Streptomyces viridochromogenes</i> Tü494 and its expression in <i>Nicotiana tabacum</i></Title_Primary><Authors_Primary>Wohlleben,W.</Authors_Primary><Authors_Primary>Arnold,W.</Authors_Primary><Authors_Primary>Broer,I.</Authors_Primary><Authors_Primary>Hillemann,D.</Authors_Primary><Authors_Primary>Strauch,E.</Authors_Primary><Authors_Primary>Punier,A.</Authors_Primary><Date_Primary>1988</Date_Primary><Keywords>Amino Acids</Keywords><Keywords>Codon</Keywords><Keywords>Escherichia coli</Keywords><Keywords>Genes</Keywords><Keywords>Plants</Keywords><Reprint>In File</Reprint><Start_Page>25</Start_Page><End_Page>37</End_Page><Periodical>Gene</Periodical><Volume>70</Volume><ZZ_JournalStdAbbrev><f name="System">Gene</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(Wohlleben et al. 1988). The Streptomycetae bacteria were first described in the early 1900’s. These organisms are generally soil-borne, although they may also be isolated from water. They are not typically pathogenic to animals including humans, and few species have been shown to be phytopathogenic ADDIN REFMGR.CITE <Refman><Cite><Author>Bradbury</Author><Year>1986</Year><RecNum>346</RecNum><IDText>Guide to plant pathogenic bacteria</IDText><MDL Ref_Type="Book, Whole"><Ref_Type>Book, Whole</Ref_Type><Ref_ID>346</Ref_ID><Title_Primary>Guide to plant pathogenic bacteria</Title_Primary><Authors_Primary>Bradbury,J.F.</Authors_Primary><Date_Primary>1986</Date_Primary><Keywords>Bacteria</Keywords><Reprint>Not in File</Reprint><Start_Page>190</Start_Page><End_Page>197</End_Page><Pub_Place>Aberystwyth</Pub_Place><Publisher>Cambridge News Ltd</Publisher><ZZ_WorkformID>2</ZZ_WorkformID></MDL></Cite><Cite><Author>Kützner</Author><Year>1981</Year><RecNum>347</RecNum><IDText>The Family Streptomycetaceae</IDText><MDL Ref_Type="Book Chapter"><Ref_Type>Book Chapter</Ref_Type><Ref_ID>347</Ref_ID><Title_Primary>The Family Streptomycetaceae</Title_Primary><Authors_Primary>Kützner,H.J.</Authors_Primary><Date_Primary>1981</Date_Primary><Keywords>Bacteria</Keywords><Reprint>Not in File</Reprint><Start_Page>2028</Start_Page><End_Page>2090</End_Page><Title_Secondary>The Prokaryotes: A Handbook on Habitats, Isolation and Identification of Bacteria</Title_Secondary><Authors_Secondary>Starr,M.P.</Authors_Secondary><Authors_Secondary>Stolp,H.</Authors_Secondary><Authors_Secondary>Trüper,H.G.</Authors_Secondary><Authors_Secondary>Ballows,A.</Authors_Secondary><Authors_Secondary>Schlegel,H.G.</Authors_Secondary><Pub_Place>Berlin</Pub_Place><Publisher>Springer Verlag</Publisher><ZZ_WorkformID>3</ZZ_WorkformID></MDL></Cite></Refman>(Kützner 1981; Bradbury 1986). Although these organisms are not used in the food industry, the pat gene from S. viridochromogenes, has been used to confer glufosinate ammonium-tolerance in a range of food producing crops. The bar gene from the closely related S. hygroscopicus produces a protein that is structurally and functionally equivalent to the protein encoded by the pat gene ADDIN REFMGR.CITE <Refman><Cite><Author>Wehrmann</Author><Year>1996</Year><RecNum>317</RecNum><IDText>The similarities of bar and pat gene products make them equally applicable for plant engineers</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>317</Ref_ID><Title_Primary>The similarities of <i>bar</i> and <i>pat</i> gene products make them equally applicable for plant engineers</Title_Primary><Authors_Primary>Wehrmann,A.</Authors_Primary><Authors_Primary>Van Vliet,A.</Authors_Primary><Authors_Primary>Opsomer,C.</Authors_Primary><Authors_Primary>Botterman,J.</Authors_Primary><Authors_Primary>Schulz,A.</Authors_Primary><Date_Primary>1996</Date_Primary><Keywords>Soybeans</Keywords><Keywords>Genes</Keywords><Keywords>Genetic Engineering</Keywords><Keywords>Escherichia coli</Keywords><Keywords>Proteins</Keywords><Keywords>Plants</Keywords><Keywords>Molecular Weight</Keywords><Keywords>Digestion</Keywords><Keywords>Human</Keywords><Reprint>In File</Reprint><Start_Page>1274</Start_Page><End_Page>1278</End_Page><Periodical>Nature Biotechnology</Periodical><Volume>14</Volume><Web_URL_Link2>file://Y:\References\GM References_in RefMan<u>\Wehrmann et al_1996_bar and pat.pdf</u></Web_URL_Link2><ZZ_JournalFull><f name="System">Nature Biotechnology</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(Wehrmann et al. 1996) and has similarly been used widely for genetic modification of crop species. 2.2.3Other organismsGenetic elements from several other organisms have been used in the genetic modification of line 4114 (refer to Table 1). These non-coding sequences are used to drive, enhance, target or terminate expression of the novel genetic material. None of the sources of these genetic elements is associated with toxic or allergenic responses in humans. The genetic elements derived from plant pathogens are not pathogenic in themselves and do not cause pathogenic symptoms in line 4114.3Molecular characterisationMolecular characterisation is necessary to provide an understanding of the genetic material introduced into the host genome and helps to frame the subsequent parts of the safety assessment. The molecular characterisation addresses three main aspects:the transformation method together with a detailed description of the DNA sequences introduced to the host genome a characterisation of the inserted DNA including any rearrangements that may have occurred as a consequence of the transformation the genetic stability of the inserted DNA and any accompanying expressed traits.Studies submitted:2011. Characterization of DP-??4114-3 Maize: Insertion Integrity, Stability, Copy Number, and Backbone Analysis. Study ID PHI-2009-015. Pioneer Hi-Bred International, Inc (unpublished).2011. Maize Event DP-004114-3 and Methods for Detection Thereof. Patent No. US2011/0154523 A1. . Segregation Analysis of Five Generations of a Maize Line Containing Event DP-004114-3. Study ID PHI-2010-197/010. Pioneer Hi-Bred International, Inc (unpublished)2015. Junction Reading Frame Analysis at the Insertion Site of maize Event DP-004114-3. Study ID PHI-R036-Y15. Pioneer Hi-Bred International, Inc (unpublished) 3.1Method used in the genetic modificationImmature embryos from line PHWWE were aseptically removed from 9 – 11 day post-pollination ears and transformed, using a disarmed strain of Agrobacterium tumefaciens (LBA4404), with the T-DNA from plasmid vector PHP27118 (see REF _Ref261348424 \h \* MERGEFORMAT Figure 2) following the method of Zhao et al ADDIN REFMGR.CITE <Refman><Cite ExcludeAuth="1"><Author>Zhao</Author><Year>2001</Year><RecNum>1701</RecNum><IDText>High throughput genetic transformation mediated by Agrobacterium tumefaciens in maize</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1701</Ref_ID><Title_Primary>High throughput genetic transformation mediated by <i>Agrobacterium tumefaciens</i> in maize</Title_Primary><Authors_Primary>Zhao,Z-y</Authors_Primary><Authors_Primary>Gu,W.</Authors_Primary><Authors_Primary>Cai,T.</Authors_Primary><Authors_Primary>Tagliani,L.</Authors_Primary><Authors_Primary>Hondred,D.</Authors_Primary><Authors_Primary>Bond,D.</Authors_Primary><Authors_Primary>Schroeder,S.</Authors_Primary><Authors_Primary>Rudert,M.</Authors_Primary><Authors_Primary>Pierce,D.</Authors_Primary><Date_Primary>2001</Date_Primary><Keywords>agrobacterium</Keywords><Keywords>maize</Keywords><Keywords>Bacteria</Keywords><Keywords>Plants</Keywords><Reprint>Not in File</Reprint><Start_Page>323</Start_Page><End_Page>333</End_Page><Periodical>Molecular Breeding</Periodical><Volume>8</Volume><Web_URL_Link2>;\References\GM References_in RefMan\Zhao et al_2001_Agrobacterium-mediated maize transformation.docx</u></Web_URL_Link2><ZZ_JournalFull><f name="System">Molecular Breeding</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(2001)After co-culturing with the Agrobacterium carrying the vector, the embryos were placed on two sequential selection media - firstly containing carbenicillin (to inhibit the growth of untransformed plant cells and excess Agrobacterium) and secondly containing plant hormones (to stimulate somatic embryogenesis) and bialaphos (glufosinate - to select for expression of the pat gene). Rooted plantlets (generation T0) with normal phenotypic characteristics, polymerase chain reaction (PCR)-verified presence of the four novel genes, confirmed presence of a single copy of the T-DNA, and negative for Agrobacterium backbone sequences were selected and transferred to soil for growth and further assessment following crossing to inbred lines. Ultimately, line 4114 was selected as the lead event.Figure SEQ Figure \* ARABIC 2: Genes and regulatory elements contained in plasmid PHP271183.2Function and regulation of introduced genesA diagram of the T-DNA insert in plasmid PHP27118 is given in Figure 3. Figure SEQ Figure \* ARABIC 3: Representation of the genetic elements in the T-DNA insert of plasmid PHP27118Information on the genetic elements in the T-DNA insert present in line 4114 is summarised in REF _Ref265059500 \h \* MERGEFORMAT Table 1. There are four cassettes plus non-functional Ti sequences comprising a total of 11,928 base pairs (bp) located between a 25 bp Left Border (LB) and a 25 bp Right Border (RB). The complete plasmid is 54,910 bp in size (i.e. the vector backbone comprises 42,932 bp). The coding regions and regulatory elements are the same as those found in the breeding stack obtained by crossing lines 1507 and 59122 (1507 x 59122).Table SEQ Table \* ARABIC 1: Description of the genetic elements contained in the T-DNA of PHP27118Genetic elementbp location on plasmidSize (bp)SourceOrient.Description & FunctionReferencesRIGHT BORDER1 - 2525Ti plasmid region26 - 4318Non-functional sequencecry1F cassetteIntervening sequence44 - 11471ubiZM1115 - 21071993Zea mays (corn)Anti-clockwisePromoter region from the polyubiquiton gene (including 5’UTR and intron)Directs transcription of the cry1F geneChristensen et al. ADDIN REFMGR.CITE <Refman><Cite ExcludeAuth="1"><Author>Christensen</Author><Year>1992</Year><RecNum>1778</RecNum><IDText>Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1778</Ref_ID><Title_Primary>Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation</Title_Primary><Authors_Primary>Christensen,A.H.</Authors_Primary><Authors_Primary>Sharrock,R.A.</Authors_Primary><Authors_Primary>Quail,P.H.</Authors_Primary><Date_Primary>1992/2</Date_Primary><Keywords>Amino Acid Sequence</Keywords><Keywords>Base Sequence</Keywords><Keywords>Blotting,Northern</Keywords><Keywords>Cells,Cultured</Keywords><Keywords>Chimera</Keywords><Keywords>Cloning,Molecular</Keywords><Keywords>Dna</Keywords><Keywords>Gene Expression Regulation</Keywords><Keywords>genetics</Keywords><Keywords>metabolism</Keywords><Keywords>Molecular Sequence Data</Keywords><Keywords>Polymers</Keywords><Keywords>Polyubiquitin</Keywords><Keywords>Promoter Regions (Genetics)</Keywords><Keywords>Protoplasts</Keywords><Keywords>Restriction Mapping</Keywords><Keywords>RNA Splicing</Keywords><Keywords>Support,Non-U.'t</Keywords><Keywords>Support,U.'t,P.H.S.</Keywords><Keywords>Temperature</Keywords><Keywords>Ubiquitins</Keywords><Keywords>Zea mays</Keywords><Reprint>In File</Reprint><Start_Page>675</Start_Page><End_Page>689</End_Page><Periodical>Plant Molecular Biology</Periodical><Volume>18</Volume><Issue>4</Issue><Address>UC-Berkeley/USDA Plant Gene Expression Center, Albany 94710</Address><Web_URL>PM:1313711</Web_URL><ZZ_JournalFull><f name="System">Plant Molecular Biology</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(1992)Intervening sequence2108 - 212922cry1F2130 - 39471818Bacillus thuringiensis var aizawaiAnti-clockwiseCodon optimised synthetic and truncated version of the coding region of the cry1F gene Chambers ADDIN REFMGR.CITE <Refman><Cite ExcludeAuth="1"><Author>Chambers</Author><Year>1991</Year><RecNum>10</RecNum><IDText>Isolation and characterisation of a novel insecticidal crystal protein gene from Bacillus thuringiensis subsp. aizawai</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>10</Ref_ID><Title_Primary>Isolation and characterisation of a novel insecticidal crystal protein gene from <i>Bacillus thuringiensis</i> subsp. <i>aizawai</i></Title_Primary><Authors_Primary>Chambers,J.A.</Authors_Primary><Authors_Primary>Jelen,A.</Authors_Primary><Authors_Primary>Pearce,G.M.</Authors_Primary><Authors_Primary>Jany,C.S.</Authors_Primary><Authors_Primary>Johnson,T.B.</Authors_Primary><Authors_Primary>Gawron-Burke,C.</Authors_Primary><Date_Primary>1991</Date_Primary><Keywords>Bacillus</Keywords><Keywords>Bacillus thuringiensis</Keywords><Reprint>Not in File</Reprint><Start_Page>3966</Start_Page><End_Page>3976</End_Page><Periodical>Journal of Bacteriology</Periodical><Volume>173</Volume><Issue>13</Issue><Web_URL_Link2>;\References\GM References_in RefMan\Chambers et al_1991_Cry1F subsp aizawai.pdf</u></Web_URL_Link2><ZZ_JournalFull><f name="System">Journal of Bacteriology</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(1991)Intervening sequence3948 - 399245ORF253993 - 4706714Agrobacterium tumefaciens pTi15955Anti-clockwiseTranscriptional terminator and polyadenylation site of open reading frame 25Barker et al. ADDIN REFMGR.CITE <Refman><Cite ExcludeAuth="1"><Author>Barker</Author><Year>1983</Year><RecNum>1779</RecNum><IDText>Nucleotide sequence of the T-DNA region from the Agrobacterium tumefaciens octopine Ti plasmid pTi15955</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1779</Ref_ID><Title_Primary>Nucleotide sequence of the T-DNA region from the <i>Agrobacterium tumefaciens</i> octopine Ti plasmid pTi15955</Title_Primary><Authors_Primary>Barker,R.F.</Authors_Primary><Authors_Primary>Idler,K.B.</Authors_Primary><Authors_Primary>Thompson D.V.</Authors_Primary><Authors_Primary>Kemp,J.D.</Authors_Primary><Date_Primary>1983</Date_Primary><Keywords>agrobacterium</Keywords><Reprint>Not in File</Reprint><Start_Page>335</Start_Page><End_Page>350</End_Page><Periodical>Plant Molecular Biology</Periodical><Volume>2</Volume><Web_URL_Link2>file://Y:\References\GM References_in RefMan<u>\Barker et al_1983_T-DNA_partial paper.docx</u></Web_URL_Link2><ZZ_JournalFull><f name="System">Plant Molecular Biology</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(1983)cry34Ab1 cassetteIntervening sequence4707 - 476559ubiZM14766 - 67581993Zea mays (corn)Anti-clockwisePromoter region from the polyubiquiton gene (including 5’UTR and intron)Directs transcription of the cry34Ab1 geneChristensen et al. ADDIN REFMGR.CITE <Refman><Cite ExcludeAuth="1"><Author>Christensen</Author><Year>1992</Year><RecNum>1778</RecNum><IDText>Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1778</Ref_ID><Title_Primary>Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation</Title_Primary><Authors_Primary>Christensen,A.H.</Authors_Primary><Authors_Primary>Sharrock,R.A.</Authors_Primary><Authors_Primary>Quail,P.H.</Authors_Primary><Date_Primary>1992/2</Date_Primary><Keywords>Amino Acid Sequence</Keywords><Keywords>Base Sequence</Keywords><Keywords>Blotting,Northern</Keywords><Keywords>Cells,Cultured</Keywords><Keywords>Chimera</Keywords><Keywords>Cloning,Molecular</Keywords><Keywords>Dna</Keywords><Keywords>Gene Expression Regulation</Keywords><Keywords>genetics</Keywords><Keywords>metabolism</Keywords><Keywords>Molecular Sequence Data</Keywords><Keywords>Polymers</Keywords><Keywords>Polyubiquitin</Keywords><Keywords>Promoter Regions (Genetics)</Keywords><Keywords>Protoplasts</Keywords><Keywords>Restriction Mapping</Keywords><Keywords>RNA Splicing</Keywords><Keywords>Support,Non-U.'t</Keywords><Keywords>Support,U.'t,P.H.S.</Keywords><Keywords>Temperature</Keywords><Keywords>Ubiquitins</Keywords><Keywords>Zea mays</Keywords><Reprint>In File</Reprint><Start_Page>675</Start_Page><End_Page>689</End_Page><Periodical>Plant Molecular Biology</Periodical><Volume>18</Volume><Issue>4</Issue><Address>UC-Berkeley/USDA Plant Gene Expression Center, Albany 94710</Address><Web_URL>PM:1313711</Web_URL><ZZ_JournalFull><f name="System">Plant Molecular Biology</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(1992)Intervening sequence6759 - 678628cry34Ab16787 - 7158372Bacillus thuringiensis non-motile strain PS149E1Anti-clockwiseSynthesised, codon-optimized version of the cry34Ab1 gene encoding the 14 kDa delta-endotoxin parasporal crystal proteinEllis et al. ADDIN REFMGR.CITE <Refman><Cite ExcludeAuth="1"><Author>Ellis</Author><Year>2002</Year><RecNum>1703</RecNum><IDText>Novel Bacillus thuringiensis binary insecticidal crystal proteins active on western corn rootworm, Diabrotica virgifera virgifera Le Conte</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1703</Ref_ID><Title_Primary>Novel <i>Bacillus thuringiensis</i> binary insecticidal crystal proteins active on western corn rootworm, <i>Diabrotica virgifera virgifera</i> Le Conte</Title_Primary><Authors_Primary>Ellis,R.T.</Authors_Primary><Authors_Primary>Stockhoff,B.A.</Authors_Primary><Authors_Primary>Stamp,L.</Authors_Primary><Authors_Primary>Schnepf,H.E.</Authors_Primary><Authors_Primary>Schwab,G.E.</Authors_Primary><Authors_Primary>Knuth,M.</Authors_Primary><Authors_Primary>Russell,J.</Authors_Primary><Authors_Primary>Cardineau,G.A.</Authors_Primary><Authors_Primary>Narva,K.E.</Authors_Primary><Date_Primary>2002</Date_Primary><Keywords>Bacillus</Keywords><Keywords>Bacillus thuringiensis</Keywords><Keywords>Proteins</Keywords><Keywords>Genes</Keywords><Keywords>Operon</Keywords><Keywords>Mutagenesis</Keywords><Keywords>Mortality</Keywords><Reprint>Not in File</Reprint><Start_Page>1137</Start_Page><End_Page>1145</End_Page><Periodical>Applied and Environmental Microbiology</Periodical><Volume>68</Volume><Issue>3</Issue><Web_URL_Link2>;\References\GM References_in RefMan\Ellis et al_2002_cry34 </u>and<u> cry35.pdf</u></Web_URL_Link2><ZZ_JournalFull><f name="System">Applied and Environmental Microbiology</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(2002); Herman et al. ADDIN REFMGR.CITE <Refman><Cite ExcludeAuth="1"><Author>Herman</Author><Year>2002</Year><RecNum>1704</RecNum><IDText>Binary insecticidal crystal protein from Bacillus thuringiensis, strain PS149B1: effects of individual protein components and mixtures in laboratory bioassays </IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1704</Ref_ID><Title_Primary>Binary insecticidal crystal protein from Bacillus thuringiensis, strain PS149B1: effects of individual protein components and mixtures in laboratory bioassays<f name="AdvMT_SY"> </f></Title_Primary><Authors_Primary>Herman,R.A.</Authors_Primary><Authors_Primary>Scherer,P.N.</Authors_Primary><Authors_Primary>Young,D.L.</Authors_Primary><Authors_Primary>Mihaliak,C.A.</Authors_Primary><Authors_Primary>Meade,T.</Authors_Primary><Authors_Primary>Woodsworth,A.T.</Authors_Primary><Authors_Primary>Stockhoff,B.A.</Authors_Primary><Authors_Primary>Narva,K.E.</Authors_Primary><Date_Primary>2002</Date_Primary><Keywords>Bacillus</Keywords><Keywords>Bacillus thuringiensis</Keywords><Keywords>Proteins</Keywords><Keywords>maize</Keywords><Keywords>Plants</Keywords><Keywords>Zea mays</Keywords><Reprint>Not in File</Reprint><Start_Page>635</Start_Page><End_Page>639</End_Page><Periodical>Journal of Economic Entomology</Periodical><Volume>95</Volume><Issue>3</Issue><Web_URL_Link2>;\References\GM References_in RefMan\Herman et al_2002_Cry34Ab1 </u>and<u> Cry35Ab1.PDF</u></Web_URL_Link2><ZZ_JournalFull><f name="System">Journal of Economic Entomology</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(2002); Moellenbeck et al. PFJlZm1hbj48Q2l0ZSBFeGNsdWRlQXV0aD0iMSI+PEF1dGhvcj5Nb2VsbGVuYmVjazwvQXV0aG9y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 REFMGR.CITE PFJlZm1hbj48Q2l0ZSBFeGNsdWRlQXV0aD0iMSI+PEF1dGhvcj5Nb2VsbGVuYmVjazwvQXV0aG9y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 EN.CITE.DATA (2001)Intervening sequence7159 - 718224pinII7183 - 7492310Solanum tuberosum (potato)Anti-clockwiseTerminator region from the proteinase inhibitor II geneDirects polyadenylation of the cry34Ab1 geneAn et al. ADDIN REFMGR.CITE <Refman><Cite ExcludeAuth="1"><Author>An</Author><Year>1989</Year><RecNum>1781</RecNum><IDText>Functional analysis of the 3' control region of the potato wound-inducible proteinase inhibitor II gene</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1781</Ref_ID><Title_Primary>Functional analysis of the 3' control region of the potato wound-inducible proteinase inhibitor II gene</Title_Primary><Authors_Primary>An,G.</Authors_Primary><Authors_Primary>Mitra,A.</Authors_Primary><Authors_Primary>Choi,H.K.</Authors_Primary><Authors_Primary>Costa,M.A.</Authors_Primary><Authors_Primary>An,K.</Authors_Primary><Authors_Primary>Thornburg,R.W.</Authors_Primary><Authors_Primary>Ryan,C.A.</Authors_Primary><Date_Primary>1989/1</Date_Primary><Keywords>Amino Acid Sequence</Keywords><Keywords>Base Sequence</Keywords><Keywords>Dna</Keywords><Keywords>Gene Expression Regulation</Keywords><Keywords>genetics</Keywords><Keywords>metabolism</Keywords><Keywords>Molecular Sequence Data</Keywords><Keywords>Potatoes</Keywords><Keywords>Promoter Regions (Genetics)</Keywords><Keywords>Protease Inhibitors</Keywords><Keywords>Regulatory Sequences,Nucleic Acid</Keywords><Keywords>RNA,Messenger</Keywords><Keywords>Support,Non-U.'t</Keywords><Keywords>Support,U.'t,Non-P.H.S.</Keywords><Keywords>Terminator Regions (Genetics)</Keywords><Reprint>Not in File</Reprint><Start_Page>115</Start_Page><End_Page>122</End_Page><Periodical>Plant Cell</Periodical><Volume>1</Volume><Issue>1</Issue><Address>Institute of Biological Chemistry, Washington State University, Pullman 99164-6340</Address><Web_URL>PM:2535459</Web_URL><Web_URL_Link2>;\References\GM References_in RefMan\An et al_1989_potato pinII gene.pdf</u></Web_URL_Link2><ZZ_JournalStdAbbrev><f name="System">Plant Cell</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(1989);Keil et al. ADDIN REFMGR.CITE <Refman><Cite ExcludeAuth="1"><Author>Keil</Author><Year>1986</Year><RecNum>183</RecNum><IDText>Primary structure of a proteinase inhibitor II gene from potato (Solanum tuberosum)</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>183</Ref_ID><Title_Primary>Primary structure of a proteinase inhibitor II gene from potato (Solanum tuberosum)</Title_Primary><Authors_Primary>Keil,M.</Authors_Primary><Authors_Primary>Sanchez-Serrano,J.</Authors_Primary><Authors_Primary>Schell,J.</Authors_Primary><Authors_Primary>Willmitzer,L.</Authors_Primary><Date_Primary>1986/7/25</Date_Primary><Keywords>Amino Acid Sequence</Keywords><Keywords>Amino Acids</Keywords><Keywords>Base Sequence</Keywords><Keywords>Dna</Keywords><Keywords>DNA Restriction Enzymes</Keywords><Keywords>Genes</Keywords><Keywords>genetics</Keywords><Keywords>Plant Proteins</Keywords><Keywords>Plants</Keywords><Keywords>Protease Inhibitors</Keywords><Keywords>Proteins</Keywords><Keywords>Solanum tuberosum</Keywords><Reprint>Not in File</Reprint><Start_Page>5641</Start_Page><End_Page>5650</End_Page><Periodical>Nucleic Acids Research</Periodical><Volume>14</Volume><Issue>14</Issue><Web_URL>PM:3016659</Web_URL><Web_URL_Link2><u>file://F:\FSANZ Common\GMO\References\GM References_in RefMan\Keil et al_1986_potato pinII gene.pdf</u></Web_URL_Link2><ZZ_JournalFull><f name="System">Nucleic Acids Research</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(1986)cry35Ab1 cassetteIntervening sequence7493 - 752230TA peroxidase7523 - 88201298Triticum aestivum (wheat)Anti-clockwisePromoter and leader sequence from the peroxidase geneDirects transcription of the cry35Ab1 geneHertig et al. ADDIN REFMGR.CITE <Refman><Cite><Author>Hertig</Author><Year>1991</Year><RecNum>22</RecNum><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>22</Ref_ID><Title_Primary>Sequence and tissue-specific expression of a putative peroxidase gene from wheat (Triticum aestivum L.)</Title_Primary><Authors_Primary>Hertig,C.</Authors_Primary><Authors_Primary>Rebmann,G.</Authors_Primary><Authors_Primary>Bull,J.</Authors_Primary><Authors_Primary>Mauch,F.</Authors_Primary><Authors_Primary>Dudler,R.</Authors_Primary><Date_Primary>1991/1</Date_Primary><Keywords>Amino Acid Sequence</Keywords><Keywords>Base Sequence</Keywords><Keywords>enzymology</Keywords><Keywords>Gene Expression</Keywords><Keywords>genetics</Keywords><Keywords>Introns</Keywords><Keywords>Molecular Sequence Data</Keywords><Keywords>Peroxidase</Keywords><Keywords>Peroxidases</Keywords><Keywords>Support,Non-U.'t</Keywords><Keywords>Transcription,Genetic</Keywords><Keywords>Triticum</Keywords><Reprint>Not in File</Reprint><Start_Page>171</Start_Page><End_Page>174</End_Page><Periodical>Plant Molecular Biology</Periodical><Volume>16</Volume><Issue>1</Issue><Address>Institute for Plant Biology, University of Zurich, Switzerland</Address><Web_URL>PM:1653627</Web_URL><Web_URL_Link2><u>file://F:\FSANZ Common\GMO\References\GM References_in RefMan\Hertig et al_1991_wheat peroxidase gene.PDF</u></Web_URL_Link2><ZZ_JournalFull><f name="System">Plant Molecular Biology</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(Hertig et al. 1991) Intervening sequence8821 - 883616cry35Ab18837 - 99881152Bacillus thuringiensis non-motile strain PS149E1Anti-clockwiseSynthesises, codon-optimized version of the cry35Ab1 gene encoding the 44 kDa delta-endotoxin parasporal crystal proteinEllis et al. ADDIN REFMGR.CITE <Refman><Cite ExcludeAuth="1"><Author>Ellis</Author><Year>2002</Year><RecNum>1703</RecNum><IDText>Novel Bacillus thuringiensis binary insecticidal crystal proteins active on western corn rootworm, Diabrotica virgifera virgifera Le Conte</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1703</Ref_ID><Title_Primary>Novel <i>Bacillus thuringiensis</i> binary insecticidal crystal proteins active on western corn rootworm, <i>Diabrotica virgifera virgifera</i> Le Conte</Title_Primary><Authors_Primary>Ellis,R.T.</Authors_Primary><Authors_Primary>Stockhoff,B.A.</Authors_Primary><Authors_Primary>Stamp,L.</Authors_Primary><Authors_Primary>Schnepf,H.E.</Authors_Primary><Authors_Primary>Schwab,G.E.</Authors_Primary><Authors_Primary>Knuth,M.</Authors_Primary><Authors_Primary>Russell,J.</Authors_Primary><Authors_Primary>Cardineau,G.A.</Authors_Primary><Authors_Primary>Narva,K.E.</Authors_Primary><Date_Primary>2002</Date_Primary><Keywords>Bacillus</Keywords><Keywords>Bacillus thuringiensis</Keywords><Keywords>Proteins</Keywords><Keywords>Genes</Keywords><Keywords>Operon</Keywords><Keywords>Mutagenesis</Keywords><Keywords>Mortality</Keywords><Reprint>Not in File</Reprint><Start_Page>1137</Start_Page><End_Page>1145</End_Page><Periodical>Applied and Environmental Microbiology</Periodical><Volume>68</Volume><Issue>3</Issue><Web_URL_Link2>;\References\GM References_in RefMan\Ellis et al_2002_cry34 </u>and<u> cry35.pdf</u></Web_URL_Link2><ZZ_JournalFull><f name="System">Applied and Environmental Microbiology</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(2002); Herman et al. ADDIN REFMGR.CITE <Refman><Cite ExcludeAuth="1"><Author>Herman</Author><Year>2002</Year><RecNum>1704</RecNum><IDText>Binary insecticidal crystal protein from Bacillus thuringiensis, strain PS149B1: effects of individual protein components and mixtures in laboratory bioassays </IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1704</Ref_ID><Title_Primary>Binary insecticidal crystal protein from Bacillus thuringiensis, strain PS149B1: effects of individual protein components and mixtures in laboratory bioassays<f name="AdvMT_SY"> </f></Title_Primary><Authors_Primary>Herman,R.A.</Authors_Primary><Authors_Primary>Scherer,P.N.</Authors_Primary><Authors_Primary>Young,D.L.</Authors_Primary><Authors_Primary>Mihaliak,C.A.</Authors_Primary><Authors_Primary>Meade,T.</Authors_Primary><Authors_Primary>Woodsworth,A.T.</Authors_Primary><Authors_Primary>Stockhoff,B.A.</Authors_Primary><Authors_Primary>Narva,K.E.</Authors_Primary><Date_Primary>2002</Date_Primary><Keywords>Bacillus</Keywords><Keywords>Bacillus thuringiensis</Keywords><Keywords>Proteins</Keywords><Keywords>maize</Keywords><Keywords>Plants</Keywords><Keywords>Zea mays</Keywords><Reprint>Not in File</Reprint><Start_Page>635</Start_Page><End_Page>639</End_Page><Periodical>Journal of Economic Entomology</Periodical><Volume>95</Volume><Issue>3</Issue><Web_URL_Link2>;\References\GM References_in RefMan\Herman et al_2002_Cry34Ab1 </u>and<u> Cry35Ab1.PDF</u></Web_URL_Link2><ZZ_JournalFull><f name="System">Journal of Economic Entomology</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(2002); Moellenbeck et al. PFJlZm1hbj48Q2l0ZSBFeGNsdWRlQXV0aD0iMSI+PEF1dGhvcj5Nb2VsbGVuYmVjazwvQXV0aG9y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 REFMGR.CITE PFJlZm1hbj48Q2l0ZSBFeGNsdWRlQXV0aD0iMSI+PEF1dGhvcj5Nb2VsbGVuYmVjazwvQXV0aG9y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 EN.CITE.DATA (2001)Intervening sequence9989 - 1001224pinII10013 - 10322310Solanum tuberosum (potato)Anti-clockwiseTerminator region from the proteinase inhibitor II geneDirects polyadenylation of the cry34Ab1 geneAn et al. ADDIN REFMGR.CITE <Refman><Cite ExcludeAuth="1"><Author>An</Author><Year>1989</Year><RecNum>1781</RecNum><IDText>Functional analysis of the 3' control region of the potato wound-inducible proteinase inhibitor II gene</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1781</Ref_ID><Title_Primary>Functional analysis of the 3' control region of the potato wound-inducible proteinase inhibitor II gene</Title_Primary><Authors_Primary>An,G.</Authors_Primary><Authors_Primary>Mitra,A.</Authors_Primary><Authors_Primary>Choi,H.K.</Authors_Primary><Authors_Primary>Costa,M.A.</Authors_Primary><Authors_Primary>An,K.</Authors_Primary><Authors_Primary>Thornburg,R.W.</Authors_Primary><Authors_Primary>Ryan,C.A.</Authors_Primary><Date_Primary>1989/1</Date_Primary><Keywords>Amino Acid Sequence</Keywords><Keywords>Base Sequence</Keywords><Keywords>Dna</Keywords><Keywords>Gene Expression Regulation</Keywords><Keywords>genetics</Keywords><Keywords>metabolism</Keywords><Keywords>Molecular Sequence Data</Keywords><Keywords>Potatoes</Keywords><Keywords>Promoter Regions (Genetics)</Keywords><Keywords>Protease Inhibitors</Keywords><Keywords>Regulatory Sequences,Nucleic Acid</Keywords><Keywords>RNA,Messenger</Keywords><Keywords>Support,Non-U.'t</Keywords><Keywords>Support,U.'t,Non-P.H.S.</Keywords><Keywords>Terminator Regions (Genetics)</Keywords><Reprint>Not in File</Reprint><Start_Page>115</Start_Page><End_Page>122</End_Page><Periodical>Plant Cell</Periodical><Volume>1</Volume><Issue>1</Issue><Address>Institute of Biological Chemistry, Washington State University, Pullman 99164-6340</Address><Web_URL>PM:2535459</Web_URL><Web_URL_Link2>;\References\GM References_in RefMan\An et al_1989_potato pinII gene.pdf</u></Web_URL_Link2><ZZ_JournalStdAbbrev><f name="System">Plant Cell</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(1989);Keil et al. ADDIN REFMGR.CITE <Refman><Cite ExcludeAuth="1"><Author>Keil</Author><Year>1986</Year><RecNum>183</RecNum><IDText>Primary structure of a proteinase inhibitor II gene from potato (Solanum tuberosum)</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>183</Ref_ID><Title_Primary>Primary structure of a proteinase inhibitor II gene from potato (Solanum tuberosum)</Title_Primary><Authors_Primary>Keil,M.</Authors_Primary><Authors_Primary>Sanchez-Serrano,J.</Authors_Primary><Authors_Primary>Schell,J.</Authors_Primary><Authors_Primary>Willmitzer,L.</Authors_Primary><Date_Primary>1986/7/25</Date_Primary><Keywords>Amino Acid Sequence</Keywords><Keywords>Amino Acids</Keywords><Keywords>Base Sequence</Keywords><Keywords>Dna</Keywords><Keywords>DNA Restriction Enzymes</Keywords><Keywords>Genes</Keywords><Keywords>genetics</Keywords><Keywords>Plant Proteins</Keywords><Keywords>Plants</Keywords><Keywords>Protease Inhibitors</Keywords><Keywords>Proteins</Keywords><Keywords>Solanum tuberosum</Keywords><Reprint>Not in File</Reprint><Start_Page>5641</Start_Page><End_Page>5650</End_Page><Periodical>Nucleic Acids Research</Periodical><Volume>14</Volume><Issue>14</Issue><Web_URL>PM:3016659</Web_URL><Web_URL_Link2><u>file://F:\FSANZ Common\GMO\References\GM References_in RefMan\Keil et al_1986_potato pinII gene.pdf</u></Web_URL_Link2><ZZ_JournalFull><f name="System">Nucleic Acids Research</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(1986)pat cassetteIntervening sequence10323 - 103674535S10368 - 10897530Cauliflower mosaic virusAnti-clockwisePromoter from the 35S geneDrives transcription of the pat geneFranck et al. ADDIN REFMGR.CITE <Refman><Cite ExcludeAuth="1"><Author>Franck</Author><Year>1980</Year><RecNum>1705</RecNum><IDText>Nucleotide sequence of Cauliflower mosiac virus DNA</IDText><MDL Ref_Type="Journal (Full)"><Ref_Type>Journal (Full)</Ref_Type><Ref_ID>1705</Ref_ID><Title_Primary>Nucleotide sequence of Cauliflower mosiac virus DNA</Title_Primary><Authors_Primary>Franck,A.</Authors_Primary><Authors_Primary>Guilley,H.</Authors_Primary><Authors_Primary>Jonard,G.</Authors_Primary><Authors_Primary>Richards,K.</Authors_Primary><Authors_Primary>Hirth,L.</Authors_Primary><Date_Primary>1980</Date_Primary><Keywords>Dna</Keywords><Keywords>analysis</Keywords><Keywords>Codon</Keywords><Keywords>Open Reading Frames</Keywords><Reprint>Not in File</Reprint><Start_Page>285</Start_Page><End_Page>294</End_Page><Periodical>Cell</Periodical><Volume>21</Volume><Issue>1</Issue><Misc_2><f name="Times New Roman">DOI: 10.1016/0092-8674(80)90136-1</f></Misc_2><Web_URL_Link2>;\References\GM References_in RefMan\Franck et al_1980_CaMV sequence.PDF</u></Web_URL_Link2><ZZ_JournalStdAbbrev><f name="System">Cell</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>32</ZZ_WorkformID></MDL></Cite></Refman>(1980); Odell et al. ADDIN REFMGR.CITE <Refman><Cite><Author>Odell</Author><Year>1985</Year><RecNum>179</RecNum><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>179</Ref_ID><Title_Primary>Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter</Title_Primary><Authors_Primary>Odell,J.T.</Authors_Primary><Authors_Primary>Nagy,F.</Authors_Primary><Authors_Primary>Chua,N.-H.</Authors_Primary><Date_Primary>1985</Date_Primary><Keywords>Dna</Keywords><Reprint>In File</Reprint><Start_Page>810</Start_Page><End_Page>812</End_Page><Periodical>Nature</Periodical><Volume>313</Volume><Web_URL_Link2><u>file://F:\FSANZ Common\GMO\References\GM References_in RefMan\Odell_1985_CaMV 35 S promoter.pdf</u></Web_URL_Link2><ZZ_JournalStdAbbrev><f name="System">Nature</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(Odell et al. 1985); Pietrzak et al. ADDIN REFMGR.CITE <Refman><Cite ExcludeAuth="1"><Author>Pietrzak</Author><Year>1986</Year><RecNum>590</RecNum><IDText>Expression in plants of two bacterial antibiotic resistance genes after protoplast transformation with a new plant expression vector</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>590</Ref_ID><Title_Primary>Expression in plants of two bacterial antibiotic resistance genes after protoplast transformation with a new plant expression vector</Title_Primary><Authors_Primary>Pietrzak,M.</Authors_Primary><Authors_Primary>Shillito,R.D.</Authors_Primary><Authors_Primary>Hohn,T.</Authors_Primary><Authors_Primary>Potrykus,I.</Authors_Primary><Date_Primary>1986</Date_Primary><Keywords>CaMV</Keywords><Keywords>CAT</Keywords><Keywords>Genes</Keywords><Keywords>npt</Keywords><Keywords>Plants</Keywords><Keywords>Rna</Keywords><Reprint>In File</Reprint><Start_Page>5857</Start_Page><End_Page>5868</End_Page><Periodical>Nucleic Acids Research</Periodical><Volume>14</Volume><Web_URL_Link2><u>file://F:\FSANZ Common\GMO\References\GM References_in RefMan\Pietrzak et al_1986_CaMV 35S.pdf</u></Web_URL_Link2><ZZ_JournalFull><f name="System">Nucleic Acids Research</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(1986)Intervening sequence10898 - 1091619pat10917 - 11468552Streptomyces viridochromogenesAnti-clockwiseCodon-optimized phosphinothricin acetyltransferase coding sequenceWohlleben et al. ADDIN REFMGR.CITE <Refman><Cite ExcludeAuth="1"><Author>Wohlleben</Author><Year>1988</Year><RecNum>351</RecNum><IDText>Nucleotide sequence of the phosphinothricin N-acetyltransferase gene from Streptomyces viridochromogenes Tü494 and its expression in Nicotiana tabacum</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>351</Ref_ID><Title_Primary>Nucleotide sequence of the phosphinothricin N-acetyltransferase gene from <i>Streptomyces viridochromogenes</i> Tü494 and its expression in <i>Nicotiana tabacum</i></Title_Primary><Authors_Primary>Wohlleben,W.</Authors_Primary><Authors_Primary>Arnold,W.</Authors_Primary><Authors_Primary>Broer,I.</Authors_Primary><Authors_Primary>Hillemann,D.</Authors_Primary><Authors_Primary>Strauch,E.</Authors_Primary><Authors_Primary>Punier,A.</Authors_Primary><Date_Primary>1988</Date_Primary><Keywords>Amino Acids</Keywords><Keywords>Codon</Keywords><Keywords>Escherichia coli</Keywords><Keywords>Genes</Keywords><Keywords>Plants</Keywords><Reprint>In File</Reprint><Start_Page>25</Start_Page><End_Page>37</End_Page><Periodical>Gene</Periodical><Volume>70</Volume><ZZ_JournalStdAbbrev><f name="System">Gene</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(1988)Intervening sequence11469 - 114882035S11489 - 11680192Terminator region from the 35S geneDirects polyadenylation of the pat geneFranck et al. ADDIN REFMGR.CITE <Refman><Cite ExcludeAuth="1"><Author>Franck</Author><Year>1980</Year><RecNum>1705</RecNum><IDText>Nucleotide sequence of Cauliflower mosiac virus DNA</IDText><MDL Ref_Type="Journal (Full)"><Ref_Type>Journal (Full)</Ref_Type><Ref_ID>1705</Ref_ID><Title_Primary>Nucleotide sequence of Cauliflower mosiac virus DNA</Title_Primary><Authors_Primary>Franck,A.</Authors_Primary><Authors_Primary>Guilley,H.</Authors_Primary><Authors_Primary>Jonard,G.</Authors_Primary><Authors_Primary>Richards,K.</Authors_Primary><Authors_Primary>Hirth,L.</Authors_Primary><Date_Primary>1980</Date_Primary><Keywords>Dna</Keywords><Keywords>analysis</Keywords><Keywords>Codon</Keywords><Keywords>Open Reading Frames</Keywords><Reprint>Not in File</Reprint><Start_Page>285</Start_Page><End_Page>294</End_Page><Periodical>Cell</Periodical><Volume>21</Volume><Issue>1</Issue><Misc_2><f name="Times New Roman">DOI: 10.1016/0092-8674(80)90136-1</f></Misc_2><Web_URL_Link2>;\References\GM References_in RefMan\Franck et al_1980_CaMV sequence.PDF</u></Web_URL_Link2><ZZ_JournalStdAbbrev><f name="System">Cell</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>32</ZZ_WorkformID></MDL></Cite></Refman>(1980); Pietrzak et al. ADDIN REFMGR.CITE <Refman><Cite ExcludeAuth="1"><Author>Pietrzak</Author><Year>1986</Year><RecNum>590</RecNum><IDText>Expression in plants of two bacterial antibiotic resistance genes after protoplast transformation with a new plant expression vector</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>590</Ref_ID><Title_Primary>Expression in plants of two bacterial antibiotic resistance genes after protoplast transformation with a new plant expression vector</Title_Primary><Authors_Primary>Pietrzak,M.</Authors_Primary><Authors_Primary>Shillito,R.D.</Authors_Primary><Authors_Primary>Hohn,T.</Authors_Primary><Authors_Primary>Potrykus,I.</Authors_Primary><Date_Primary>1986</Date_Primary><Keywords>CaMV</Keywords><Keywords>CAT</Keywords><Keywords>Genes</Keywords><Keywords>npt</Keywords><Keywords>Plants</Keywords><Keywords>Rna</Keywords><Reprint>In File</Reprint><Start_Page>5857</Start_Page><End_Page>5868</End_Page><Periodical>Nucleic Acids Research</Periodical><Volume>14</Volume><Web_URL_Link2><u>file://F:\FSANZ Common\GMO\References\GM References_in RefMan\Pietrzak et al_1986_CaMV 35S.pdf</u></Web_URL_Link2><ZZ_JournalFull><f name="System">Nucleic Acids Research</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(1986)Intervening sequence11681 - 11874194Ti plasmid region11875 - 1195379Non-functional sequenceLEFT BORDER11954 - 11978253.2.1cry1F expression cassetteThe bacterial cry1F gene sequence has been shown to provide high levels of protection against certain insect pests when it is expressed in plants. The gene encodes one of the family of Bt insecticidal proteins, Cry1F, that specifically inhibits European and southwestern corn borer insects, black cutworm and armyworms.The introduced cry1F gene has been re-synthesised in the laboratory prior to transformation to optimise expression levels in the plant; naturally occurring Bt genes tend to be A:T rich, while plant genes have higher G:C contentThe corresponding amino acid sequence of the Cry1F protein is unchanged by the modified DNA sequence, except for one change at the carboxy terminus of the protein. A leucine residue occurs in place of a phenylalanine residue at position 604 of the 605 amino acids of the plant expressed protein. This single amino acid change results from an intended nucleotide change required to facilitate processing steps in the laboratory. The leucine substitution represents a conservative change in terms of the naturally occurring amino acid at the corresponding position in other Bt proteins. Under the regulation of the constitutive Ubi-1 promoter from corn, including the 5’ untranslated region (UTR) and intron associated with the native polyubiquiton promoter, expression of the cry1F gene would be expected in all parts of the plant, conferring insect protection at the whole plant level. Transcription is terminated by the polyadenylation signal from ORF25 of Agrobacterium tumefaciens.The promoter, coding region and terminator in this cassette are identical to the corresponding elements in line 1507 ADDIN REFMGR.CITE <Refman><Cite><Author>FSANZ</Author><Year>2003</Year><RecNum>354</RecNum><IDText>Application A446: Food derived from Insect-Protected and Glufosinate Ammonium-Tolerant Corn Line 1507</IDText><MDL Ref_Type="Report"><Ref_Type>Report</Ref_Type><Ref_ID>354</Ref_ID><Title_Primary>Application A446: Food derived from Insect-Protected and Glufosinate Ammonium-Tolerant Corn Line 1507</Title_Primary><Authors_Primary>FSANZ</Authors_Primary><Date_Primary>2003</Date_Primary><Reprint>Not in File</Reprint><Publisher>Report prepared by Food Standards Australia New Zealand</Publisher><Web_URL><u>;(FSANZ 2003). The UbiZM1 promoter in line 4114 is described as being 7 bp longer than the same promoter in line 1507 but a more recent sequence analysis showed that the 7 bp previously identified as a polylinker in line 1507 is in fact a part of the UbiZM1 promoter.3.2.2cry34Ab1 and cry35Ab1 expression cassettesThe genes encode the proteins Cry34Ab1 (123 amino acids) and Cry35Ab1 (383 amino acids). Both proteins are required for maximal activity against western corn rootworm PFJlZm1hbj48Q2l0ZT48QXV0aG9yPkVsbGlzPC9BdXRob3I+PFllYXI+MjAwMjwvWWVhcj48UmVj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ADDIN REFMGR.CITE PFJlZm1hbj48Q2l0ZT48QXV0aG9yPkVsbGlzPC9BdXRob3I+PFllYXI+MjAwMjwvWWVhcj48UmVj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ADDIN EN.CITE.DATA (Ellis et al. 2002; Herman et al. 2002). The transgenes that encode these proteins were optimised for expression in corn plants. The proteins encoded by the synthetic transgenes are identical in sequence to the native Bt crystal proteins. The cry34Ab1 gene is regulated by the ubiquitin promoter from Zea mays and the Solanum tuberosum proteinase inhibitor terminator. The cry35Ab1 gene is regulated by the wheat peroxidase gene promoter and the Solanum tuberosum proteinase inhibitor terminator. The promoter, coding region and terminator in each cassette are identical to the corresponding elements in line 59122 ADDIN REFMGR.CITE <Refman><Cite><Author>FSANZ</Author><Year>2005</Year><RecNum>355</RecNum><IDText>Application A543: Food derived from Insect-Protected, Glufosinate Ammonium-Tolerant Corn Line DAS-59122-7</IDText><MDL Ref_Type="Report"><Ref_Type>Report</Ref_Type><Ref_ID>355</Ref_ID><Title_Primary>Application A543: Food derived from Insect-Protected, Glufosinate Ammonium-Tolerant Corn Line DAS-59122-7</Title_Primary><Authors_Primary>FSANZ</Authors_Primary><Date_Primary>2005</Date_Primary><Reprint>Not in File</Reprint><Publisher>Report prepared by Food Standards Australia New Zealand</Publisher><Web_URL><u>;(FSANZ 2005). Again, apparent small discrepancies between the sizes of elements in line 4114 and in line 59122 are due to more sophisticated recent sequence analysis3.2.3pat expression cassetteAs with the genes in the other expression cassettes, the codon usage pattern of the native Streptomyces pat gene has been modified in the laboratory prior to introduction into the plant. The amino acid sequence of the resulting PAT protein however is not changed. The gene is under the regulation of the constitutive promoter and terminator from Cauliflower mosaic virus (CaMV) and therefore the new protein is expected to be expressed in all parts of the plant, including the grain. The promoter, coding region and terminator are identical to the corresponding elements in both lines 1507 and 59122 with, again, apparent small discrepancies being due to more sophisticated recent sequence analysis.3.3Breeding of corn line DP-004114-3The breeding pedigree for the various generations is given in Figure 4.From a single T0 plant, breeding then proceeded in order to produce specific generations that were used in characterisation and analysis (as indicated in Table 2). Pioneer proprietary inbreds PH1B5, PHR03, PHTFE, PHNAR, and PH09B were used in crossing and backcrossing steps and, because of the complexity of the crossing, a number of different non-GM lines were used as controls, depending on the particular generation(s) used for analysis. In addition, since line 4114 contains the same traits as the approved GM lines 1507 and 59122, these two lines and the breeding stack produced from the two lines were used as comparators for the protein expression analysis.Figure SEQ Figure \* ARABIC 4: Breeding diagram for corn line DP-004114-3Table SEQ Table \* ARABIC 2: Line 4114 generations used for various analysesAnalysisLine 4114 generation usedControl(s) usedReference comparatorsMolecular characterisation (Section 3.4)T2, T3, BC3F12, BC3F22PH09B; PHWWEGenetic stability (Section 3.5)T2, T3, BC3F12, BC3F22PH09B, PHWWEMendelian inheritance (Section 3.5)F1*1, BC2F11, BC3F11, BC2F12, BC3F12N/AProtein expression levels in plant parts (Section 4.1.3)F1*5line 1507; line 59122; 1507 x 59122Protein characterisation (Section 4.1.4)F1*5Non-GM, near-isoline1507 x 59122Compositional analysis (Section 5)F1*5PHNAR x PHTFEPioneer? hybrids34M94, 33G26,33J24, 3394, 38B85,37Y12, 34A15, and34P883.4Characterisation of the genetic modification in the plantA range of analyses were undertaken to characterise the genetic modification in line 4114. These analyses focussed on the nature of the insertion of the introduced genetic elements and whether any unintended genetic re-arrangements may have occurred as a consequence of the transformation procedure. 3.4.1Transgene copy number, insertion integrity and plasmid backbone analysisTotal genomic DNA from pooled leaves of plants of theT3 generation from verified line 4114 (using lateral flow immunoassay to confirm expression of the proteins, and event-specific PCR analysis) and from the negative controls (PHO9B and PHWWE) was used for Southern blots to determine the number of T-DNA insertions and the sequence integrity of the introduced DNA in line 4114, and test for the presence or absence of plasmid vector backbone sequences. Positive controls (DNA from PHO9B and PHWWE spiked with digested plasmid PHP27118) were also included in the Southern blot analysis.Southern analysis of Bcl I-digested DNA was used to determine insert copy number in line 4114. The Bcl I restriction enzyme has one recognition site within the T-DNA of PHP27118 thereby cutting it into two fragments. Digested DNA fragments were separated and transferred to a membrane for hybridisation with 11 different digoxigenin (DIG)-labellled probes specific to each of the genetic elements within the PHP27118 T-DNA. Two anomalous results were noted. Firstly, the detection of an extra fragment in digested DNA from line 4114, PHWWE and PHO9B when a ubiZM1 probe was used, was consistent with the presence of native copies of the ubiZM1 promoter and intron. Secondly, the occurrence of an unusual banding pattern in Bcl I-digested PHP27118 plasmid DNA was explained by the methylation of the DNA adenine methylase (Dam) recognition sequence in all Bcl1 sites on the plasmid. The effect of this would be to block digestion by Bcl I. For all other results, the expected sizes of two fragments were obtained thereby confirming that there is integration of a single copy of the T-DNA within the genome of line 4114.Southern blot analysis of Hind III-digested DNA (three sites located within the T-DNA) from line 4114 was used to confirm the integrity of the T-DNA insert. The same 11 probes as used for the copy number analysis were used. As for the Bcl I digestion, an extra fragment associated with the ubiZM1 probes reflected the presence of native sequences. The results indicated the predicted size fragments were detected and hence that the single insert contained the intact T-DNA from plasmid PHP27118 without truncation, rearrangement or deletion of nucleotide sequences.To test whether plasmid backbone DNA is present in line 4114, Southern analysis of Bcl I-digested DNA obtained from 4 generations (T2, T3, BC3F1*3, and BC3F2*2) from verified plants was undertaken. Five DIG-labelled probes spanning the backbone were used. No hybridisation fragments were detected, thus confirming the lack of integration of any plasmid backbone sequences.3.4.2Insert organisation and sequenceThe experimental work to determine the organisation and sequence of the insert in line 4114 is available in a patent ADDIN REFMGR.CITE <Refman><Cite><Author>Diehn</Author><Year>2011</Year><RecNum>1707</RecNum><IDText>Maize event DP-004114-3 and methods for detection thereof</IDText><MDL Ref_Type="Patent"><Ref_Type>Patent</Ref_Type><Ref_ID>1707</Ref_ID><Title_Primary>Maize event DP-004114-3 and methods for detection thereof</Title_Primary><Authors_Primary>Diehn,S.</Authors_Primary><Authors_Primary>Lu,A.L.</Authors_Primary><Authors_Primary>Nowatzki,T.M.</Authors_Primary><Authors_Primary>Nubel,D.S.</Authors_Primary><Authors_Primary>Pascual,M.A.</Authors_Primary><Authors_Primary>Register III,J.C.</Authors_Primary><Authors_Primary>Scelonge,C.J.</Authors_Primary><Authors_Primary>Young,J.K.</Authors_Primary><Authors_Primary>Crowgey,E.L.</Authors_Primary><Date_Primary>2011</Date_Primary><Keywords>maize</Keywords><Keywords>methods</Keywords><Keywords>Dna</Keywords><Keywords>Plants</Keywords><Reprint>Not in File</Reprint><Issue>US 2011/0154523 A1</Issue><Web_URL><u>;\References\GM References_in RefMan\Diehn et al_2011_Maize event DP004114-3 patent.pdf</u></Web_URL_Link2><ZZ_WorkformID>22</ZZ_WorkformID></MDL></Cite></Refman>(Diehn et al. 2011). Genomic DNA was obtained from verified leaf tissue and the samples were used to characterise the DNA sequence in the transgene insertion and its flanking border regions. Six overlapping polymerase chain reaction (PCR) fragments spanning the inserted sequences and border regions in event DP-004114-3 were amplified, purified and then cloned into a bacterial vector. For each fragment, the DNA from vector colonies was sequenced individually and the sequences were aligned to obtain a consensus sequence. Commercially available software (Sequencher?) was then used to assemble the consensus sequences to obtain a final sequence for the insert. Sequence annotation was performed using Vector NTI? 9.1.0 software by comparing the T-DNA insert sequences from line 4114 with sequences in the T-DNA of plasmid PHP27118.In order to confirm that the 5’ and 3’ border regions are of corn origin, PCR amplification and sequencing of the border regions from both line 4114 and the PHWEE control was carried out and the sequences were compared.The sequence of the insert and border regions was determined and 16,752 bp of line 4114 genomic sequence was confirmed ADDIN REFMGR.CITE <Refman><Cite><Author>Diehn</Author><Year>2011</Year><RecNum>1707</RecNum><IDText>Maize event DP-004114-3 and methods for detection thereof</IDText><MDL Ref_Type="Patent"><Ref_Type>Patent</Ref_Type><Ref_ID>1707</Ref_ID><Title_Primary>Maize event DP-004114-3 and methods for detection thereof</Title_Primary><Authors_Primary>Diehn,S.</Authors_Primary><Authors_Primary>Lu,A.L.</Authors_Primary><Authors_Primary>Nowatzki,T.M.</Authors_Primary><Authors_Primary>Nubel,D.S.</Authors_Primary><Authors_Primary>Pascual,M.A.</Authors_Primary><Authors_Primary>Register III,J.C.</Authors_Primary><Authors_Primary>Scelonge,C.J.</Authors_Primary><Authors_Primary>Young,J.K.</Authors_Primary><Authors_Primary>Crowgey,E.L.</Authors_Primary><Date_Primary>2011</Date_Primary><Keywords>maize</Keywords><Keywords>methods</Keywords><Keywords>Dna</Keywords><Keywords>Plants</Keywords><Reprint>Not in File</Reprint><Issue>US 2011/0154523 A1</Issue><Web_URL><u>;\References\GM References_in RefMan\Diehn et al_2011_Maize event DP004114-3 patent.pdf</u></Web_URL_Link2><ZZ_WorkformID>22</ZZ_WorkformID></MDL></Cite></Refman>(Diehn et al. 2011). This comprised 2,398 bp of the 5′ genomic border sequence, 2,405 bp of the 3′ genomic border sequence, and 11,949 bp of inserted T-DNA from PHP27118. The inserted T-DNA in line 4114 was found to have a 29 bp deletion on the RB) end and a 24 bp deletion on the LB end; this truncation of the border sequences is not uncommon for Agrobacterium-mediated transformation events ADDIN REFMGR.CITE <Refman><Cite><Author>Kim</Author><Year>2007</Year><RecNum>1747</RecNum><IDText>Genome-wide analysis of Agrobacterium T-DNA integration sites in the Arabidopsis genome generated under non-selective conditions</IDText><MDL Ref_Type="Journal (Full)"><Ref_Type>Journal (Full)</Ref_Type><Ref_ID>1747</Ref_ID><Title_Primary>Genome-wide analysis of <i>Agrobacterium</i> T-DNA integration sites in the Arabidopsis genome generated under non-selective conditions</Title_Primary><Authors_Primary>Kim,S.-I.</Authors_Primary><Authors_Primary>Veena</Authors_Primary><Authors_Primary>Gelvin,S.B.</Authors_Primary><Date_Primary>2007</Date_Primary><Keywords>analysis</Keywords><Keywords>agrobacterium</Keywords><Keywords>Arabidopsis</Keywords><Keywords>Plants</Keywords><Keywords>Dna</Keywords><Keywords>Methylation</Keywords><Reprint>Not in File</Reprint><Start_Page>779</Start_Page><End_Page>791</End_Page><Periodical>The Plant Journal</Periodical><Volume>51</Volume><Misc_2><f name="Palatino-Bold">doi: 10.1111/j.1365-313X.2007.03183.x</f></Misc_2><Web_URL_Link2>;\References\GM References_in RefMan\Kim et al_2007_T-DNA integration.pdf</u></Web_URL_Link2><ZZ_JournalFull><f name="System">The Plant Journal</f></ZZ_JournalFull><ZZ_WorkformID>32</ZZ_WorkformID></MDL></Cite></Refman>(Kim et al. 2007). The comparison of border region sequences from line 4114 and the control confirmed they are of corn genomic origin.3.4.3Open reading frame (ORF) analysisSequences spanning the 5’ and 3’ junctions of the insert were translated from start codon to stop codon (TGA, TAG, TAA) in all six reading frames, and encoding sequences of 30 or more amino acids. ORFs shorter than 29 amino acids were not evaluated since a minimum 35% identity requires at least a match of 29 amino acids over 80 amino acids. The 35% identity is a recommended criterion for indicating potential allergenicity ADDIN REFMGR.CITE <Refman><Cite><Author>Codex</Author><Year>2009</Year><RecNum>1656</RecNum><IDText>Foods derived from modern biotechnology, 2nd edition</IDText><MDL Ref_Type="Report"><Ref_Type>Report</Ref_Type><Ref_ID>1656</Ref_ID><Title_Primary>Foods derived from modern biotechnology, 2nd edition</Title_Primary><Authors_Primary>Codex</Authors_Primary><Date_Primary>2009</Date_Primary><Keywords>Biotechnology</Keywords><Reprint>Not in File</Reprint><Pub_Place>Rome</Pub_Place><Publisher>Codex Alimentarius Commission, Joint FAO/WHO Food Standards Programme, Food and Agriculture Organization of the United Nations</Publisher><Web_URL><u>;\References\GM References_in RefMan\Codex_2009_food derived from modern biotech.pdf</u></Web_URL_Link2><ZZ_WorkformID>24</ZZ_WorkformID></MDL></Cite></Refman>(Codex 2009). A single ORF (putatively encoding a sequence of 99 amino acids) was identified spanning the 5’ genomic border. No analysis was done to determine whether any potential regulatory elements were associated with it.The putative polypeptide was analysed using a bioinformatic strategy to determine similarity to known protein toxins or allergens (refer to Section 4.1.6).3.5Stability of the genetic changes in line 4114The concept of stability encompasses both the genetic and phenotypic stability of the introduced trait over a number of generations. Genetic stability refers to maintenance of the modification (as produced in the initial transformation event) over successive generations. It is best assessed by molecular techniques, such as Southern blot analysis. Phenotypic stability refers to the expressed trait remaining unchanged over successive generations. It is often quantified by a trait inheritance analysis to determine Mendelian heritability via assay techniques (chemical, molecular, visual).The genetic stability of event DP-004114-3 was evaluated by Southern blot analysis of Bcl I-digested genomic DNA from plants from four generations (T2, T3, BC3F12, BC3F22). Four DIG-labelled probes corresponding to the four coding regions in the T-DNA insert were used for hybridisation. The expected hybridisation patterns were obtained thereby indicating the stable inheritance of the insert.Phenotypic stability was assessed by determining the segregation of the cry1F, cry34Ab1, cry35Ab1 and pat genes within five generations (F1*1, BC2F11, BC3F11, BC2F12, BC3F1*2). Since it was demonstrated that the insert resides at a single locus within the genome of line 4114, the expectation would be that the genetic material within it would be inherited according to Mendelian principles. Leaf punches from individual plants from each generation were analysed by event-specific PCR for the presence of the T-DNA insert and by gene-specific PCR for the presence of each of the four genes. In addition, plants were sprayed with glufosinate to evaluate whether they possessed the herbicide-tolerance phenotype. In all cases, a plant that tested positive for the T-DNA insert also tested positive for the presence of the cry1F, cry34Ab1, cry35Ab1 and pat genes and for a positive herbicide tolerance phenotype. Chi-squared (Χ2) analysis was used to compare the observed segregation data to the hypothesised ratio of 1:1 (positive:negative). The results (Table 3) indicated that there were no significant differences between the observed and expected segregation ratios in any of the generations. This both supported the conclusion that the T-DNA resides at a single locus and showed that the T-DNA is inherited according to Mendelian principles.Table SEQ Table \* ARABIC 3: Segregation of the DP-004114-3 T-DNA sequences over five generationsGenerationTotal plantsRatio positive:negativeΧ2Probability (P)1ObservedExpectedF1*1981:0.881:10.3670.545BC2F111001:1.081:10.1600.689BC3F111001:1.121:10.3600.549BC2F121001:0.881:10.3600.549BC3F1*21951:1.241:11.620.1321Statistical significance is when P≤0.053.6Antibiotic resistance marker genesNo antibiotic marker genes are present in line 4114. The insert sequence analysis (Section 3.4.1) showed that no plasmid backbone has been integrated into the 4114 genome during transformation, i.e. the spc gene, which was used as a bacterial selectable marker gene, is not present in line 4114.3.7Conclusion Corn line 4114 contains four expression cassettes: Comprehensive molecular analyses of line 4114 indicate there is a single insertion site comprising a single, complete copy of each of the cry1F, cry34Ab1, cry35Ab1, and pat genes. The introduced genetic elements are stably inherited from one generation to the next. There are no antibiotic resistance marker genes present in the line and no plasmid backbone has been incorporated into the transgenic locus.4Characterisation and safety assessment of new substances4.1Newly expressed proteinsIn considering the safety of novel proteins it is important to note that a large and diverse range of proteins are ingested as part of the normal human diet without any adverse effects, although a small number have the potential to impair health, e.g., because they are allergens or anti-nutrients ADDIN REFMGR.CITE <Refman><Cite><Author>Delaney</Author><Year>2008</Year><RecNum>288</RecNum><IDText>Evaluation of protein safety in the context of agricultural biotechnology</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>288</Ref_ID><Title_Primary>Evaluation of protein safety in the context of agricultural biotechnology</Title_Primary><Authors_Primary>Delaney,B.</Authors_Primary><Authors_Primary>Astwood,J.D.</Authors_Primary><Authors_Primary>Cunny,H.</Authors_Primary><Authors_Primary>Eichen Conn,R.</Authors_Primary><Authors_Primary>Herouet-Guicheney,C.</Authors_Primary><Authors_Primary>MacIntosh,S.</Authors_Primary><Authors_Primary>Meyer,L.S.</Authors_Primary><Authors_Primary>Privalle,L.S.</Authors_Primary><Authors_Primary>Gao,Y.</Authors_Primary><Authors_Primary>Mattsson,J.</Authors_Primary><Authors_Primary>Levine,M.</Authors_Primary><Authors_Primary>ILSI</Authors_Primary><Date_Primary>2008</Date_Primary><Keywords>Safety</Keywords><Keywords>Proteins</Keywords><Keywords>methods</Keywords><Keywords>Amino Acid Sequence</Keywords><Reprint>In File</Reprint><Start_Page>S71</Start_Page><End_Page>S97</End_Page><Periodical>Food and Chemical Toxicology</Periodical><Volume>46</Volume><Web_URL_Link3>file://Y:\References\GM References_in RefMan<u>\Delaney et al_2008_Protein Safety.pdf</u></Web_URL_Link3><ZZ_JournalFull><f name="System">Food and Chemical Toxicology</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(Delaney et al. 2008). As proteins perform a wide variety of functions, different possible effects have to be considered during the safety assessment including potential toxic, anti-nutritional and allergenic effects. To effectively identify any potential hazards requires knowledge of the characteristics, concentration and localisation of all novel proteins expressed in the organism as well as a detailed understanding of their biochemical function and phenotypic effects. It is also important to determine if the novel protein is expressed as expected, including whether any post-translational modifications have occurred.Two types of novel proteins were considered:Those that were expected to be directly produced as a result of the translation of the introduced genes. A number of different analyses were done to characterise these proteins and determine in planta expression.Those that may be potentially translated as a result of the creation of ORFs during the transformation process (see Section 3.4.3).4.1.1Cry proteinsCrystal (Cry) proteins produced by Bacillus thuringiensis (Bt) are classified by their primary amino acid sequence and more than 500 different cry gene sequences have been classified into 73 groups (Cry1–Cry73) The largest family is the 3D-Cry group and contains proteins subdivided into further groups based on their specificity for target insects; Cry 3 proteins (such as Cry34Ab1 and Cry35Ab1) act specifically on Coleopterans while Cry1 proteins (such as Cry1F) act specifically on Lepidopterans ADDIN REFMGR.CITE <Refman><Cite><Author>H?fte</Author><Year>1989</Year><RecNum>365</RecNum><IDText>Insecticidal crystal proteins of Bacillus thuringiensis</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>365</Ref_ID><Title_Primary>Insecticidal crystal proteins of <i>Bacillus thuringiensis</i></Title_Primary><Authors_Primary>Höfte,H.</Authors_Primary><Authors_Primary>Whiteley,H.R.</Authors_Primary><Date_Primary>1989</Date_Primary><Keywords>Proteins</Keywords><Keywords>Bacillus</Keywords><Keywords>Bacillus thuringiensis</Keywords><Reprint>In File</Reprint><Start_Page>242</Start_Page><End_Page>255</End_Page><Periodical>Microbiological Reviews</Periodical><Volume>53</Volume><Web_URL><u>:\References\GM References_in RefMan<u>\Hofte </u>and<u> Whiteley_1989_Cry proteins.pdf</u></Web_URL_Link2><ZZ_JournalFull><f name="System">Microbiological Reviews</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(H?fte and Whiteley 1989). The primary action of Cry toxins is to lyse midgut epithelial cells in the target insect by forming pores in the apical microvilli membrane of the cells, which subsequently leads to ion leakage and cell lysis. The crystal inclusions ingested by susceptible larvae dissolve in the alkaline environment of the gut, and the solubilised inactive protoxins are cleaved by midgut proteases yielding 60-70 kDa protease resistant core toxins ADDIN REFMGR.CITE <Refman><Cite><Author>Bravo</Author><Year>2007</Year><RecNum>360</RecNum><IDText>Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>360</Ref_ID><Title_Primary>Mode of action of <i>Bacillus thuringiensis</i> Cry and Cyt toxins and their potential for insect control</Title_Primary><Authors_Primary>Bravo,A.</Authors_Primary><Authors_Primary>Gill,S.S.</Authors_Primary><Authors_Primary>Soberón,M.</Authors_Primary><Date_Primary>2007</Date_Primary><Keywords>Bacillus</Keywords><Keywords>Bacillus thuringiensis</Keywords><Keywords>Proteins</Keywords><Keywords>Insects</Keywords><Keywords>Lepidoptera</Keywords><Keywords>Coleoptera</Keywords><Keywords>Insect Control</Keywords><Keywords>Evolution</Keywords><Keywords>Human</Keywords><Reprint>In File</Reprint><Start_Page>423</Start_Page><End_Page>435</End_Page><Periodical>Toxicon</Periodical><Volume>49</Volume><Web_URL><u> name="System">Toxicon</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(Bravo et al. 2007). Toxin activation involves the proteolytic removal of an N-terminal peptide. The activated toxin then binds to specific receptors on the brush border membrane of the midgut epithelium columnar cells PFJlZm1hbj48Q2l0ZT48QXV0aG9yPkFyb25zb248L0F1dGhvcj48WWVhcj4yMDAxPC9ZZWFyPjxS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ADDIN REFMGR.CITE PFJlZm1hbj48Q2l0ZT48QXV0aG9yPkFyb25zb248L0F1dGhvcj48WWVhcj4yMDAxPC9ZZWFyPjxS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ADDIN EN.CITE.DATA (Hofmann et al. 1988; Aronson and Shai 2001) before inserting into the membrane. Toxin insertion leads to formation of lytic pores in microvilli apical membranes PFJlZm1hbj48Q2l0ZT48QXV0aG9yPmRlIE1hYWdkPC9BdXRob3I+PFllYXI+MjAwMTwvWWVhcj48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ADDIN REFMGR.CITE PFJlZm1hbj48Q2l0ZT48QXV0aG9yPmRlIE1hYWdkPC9BdXRob3I+PFllYXI+MjAwMTwvWWVhcj48
UmVjTnVtPjM3NjwvUmVjTnVtPjxJRFRleHQ+SG93IEJhY2lsbHVzIHRodXJpbmdpZW5zaXMgaGFz
IGV2b2x2ZWQgc3BlY2lmaWMgdG94aW5zIHRvIGNvbG9uaXplIHRoZSBpbnNlY3Qgd29ybGQ8L0lE
VGV4dD48TURMIFJlZl9UeXBlPSJKb3VybmFsIj48UmVmX1R5cGU+Sm91cm5hbDwvUmVmX1R5cGU+
PFJlZl9JRD4zNzY8L1JlZl9JRD48VGl0bGVfUHJpbWFyeT5Ib3cgPGk+QmFjaWxsdXMgdGh1cmlu
Z2llbnNpczwvaT4gaGFzIGV2b2x2ZWQgc3BlY2lmaWMgdG94aW5zIHRvIGNvbG9uaXplIHRoZSBp
bnNlY3Qgd29ybGQ8L1RpdGxlX1ByaW1hcnk+PEF1dGhvcnNfUHJpbWFyeT5kZSBNYWFnZCxSLkEu
PC9BdXRob3JzX1ByaW1hcnk+PEF1dGhvcnNfUHJpbWFyeT5CcmF2byxBLjwvQXV0aG9yc19Qcmlt
YXJ5PjxBdXRob3JzX1ByaW1hcnk+Q3JpY2ttb3JlLE4uPC9BdXRob3JzX1ByaW1hcnk+PERhdGVf
UHJpbWFyeT4yMDAxPC9EYXRlX1ByaW1hcnk+PEtleXdvcmRzPkJhY2lsbHVzPC9LZXl3b3Jkcz48
S2V5d29yZHM+QmFjaWxsdXMgdGh1cmluZ2llbnNpczwvS2V5d29yZHM+PEtleXdvcmRzPkJhY3Rl
cmlhPC9LZXl3b3Jkcz48S2V5d29yZHM+SW5zZWN0czwvS2V5d29yZHM+PEtleXdvcmRzPlByb3Rl
aW5zPC9LZXl3b3Jkcz48UmVwcmludD5JbiBGaWxlPC9SZXByaW50PjxTdGFydF9QYWdlPjE5Mzwv
U3RhcnRfUGFnZT48RW5kX1BhZ2U+MTk5PC9FbmRfUGFnZT48UGVyaW9kaWNhbD5UcmVuZHMgaW4g
R2VuZXRpY3M8L1BlcmlvZGljYWw+PFZvbHVtZT4xNzwvVm9sdW1lPjxXZWJfVVJMPjx1Pmh0dHA6
Ly93d3cuYmlvdGVjaC1pbmZvLm5ldC9CdF9UaUcucGRmPC91PjwvV2ViX1VSTD48V2ViX1VSTF9M
aW5rMj5maWxlOi8vWTpcUmVmZXJlbmNlc1xHTSBSZWZlcmVuY2VzX2luIFJlZk1hbjx1PlxkZSBN
YWFnZCBldCBhbF8yMDAxX0J0IHRveGlucy5wZGY8L3U+PC9XZWJfVVJMX0xpbmsyPjxaWl9Kb3Vy
bmFsRnVsbD48ZiBuYW1lPSJTeXN0ZW0iPlRyZW5kcyBpbiBHZW5ldGljczwvZj48L1paX0pvdXJu
YWxGdWxsPjxaWl9Xb3JrZm9ybUlEPjE8L1paX1dvcmtmb3JtSUQ+PC9NREw+PC9DaXRlPjxDaXRl
PjxBdXRob3I+QXJvbnNvbjwvQXV0aG9yPjxZZWFyPjIwMDE8L1llYXI+PFJlY051bT4zNzc8L1Jl
Y051bT48SURUZXh0PldoeSBCYWNpbGx1cyB0aHVyaW5naWVuc2lzIGluc2VjdGljaWRhbCB0b3hp
bnMgYXJlIHNvIGVmZmVjdGl2ZTogdW5pcXVlIGZlYXR1cmVzIG9mIHRoZWlyIG1vZGUgb2YgYWN0
aW9uPC9JRFRleHQ+PE1ETCBSZWZfVHlwZT0iSm91cm5hbCI+PFJlZl9UeXBlPkpvdXJuYWw8L1Jl
Zl9UeXBlPjxSZWZfSUQ+Mzc3PC9SZWZfSUQ+PFRpdGxlX1ByaW1hcnk+V2h5IDxpPkJhY2lsbHVz
IHRodXJpbmdpZW5zaXM8L2k+IGluc2VjdGljaWRhbCB0b3hpbnMgYXJlIHNvIGVmZmVjdGl2ZTog
dW5pcXVlIGZlYXR1cmVzIG9mIHRoZWlyIG1vZGUgb2YgYWN0aW9uPC9UaXRsZV9QcmltYXJ5PjxB
dXRob3JzX1ByaW1hcnk+QXJvbnNvbixBLkkuPC9BdXRob3JzX1ByaW1hcnk+PEF1dGhvcnNfUHJp
bWFyeT5TaGFpLFkuPC9BdXRob3JzX1ByaW1hcnk+PERhdGVfUHJpbWFyeT4yMDAxPC9EYXRlX1By
aW1hcnk+PEtleXdvcmRzPkJhY3RlcmlhPC9LZXl3b3Jkcz48S2V5d29yZHM+SW5zZWN0czwvS2V5
d29yZHM+PEtleXdvcmRzPkJhY2lsbHVzPC9LZXl3b3Jkcz48S2V5d29yZHM+QmFjaWxsdXMgdGh1
cmluZ2llbnNpczwvS2V5d29yZHM+PFJlcHJpbnQ+SW4gRmlsZTwvUmVwcmludD48U3RhcnRfUGFn
ZT4xPC9TdGFydF9QYWdlPjxFbmRfUGFnZT44PC9FbmRfUGFnZT48UGVyaW9kaWNhbD5GRU1TIE1p
Y3JvYmlvbG9neSBMZXR0ZXJzPC9QZXJpb2RpY2FsPjxWb2x1bWU+MTk1PC9Wb2x1bWU+PFdlYl9V
Ukw+PHU+aHR0cDovL3d3dzMuaW50ZXJzY2llbmNlLndpbGV5LmNvbS9jZ2ktYmluL2Z1bGx0ZXh0
LzExOTAyMDMyMi9QREZTVEFSVDwvdT48L1dlYl9VUkw+PFdlYl9VUkxfTGluazI+ZmlsZTovL1k6
XFJlZmVyZW5jZXNcR00gUmVmZXJlbmNlc19pbiBSZWZNYW48dT5cQXJvbnNvbiA8L3U+YW5kPHU+
IFNoYWlfMjAwMV9CdCB0b3hpbnMucGRmPC91PjwvV2ViX1VSTF9MaW5rMj48WlpfSm91cm5hbEZ1
bGw+PGYgbmFtZT0iU3lzdGVtIj5GRU1TIE1pY3JvYmlvbG9neSBMZXR0ZXJzPC9mPjwvWlpfSm91
cm5hbEZ1bGw+PFpaX1dvcmtmb3JtSUQ+MTwvWlpfV29ya2Zvcm1JRD48L01ETD48L0NpdGU+PC9S
ZWZtYW4+
ADDIN EN.CITE.DATA (de Maagd et al. 2001; Aronson and Shai 2001) and eventually to cell lysis and disruption of the gut epithelium. The septicaemia that inevitably follows may be mediated by an influx of enteric bacteria into the haemocoel ADDIN REFMGR.CITE <Refman><Cite><Author>Broderick</Author><Year>2006</Year><RecNum>379</RecNum><IDText>Midgut bacteria required for Bacillus thuringiensis insecticidal activity</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>379</Ref_ID><Title_Primary>Midgut bacteria required for <i>Bacillus thuringiensis</i> insecticidal activity</Title_Primary><Authors_Primary>Broderick,N.A.</Authors_Primary><Authors_Primary>Raffa,K.F.</Authors_Primary><Authors_Primary>Handelsman,J.</Authors_Primary><Date_Primary>2006</Date_Primary><Keywords>Bacillus</Keywords><Keywords>Bacillus thuringiensis</Keywords><Keywords>Insects</Keywords><Keywords>Human</Keywords><Keywords>Bacteria</Keywords><Keywords>Proteins</Keywords><Keywords>Plants</Keywords><Keywords>Starvation</Keywords><Keywords>Mortality</Keywords><Reprint>In File</Reprint><Start_Page>15196</Start_Page><End_Page>15199</End_Page><Periodical>Proceedings of the National Academy of Sciences</Periodical><Volume>103</Volume><Web_URL><u>:\References\GM References_in RefMan<u>\Broderick et al_2006_Bt activity.pdf</u></Web_URL_Link2><ZZ_JournalFull><f name="System">Proceedings of the National Academy of Sciences</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(Broderick et al. 2006).4.1.1.1Cry1FIn its native form, Cry 1F is produced in B. thuringiensis as a large protoxin of 1174 amino acids. Following solubilisation and proteolytic processing in the gut of susceptible insect larvae, the active toxin moiety corresponds to approximately 600 amino acids at the N-terminal end of the full-length protein. Although precise cleavage has not been shown, the activated toxin is estimated to correspond to amino acids 28-612, based on laboratory data and computer simulations. Therefore, to confer insect resistance, a truncated form of the cry1F gene, encoding only the active toxin moiety, was inserted into the corn plants. The truncated Cry1F protein present in line 4114 should be identical to amino acids 1-605 of the N-terminal domain of the native Cry1F protoxin with the exception of a single amino acid substitution, leucine in place of phenylalanine, at position 604 (F604L – see Figure 5). The amino acid change was made to facilitate production in the laboratory of large quantities of a microbially-produced Cry1F/Cry1A(b) chimeric protein that was used as a source of the Cry1F moiety required for safety studies. The chimeric protein is a fusion of the gene sequence coding for the Cry1A(b) C-terminal domain with the gene sequence coding for the Cry1F core toxin. The decision to use F604L substitution was based on the occurrence of leucine in the homologous position of other Cry1 proteins, and is therefore a conservative substitution. The predicted molecular weight is 68 kDa.Cry1F provides control of not only European corn borer and southwestern corn borer, but also provides protection against damaging infestations of other lepidopteran pests including sugarcane borer, fall armyworm, black cutworm, and western bean cutworm ADDIN REFMGR.CITE <Refman><Cite><Author>Siebert</Author><Year>2014</Year><RecNum>1700</RecNum><IDText>Efficacy of Cry1F insecticidal protein in maize and cotton for control of fall armyworm (Lepidoptera: Noctuidae)</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1700</Ref_ID><Title_Primary>Efficacy of Cry1F insecticidal protein in maize and cotton for control of fall armyworm (Lepidoptera: Noctuidae)</Title_Primary><Authors_Primary>Siebert,M.W.</Authors_Primary><Authors_Primary>Babock,J.M.</Authors_Primary><Authors_Primary>Nolting,S.</Authors_Primary><Authors_Primary>Santos,A.C.</Authors_Primary><Authors_Primary>Adamczyk,J.J.</Authors_Primary><Authors_Primary>Neese,P.A.</Authors_Primary><Authors_Primary>King,J.E.</Authors_Primary><Authors_Primary>Jenkins,J.N.</Authors_Primary><Authors_Primary>McCarty,J.</Authors_Primary><Authors_Primary>Lorenz,G.M.</Authors_Primary><Authors_Primary>Fromme,D.D.</Authors_Primary><Authors_Primary>Lassiter,R.B.</Authors_Primary><Date_Primary>2014</Date_Primary><Keywords>maize</Keywords><Keywords>Lepidoptera</Keywords><Keywords>United States</Keywords><Keywords>Insecticides</Keywords><Keywords>Fruit</Keywords><Reprint>Not in File</Reprint><Start_Page>555</Start_Page><End_Page>565</End_Page><Periodical>Florida Entomologist</Periodical><Volume>91</Volume><Issue>4</Issue><Web_URL_Link2>;\References\GM References_in RefMan\Siebert et al_2008_Cry1F.pdf</u></Web_URL_Link2><ZZ_JournalFull><f name="System">Florida Entomologist</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(Siebert et al. 2014)Figure SEQ Figure \* ARABIC 5: Amino acid sequence of the Cry1F protein4.1.1.2Cry34Ab1 and Cry35Ab1Both proteins are required together for mortality of the corn rootworm larvae. Although the Cry34Ab1 protein is active alone in corn rootworm larvae when applied at high concentrations in bioassays, transgenic plants which expressed only the Cry34Ab1 protein do not control western corn rootworm. The activity of the Cry34Ab1 protein in bioassays is greatly potentiated by Cry35Ab1. The Cry35Ab1 protein alone is not active against corn rootworm. In vivo, only a small quantity of Cry35Ab1 is needed in the Cry34/35Ab1 insecticidal crystal protein (ICP). Therefore, the majority of the activity seen with mixtures of Cry34Ab1 and Cry35Ab1 may be explained by the concentration of the Cry34Ab1 protein.It is not known exactly how the Cry34/35Ab1 ICP exerts its toxicity. Histological studies have shown that the ICP causes disruption of the western corn rootworm larval mid-gut membranes. In experiments using artificial membranes, the ICP produces ion channels or pores which is at least partially responsible for the disruption of the synthetic membranes ADDIN REFMGR.CITE <Refman><Cite><Author>Masson</Author><Year>2004</Year><RecNum>62</RecNum><IDText>A novel Bacillus thuringiensis (PS149B1) containing a Cry34Ab1/Cry35Ab1 binary toxin specific for the western corn rootworm Diabrotica virgifera virgifera LeConte forms ion channels in lipid membranes</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>62</Ref_ID><Title_Primary>A novel Bacillus thuringiensis (PS149B1) containing a Cry34Ab1/Cry35Ab1 binary toxin specific for the western corn rootworm Diabrotica virgifera virgifera LeConte forms ion channels in lipid membranes</Title_Primary><Authors_Primary>Masson,L.</Authors_Primary><Authors_Primary>Schwab,G.</Authors_Primary><Authors_Primary>Mazza,A.</Authors_Primary><Authors_Primary>Brousseau,R.</Authors_Primary><Authors_Primary>Potvin,L.</Authors_Primary><Authors_Primary>Schwartz,J.L.</Authors_Primary><Date_Primary>2004/9/28</Date_Primary><Keywords>Bacillus thuringiensis</Keywords><Keywords>Proteins</Keywords><Keywords>Recombinant Proteins</Keywords><Reprint>Not in File</Reprint><Start_Page>12349</Start_Page><End_Page>12357</End_Page><Periodical>Biochemistry</Periodical><Volume>43</Volume><Issue>38</Issue><Address>Biotechnology Research Institute, National Research Council, Montreal, Quebec, Canada H4P 2R2</Address><Web_URL>PM:15379574</Web_URL><ZZ_JournalStdAbbrev><f name="System">Biochemistry</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(Masson et al. 2004). The formation of ion channels in artificial membranes has also been reported for Cry34Ab1 PFJlZm1hbj48Q2l0ZT48QXV0aG9yPkJhdW08L0F1dGhvcj48WWVhcj4yMDA0PC9ZZWFyPjxSZWNO
dW0+NjM8L1JlY051bT48SURUZXh0PkJpbmFyeSB0b3hpbnMgZnJvbSBCYWNpbGx1cyB0aHVyaW5n
aWVuc2lzIGFjdGl2ZSBhZ2FpbnN0IHRoZSB3ZXN0ZXJuIGNvcm4gcm9vdHdvcm0sIERpYWJyb3Rp
Y2EgdmlyZ2lmZXJhIHZpcmdpZmVyYSBMZUNvbnRlPC9JRFRleHQ+PE1ETCBSZWZfVHlwZT0iSm91
cm5hbCI+PFJlZl9UeXBlPkpvdXJuYWw8L1JlZl9UeXBlPjxSZWZfSUQ+NjM8L1JlZl9JRD48VGl0
bGVfUHJpbWFyeT5CaW5hcnkgdG94aW5zIGZyb20gQmFjaWxsdXMgdGh1cmluZ2llbnNpcyBhY3Rp
dmUgYWdhaW5zdCB0aGUgd2VzdGVybiBjb3JuIHJvb3R3b3JtLCA8aT5EaWFicm90aWNhIHZpcmdp
ZmVyYSB2aXJnaWZlcmE8L2k+IExlQ29udGU8L1RpdGxlX1ByaW1hcnk+PEF1dGhvcnNfUHJpbWFy
eT5CYXVtLEouQS48L0F1dGhvcnNfUHJpbWFyeT48QXV0aG9yc19QcmltYXJ5PkNodSxDLi1SLjwv
QXV0aG9yc19QcmltYXJ5PjxBdXRob3JzX1ByaW1hcnk+UnVwYXIsTS48L0F1dGhvcnNfUHJpbWFy
eT48QXV0aG9yc19QcmltYXJ5PkJyb3duLEcuUi48L0F1dGhvcnNfUHJpbWFyeT48QXV0aG9yc19Q
cmltYXJ5PkRvbm92YW4sVy5QLjwvQXV0aG9yc19QcmltYXJ5PjxBdXRob3JzX1ByaW1hcnk+SHVl
c2luZyxKLkUuPC9BdXRob3JzX1ByaW1hcnk+PEF1dGhvcnNfUHJpbWFyeT5JbGFnYW4sTy48L0F1
dGhvcnNfUHJpbWFyeT48QXV0aG9yc19QcmltYXJ5Pk1hbHZhcixULk0uPC9BdXRob3JzX1ByaW1h
cnk+PEF1dGhvcnNfUHJpbWFyeT5QbGVhdSxNLjwvQXV0aG9yc19QcmltYXJ5PjxBdXRob3JzX1By
aW1hcnk+V2FsdGVycyxNLjwvQXV0aG9yc19QcmltYXJ5PjxBdXRob3JzX1ByaW1hcnk+VmF1Z2hu
LFQuPC9BdXRob3JzX1ByaW1hcnk+PERhdGVfUHJpbWFyeT4yMDA0Lzg8L0RhdGVfUHJpbWFyeT48
S2V5d29yZHM+QW1pbm8gQWNpZCBTZXF1ZW5jZTwvS2V5d29yZHM+PEtleXdvcmRzPkFuaW1hbHM8
L0tleXdvcmRzPjxLZXl3b3Jkcz5CYWNpbGx1cyB0aHVyaW5naWVuc2lzPC9LZXl3b3Jkcz48S2V5
d29yZHM+QmFjdGVyaWFsIFByb3RlaW5zPC9LZXl3b3Jkcz48S2V5d29yZHM+QmFjdGVyaWFsIFRv
eGluczwvS2V5d29yZHM+PEtleXdvcmRzPkJhc2UgU2VxdWVuY2U8L0tleXdvcmRzPjxLZXl3b3Jk
cz5jaGVtaXN0cnk8L0tleXdvcmRzPjxLZXl3b3Jkcz5DbG9uaW5nLE1vbGVjdWxhcjwvS2V5d29y
ZHM+PEtleXdvcmRzPkNvbGVvcHRlcmE8L0tleXdvcmRzPjxLZXl3b3Jkcz5FbmRvdG94aW5zPC9L
ZXl3b3Jkcz48S2V5d29yZHM+Z2VuZXRpY3M8L0tleXdvcmRzPjxLZXl3b3Jkcz5ncm93dGggJmFt
cDsgZGV2ZWxvcG1lbnQ8L0tleXdvcmRzPjxLZXl3b3Jkcz5MYXJ2YTwvS2V5d29yZHM+PEtleXdv
cmRzPm1ldGFib2xpc208L0tleXdvcmRzPjxLZXl3b3Jkcz5Nb2xlY3VsYXIgU2VxdWVuY2UgRGF0
YTwvS2V5d29yZHM+PEtleXdvcmRzPk9wZXJvbjwvS2V5d29yZHM+PEtleXdvcmRzPnBhcmFzaXRv
bG9neTwvS2V5d29yZHM+PEtleXdvcmRzPlBlc3QgQ29udHJvbCxCaW9sb2dpY2FsPC9LZXl3b3Jk
cz48S2V5d29yZHM+UHJvdGVpbnM8L0tleXdvcmRzPjxLZXl3b3Jkcz5TZXF1ZW5jZSBBbmFseXNp
cyxETkE8L0tleXdvcmRzPjxLZXl3b3Jkcz50b3hpY2l0eTwvS2V5d29yZHM+PEtleXdvcmRzPlpl
YSBtYXlzPC9LZXl3b3Jkcz48S2V5d29yZHM+QmFjaWxsdXM8L0tleXdvcmRzPjxSZXByaW50Pk5v
dCBpbiBGaWxlPC9SZXByaW50PjxTdGFydF9QYWdlPjQ4ODk8L1N0YXJ0X1BhZ2U+PEVuZF9QYWdl
PjQ4OTg8L0VuZF9QYWdlPjxQZXJpb2RpY2FsPkFwcGxpZWQgYW5kIEVudmlyb25tZW50YWwgTWlj
cm9iaW9sb2d5PC9QZXJpb2RpY2FsPjxWb2x1bWU+NzA8L1ZvbHVtZT48SXNzdWU+ODwvSXNzdWU+
PEFkZHJlc3M+TW9uc2FudG8gQ29tcGFueSwgNzAwIENoZXN0ZXJmaWVsZCBQa3d5LiBXZXN0LCBD
aGVzdGVyZmllbGQsIE1PIDYzMDE3LTE3MzIsIFVTQS4gSmFtZXMuQS5CYXVtQG1vbnNhbnRvLmNv
bTwvQWRkcmVzcz48V2ViX1VSTD5QTToxNTI5NDgyODwvV2ViX1VSTD48WlpfSm91cm5hbEZ1bGw+
PGYgbmFtZT0iU3lzdGVtIj5BcHBsaWVkIGFuZCBFbnZpcm9ubWVudGFsIE1pY3JvYmlvbG9neTwv
Zj48L1paX0pvdXJuYWxGdWxsPjxaWl9Xb3JrZm9ybUlEPjE8L1paX1dvcmtmb3JtSUQ+PC9NREw+
PC9DaXRlPjwvUmVmbWFuPgB=
ADDIN REFMGR.CITE PFJlZm1hbj48Q2l0ZT48QXV0aG9yPkJhdW08L0F1dGhvcj48WWVhcj4yMDA0PC9ZZWFyPjxSZWNO
dW0+NjM8L1JlY051bT48SURUZXh0PkJpbmFyeSB0b3hpbnMgZnJvbSBCYWNpbGx1cyB0aHVyaW5n
aWVuc2lzIGFjdGl2ZSBhZ2FpbnN0IHRoZSB3ZXN0ZXJuIGNvcm4gcm9vdHdvcm0sIERpYWJyb3Rp
Y2EgdmlyZ2lmZXJhIHZpcmdpZmVyYSBMZUNvbnRlPC9JRFRleHQ+PE1ETCBSZWZfVHlwZT0iSm91
cm5hbCI+PFJlZl9UeXBlPkpvdXJuYWw8L1JlZl9UeXBlPjxSZWZfSUQ+NjM8L1JlZl9JRD48VGl0
bGVfUHJpbWFyeT5CaW5hcnkgdG94aW5zIGZyb20gQmFjaWxsdXMgdGh1cmluZ2llbnNpcyBhY3Rp
dmUgYWdhaW5zdCB0aGUgd2VzdGVybiBjb3JuIHJvb3R3b3JtLCA8aT5EaWFicm90aWNhIHZpcmdp
ZmVyYSB2aXJnaWZlcmE8L2k+IExlQ29udGU8L1RpdGxlX1ByaW1hcnk+PEF1dGhvcnNfUHJpbWFy
eT5CYXVtLEouQS48L0F1dGhvcnNfUHJpbWFyeT48QXV0aG9yc19QcmltYXJ5PkNodSxDLi1SLjwv
QXV0aG9yc19QcmltYXJ5PjxBdXRob3JzX1ByaW1hcnk+UnVwYXIsTS48L0F1dGhvcnNfUHJpbWFy
eT48QXV0aG9yc19QcmltYXJ5PkJyb3duLEcuUi48L0F1dGhvcnNfUHJpbWFyeT48QXV0aG9yc19Q
cmltYXJ5PkRvbm92YW4sVy5QLjwvQXV0aG9yc19QcmltYXJ5PjxBdXRob3JzX1ByaW1hcnk+SHVl
c2luZyxKLkUuPC9BdXRob3JzX1ByaW1hcnk+PEF1dGhvcnNfUHJpbWFyeT5JbGFnYW4sTy48L0F1
dGhvcnNfUHJpbWFyeT48QXV0aG9yc19QcmltYXJ5Pk1hbHZhcixULk0uPC9BdXRob3JzX1ByaW1h
cnk+PEF1dGhvcnNfUHJpbWFyeT5QbGVhdSxNLjwvQXV0aG9yc19QcmltYXJ5PjxBdXRob3JzX1By
aW1hcnk+V2FsdGVycyxNLjwvQXV0aG9yc19QcmltYXJ5PjxBdXRob3JzX1ByaW1hcnk+VmF1Z2hu
LFQuPC9BdXRob3JzX1ByaW1hcnk+PERhdGVfUHJpbWFyeT4yMDA0Lzg8L0RhdGVfUHJpbWFyeT48
S2V5d29yZHM+QW1pbm8gQWNpZCBTZXF1ZW5jZTwvS2V5d29yZHM+PEtleXdvcmRzPkFuaW1hbHM8
L0tleXdvcmRzPjxLZXl3b3Jkcz5CYWNpbGx1cyB0aHVyaW5naWVuc2lzPC9LZXl3b3Jkcz48S2V5
d29yZHM+QmFjdGVyaWFsIFByb3RlaW5zPC9LZXl3b3Jkcz48S2V5d29yZHM+QmFjdGVyaWFsIFRv
eGluczwvS2V5d29yZHM+PEtleXdvcmRzPkJhc2UgU2VxdWVuY2U8L0tleXdvcmRzPjxLZXl3b3Jk
cz5jaGVtaXN0cnk8L0tleXdvcmRzPjxLZXl3b3Jkcz5DbG9uaW5nLE1vbGVjdWxhcjwvS2V5d29y
ZHM+PEtleXdvcmRzPkNvbGVvcHRlcmE8L0tleXdvcmRzPjxLZXl3b3Jkcz5FbmRvdG94aW5zPC9L
ZXl3b3Jkcz48S2V5d29yZHM+Z2VuZXRpY3M8L0tleXdvcmRzPjxLZXl3b3Jkcz5ncm93dGggJmFt
cDsgZGV2ZWxvcG1lbnQ8L0tleXdvcmRzPjxLZXl3b3Jkcz5MYXJ2YTwvS2V5d29yZHM+PEtleXdv
cmRzPm1ldGFib2xpc208L0tleXdvcmRzPjxLZXl3b3Jkcz5Nb2xlY3VsYXIgU2VxdWVuY2UgRGF0
YTwvS2V5d29yZHM+PEtleXdvcmRzPk9wZXJvbjwvS2V5d29yZHM+PEtleXdvcmRzPnBhcmFzaXRv
bG9neTwvS2V5d29yZHM+PEtleXdvcmRzPlBlc3QgQ29udHJvbCxCaW9sb2dpY2FsPC9LZXl3b3Jk
cz48S2V5d29yZHM+UHJvdGVpbnM8L0tleXdvcmRzPjxLZXl3b3Jkcz5TZXF1ZW5jZSBBbmFseXNp
cyxETkE8L0tleXdvcmRzPjxLZXl3b3Jkcz50b3hpY2l0eTwvS2V5d29yZHM+PEtleXdvcmRzPlpl
YSBtYXlzPC9LZXl3b3Jkcz48S2V5d29yZHM+QmFjaWxsdXM8L0tleXdvcmRzPjxSZXByaW50Pk5v
dCBpbiBGaWxlPC9SZXByaW50PjxTdGFydF9QYWdlPjQ4ODk8L1N0YXJ0X1BhZ2U+PEVuZF9QYWdl
PjQ4OTg8L0VuZF9QYWdlPjxQZXJpb2RpY2FsPkFwcGxpZWQgYW5kIEVudmlyb25tZW50YWwgTWlj
cm9iaW9sb2d5PC9QZXJpb2RpY2FsPjxWb2x1bWU+NzA8L1ZvbHVtZT48SXNzdWU+ODwvSXNzdWU+
PEFkZHJlc3M+TW9uc2FudG8gQ29tcGFueSwgNzAwIENoZXN0ZXJmaWVsZCBQa3d5LiBXZXN0LCBD
aGVzdGVyZmllbGQsIE1PIDYzMDE3LTE3MzIsIFVTQS4gSmFtZXMuQS5CYXVtQG1vbnNhbnRvLmNv
bTwvQWRkcmVzcz48V2ViX1VSTD5QTToxNTI5NDgyODwvV2ViX1VSTD48WlpfSm91cm5hbEZ1bGw+
PGYgbmFtZT0iU3lzdGVtIj5BcHBsaWVkIGFuZCBFbnZpcm9ubWVudGFsIE1pY3JvYmlvbG9neTwv
Zj48L1paX0pvdXJuYWxGdWxsPjxaWl9Xb3JrZm9ybUlEPjE8L1paX1dvcmtmb3JtSUQ+PC9NREw+
PC9DaXRlPjwvUmVmbWFuPgB=
ADDIN EN.CITE.DATA (Baum et al. 2004). Meaningful in vivo activity with the ICP has only been observed in a subset of coleopteran larvae (corn rootworm). In vivo activity has not been found in adult corn rootworms, a corn aphid species or certain lepidopteran pests, indicating selective activity for corn rootworm larvae. Cry34Ab1 and Cry35Ab1 have not been observed to associate to form a hetero-dimer.The amino acid sequences of the Cry34Ab1 (mw = 14 kDa) and Cry35Ab1 (mw = 44 kDa) proteins are given in Figure 6.Figure SEQ Figure \* ARABIC 6: Amino acid sequences of the Cry34Ab1 and Cry35Ab1 proteins4.1.2PATThe PAT protein consists of 183 amino acids (Figure 7), has a molecular weight of 21 kDa, and exhibits a high degree of enzyme specificity; recognising only one substrate. PAT functions by detoxifying phosphinothricin (PPT), the active constituent of glufosinate ammonium herbicides. PPT acts by inhibiting the endogenous enzyme glutamine synthetase, an enzyme involved in amino acid biosynthesis in plant cells. By inhibiting this enzyme, PPT causes rapid accumulation of ammonia in the plant cell, leading to plant death. In transformed corn plants, the introduced PAT enzyme chemically inactivates the PPT by acetylation of the free ammonia group, giving rise to herbicide tolerance in the whole plant.Figure SEQ Figure \* ARABIC 7: Amino acid sequence of the PAT protein4.1.3Protein expression in the tissues of line 4114Study submitted:2011. Expressed Trait Protein Concentration of a Maize Line Containing Events DP-004114-3, DAS-01507-1, DAS-59122-7, and Combined Trait Product DAS-01507 x DAS-59122-7: US and Canada Test Sites. Study ID PHI-2010-059. Pioneer Hi-Bred International, Inc (unpublished).Plants of line 4114 (generation F1*5 – since F1 hybrid seed is representative of seed that would be commercially planted) were grown from verified seed lots at five field sites in the USA and Canada during the 2010 growing season. Since the genetic material in line 4114 is equivalent to that contributed by lines 1507 and 59122 in the commercial breeding stack, plants of both parents and the stack (all grown from verified seed) were also analysed to provide comparative data.Three of the planting sites overlapped with those used for the compositional analysis described in Section 5.2. There were four replicated plots at each site planted in a randomised complete-block design. Maintenance fertiliser and pesticide were applied to all plots as appropriate to the site conditions.Four replicated samples from different plants were taken at various stages of growth (Table 4) to give a total of 20 samples for each tissue and time point. Levels of Cry1F, Cry34Ab1, Cry35Ab1 and PAT were determined for each sample type using a validated enzyme linked immunosorbent assay (ELISA). For each protein, plates were pre-incubated with an appropriate-specific antibody to bind the protein to the plate wells. The protein was then detected by incubation with a different specific antibody conjugated to horseradish peroxidase followed by addition of substrate specific to the enzyme. Plates were analysed on a microplate spectrophotometer, and commercial software (SoftMax Pro GxP, Molecular Devices) was used to convert optical density values to protein concentrationThe results, averaged over all sites, are given in Table 4.Table SEQ Table \* ARABIC 4: Cry1F, Cry34Ab1, Cry35Ab1 and PAT protein content in line 4114 parts at different growth stages (averaged across 5 sites)Tissue/Growth stage1Cry1F ?g/g dwCry34Ab1 ug/g dwCry35Ab1 ?g/g dwPAT ?g/g dwMeanRangeMeanRangeMeanRangeMeanRangeLeaf V6117.8 - 172117 - 252717 - 3894.2 - 14Leaf V99.75.3 - 142622 - 313328 - 399.84.8 - 15Leaf R1137.2 - 285036 - 846843 - 130145 - 24Leaf R43419 - 5611066 - 1409066 - 110115.7 - 20Leaf R620.32 - 21194.2 - 667241 - 1100.52<LOQ - 2Root V64.20.69 - 6179 - 23139 - 200.440.14 – 0.78Root V951.3 – 7.52113 - 28137.8 - 190.650.39 – 0.9Root R15.53.9 – 7.8198.7 - 309.24.2 - 150.440.3 – 0.72Root R43.82.3 – 5.7237.5 - 366.42.1 - 120.16<LOQ – 0.39Root R63.81.4 – 6.3185.4 - 546.92.2 - 140.13<LOQ – 0.66Whole plant V9128.6 - 152318 - 267658 - 1008.76.6 - 11Whole Plant R19.97.8 - 133224 - 426644 - 1004.93.2 – 7.4Whole Plant R64.12.4 – 9.43620 - 622113 - 540.09<LOQ – 0.76Pollen R13519 - 499.24.7 - 160.34<LOQ – 0.53< LOQ2<LOQForage3 R47.85.6 - 115236 - 642918 - 521.9 1.1 – 2.8Grain R63.32.3 – 7.22414 - 391.10.54 – 2.3<LOQ<LOQ1For information on corn growth stages see e.g. Ransom & Endres ADDIN REFMGR.CITE <Refman><Cite ExcludeAuth="1"><Author>Ransom</Author><Year>2014</Year><RecNum>1145</RecNum><IDText>Corn growth and management quick guide</IDText><MDL Ref_Type="Pamphlet"><Ref_Type>Pamphlet</Ref_Type><Ref_ID>1145</Ref_ID><Title_Primary>Corn growth and management quick guide</Title_Primary><Authors_Primary>Ransom,J.</Authors_Primary><Authors_Primary>Endres,G.J.</Authors_Primary><Date_Primary>2014</Date_Primary><Reprint>In File</Reprint><Publisher>North Dakota State University Extension Service</Publisher><Misc_1>A-1173</Misc_1><Web_URL><u>:\References\GM References_in RefMan<u>\Ransom </u>and<u> Endres_2014_corn stages.pdf</u></Web_URL_Link2><ZZ_WorkformID>21</ZZ_WorkformID></MDL></Cite></Refman>(2014)2LOQ = limit of quantitation3Forage is the above ground plant parts used for animal feed.For Cry34Ab1, Cry35Ab1 and PAT, mean levels were lowest in the pollen (9.2, 0.34 ?g/g dry weight and <LOQ respectively). The highest levels of these three proteins were in the leaf samples at either the R1 or R4 stages. In contrast, the level of Cry1F was lowest in leaf tissue at R6 (the stage at which grain is harvested) and highest in pollen (35 ?g/g dry weight).The levels of the four proteins in line 4114 were compared to levels in line 1507, line 59122 and the breeding stack of these parental lines, by dividing the levels in line 4114 by the respective protein levels in line 1507, line 59122 and1507 x 59122 to provide an ‘expression ratio’ (see Table 5) i.e. a ratio of close to 1 indicated comparable levels. For all proteins, the levels in line 4114 were comparable to, or lower than, the levels in the other lines with the following exceptions (see yellow highlight in Table 5):Cry1F was higher in pollen and R6 leaf of line 4114 than in the same tissues from either comparator line.Cry34Ab1 was higher in R6 leaf of line 4114 than in R6 leaf of either comparator line.Cry35Ab1 was higher in R6 leaf of line 4114 than in R6 leaf of line 59122PAT was higher in R6 leaf of line 4114 than in all three comparator lines.PAT was higher in all tissue types (except R6 root, pollen and grain.) of line 4114 than in the same tissue types of line1507.These differences are likely to be explained by event-to-event variation and do not impact on food safety where the grain is the major source of food products. Table SEQ Table \* ARABIC 5: Summary of line 4114 protein concentrations as a ratio of line 1507, line 59122 and 1507 x 591224.1.4Characterisation of the plant-produced proteinsThe expected characteristics of the four expressed novel proteins are discussed in Sections 4.1.1. and 4.1.2. A translation of the DNA insert in line 4114 indicated that the amino acid sequences of the encoded Cry1F, Cry34Ab1, Cry35Ab1 and PAT proteins are as provided in Figures 5-7 and are identical to the sequences found in lines 1507 and 59122. A full characterisation of the proteins in planta had been done for lines 1507 and 59122 ADDIN REFMGR.CITE <Refman><Cite><Author>FSANZ</Author><Year>2003</Year><RecNum>354</RecNum><IDText>Application A446: Food derived from Insect-Protected and Glufosinate Ammonium-Tolerant Corn Line 1507</IDText><MDL Ref_Type="Report"><Ref_Type>Report</Ref_Type><Ref_ID>354</Ref_ID><Title_Primary>Application A446: Food derived from Insect-Protected and Glufosinate Ammonium-Tolerant Corn Line 1507</Title_Primary><Authors_Primary>FSANZ</Authors_Primary><Date_Primary>2003</Date_Primary><Reprint>Not in File</Reprint><Publisher>Report prepared by Food Standards Australia New Zealand</Publisher><Web_URL><u> A543: Food derived from Insect-Protected, Glufosinate Ammonium-Tolerant Corn Line DAS-59122-7</IDText><MDL Ref_Type="Report"><Ref_Type>Report</Ref_Type><Ref_ID>355</Ref_ID><Title_Primary>Application A543: Food derived from Insect-Protected, Glufosinate Ammonium-Tolerant Corn Line DAS-59122-7</Title_Primary><Authors_Primary>FSANZ</Authors_Primary><Date_Primary>2005</Date_Primary><Reprint>Not in File</Reprint><Publisher>Report prepared by Food Standards Australia New Zealand</Publisher><Web_URL><u>;(FSANZ 2003; FSANZ 2005) and therefore for line 4114 it was only necessary to confirm whether the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins expressed in line 4114 are equivalent to those expressed in the breeding stack 1507 x 59122. Accordingly, the Applicant used a Western blot analysis to do this.Study submitted:2011. Expressed trait protein concentration of a maize line containing events DP-004114-3, DAS-01507-1, DAS-59122-7, and a combined trait product DAS-01507-1 x DAS-59122-7: US and Canada test sites. Study ID PHI-2010-059. Pioneer Hi-Bred International, Inc (unpublished).2011. Western Blot Analysis of Maize Lines Containing Event DP-??4114-3 and Combined Trait Product DAS-?15?7-1xDAS-59122-7. Study ID PHI-2011-067. Pioneer Hi-Bred International, Inc (unpublished).Leaf samples were collected at the R1 growth stage from plants of each of line 4114 (generation F1*5), the breeding stack 1507 x 59122 and a verified non-GM control line. Crude extracts of proteins were prepared by extraction in buffer, before separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by transfer to polyvinylidene fluoride membranes. Each novel protein was then visualised following incubation in an appropriate monoclonal mouse primary antibody and a horseradish peroxidase-linked anti-mouse secondary antibody. There was no detection of any of the novel proteins in the samples from the control.For Cry1F, the samples from both line 4114 and the breeding stack showed a double banding pattern with sizes of approximately 60 kDa and 62 kDa. This doublet was not unexpected since it had also been observed for the Cry1F protein in line 1507 where the following explanation was given in the safety assessment ADDIN REFMGR.CITE <Refman><Cite><Author>FSANZ</Author><Year>2003</Year><RecNum>354</RecNum><IDText>Application A446: Food derived from Insect-Protected and Glufosinate Ammonium-Tolerant Corn Line 1507</IDText><MDL Ref_Type="Report"><Ref_Type>Report</Ref_Type><Ref_ID>354</Ref_ID><Title_Primary>Application A446: Food derived from Insect-Protected and Glufosinate Ammonium-Tolerant Corn Line 1507</Title_Primary><Authors_Primary>FSANZ</Authors_Primary><Date_Primary>2003</Date_Primary><Reprint>Not in File</Reprint><Publisher>Report prepared by Food Standards Australia New Zealand</Publisher><Web_URL><u>;(FSANZ 2003) - “The results of the analyses of Cry1F protein expression in plant tissues demonstrated that under denaturing conditions the Cry1F protein was detected as two bands with almost identical mobility (a doublet) of approximately 65 to 68 kDa in leaf, pollen, grain and whole plant tissue”. No other bands indicative of a partial Cry1F protein or a fusion protein of greater molecular size were observed. Due to the presence of the known enzyme cleavage sites near the amino-terminus of the protein, the doublet most likely resulted from limited N-terminal processing by a plant protease with trypsin-like specificity. It is noted that the protein obtained from a bacterial source, and used as a standard, appears as a single immunoreactive band. Importantly, the Western blot confirms the equivalent immunoreactivity of the Cry1F protein from line 4114 and the breeding stack.Similarly, for the Cry35Ab1 protein, a doublet (approximately 40 kDa and 44 kDa) was also observed in samples from both line 4114 and the breeding stack. Again, this doublet was also noted for the Cry35Ab1 protein in line 59122 ADDIN REFMGR.CITE <Refman><Cite><Author>FSANZ</Author><Year>2005</Year><RecNum>355</RecNum><IDText>Application A543: Food derived from Insect-Protected, Glufosinate Ammonium-Tolerant Corn Line DAS-59122-7</IDText><MDL Ref_Type="Report"><Ref_Type>Report</Ref_Type><Ref_ID>355</Ref_ID><Title_Primary>Application A543: Food derived from Insect-Protected, Glufosinate Ammonium-Tolerant Corn Line DAS-59122-7</Title_Primary><Authors_Primary>FSANZ</Authors_Primary><Date_Primary>2005</Date_Primary><Reprint>Not in File</Reprint><Publisher>Report prepared by Food Standards Australia New Zealand</Publisher><Web_URL><u>;(FSANZ 2005) – “the full length upper band protein of the 44-kDa is originally expressed. However, the protein is susceptible to the proteases in plant cells, resulting in the lower band form”. As for the Cry1F protein, there was only a single immunoreactive band for the bacterially-produced protein standard but, importantly the Western blot confirms the equivalent immunoreactivity of the Cry35Ab1 protein from line 4114 and the breeding stack.The Western blots for both the Cry34Ab1 and PAT proteins showed single bands at approximately 14 kDa and 21 kDa respectively.Overall, the results confirm that the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins expressed in line 4114 have the approximate molecular weights expected of the proteins (within the limitations of a Western blot for estimating molecular weight) and that each protein has the same immunoreactivity as the equivalent protein expressed in the 1507 x 59122 breeding stack.4.1.5The safety of the introduced proteinsAll of the introduced novel proteins have been assessed by FSANZ in a number of previous applications and all have been considered safe. Cry1F occurs in line 1507 which was assessed in Application A446 ADDIN REFMGR.CITE <Refman><Cite><Author>FSANZ</Author><Year>2003</Year><RecNum>354</RecNum><IDText>Application A446: Food derived from Insect-Protected and Glufosinate Ammonium-Tolerant Corn Line 1507</IDText><MDL Ref_Type="Report"><Ref_Type>Report</Ref_Type><Ref_ID>354</Ref_ID><Title_Primary>Application A446: Food derived from Insect-Protected and Glufosinate Ammonium-Tolerant Corn Line 1507</Title_Primary><Authors_Primary>FSANZ</Authors_Primary><Date_Primary>2003</Date_Primary><Reprint>Not in File</Reprint><Publisher>Report prepared by Food Standards Australia New Zealand</Publisher><Web_URL><u>;(FSANZ 2003).Cry34Ab1 and Cry35Ab1 occur in line 59122 which was assessed in Application A543 ADDIN REFMGR.CITE <Refman><Cite><Author>FSANZ</Author><Year>2005</Year><RecNum>355</RecNum><IDText>Application A543: Food derived from Insect-Protected, Glufosinate Ammonium-Tolerant Corn Line DAS-59122-7</IDText><MDL Ref_Type="Report"><Ref_Type>Report</Ref_Type><Ref_ID>355</Ref_ID><Title_Primary>Application A543: Food derived from Insect-Protected, Glufosinate Ammonium-Tolerant Corn Line DAS-59122-7</Title_Primary><Authors_Primary>FSANZ</Authors_Primary><Date_Primary>2005</Date_Primary><Reprint>Not in File</Reprint><Publisher>Report prepared by Food Standards Australia New Zealand</Publisher><Web_URL><u>;(FSANZ 2005).The PAT protein, encoded by either the pat or bar genes ADDIN REFMGR.CITE <Refman><Cite><Author>Wehrmann</Author><Year>1996</Year><RecNum>317</RecNum><IDText>The similarities of bar and pat gene products make them equally applicable for plant engineers</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>317</Ref_ID><Title_Primary>The similarities of <i>bar</i> and <i>pat</i> gene products make them equally applicable for plant engineers</Title_Primary><Authors_Primary>Wehrmann,A.</Authors_Primary><Authors_Primary>Van Vliet,A.</Authors_Primary><Authors_Primary>Opsomer,C.</Authors_Primary><Authors_Primary>Botterman,J.</Authors_Primary><Authors_Primary>Schulz,A.</Authors_Primary><Date_Primary>1996</Date_Primary><Keywords>Soybeans</Keywords><Keywords>Genes</Keywords><Keywords>Genetic Engineering</Keywords><Keywords>Escherichia coli</Keywords><Keywords>Proteins</Keywords><Keywords>Plants</Keywords><Keywords>Molecular Weight</Keywords><Keywords>Digestion</Keywords><Keywords>Human</Keywords><Reprint>In File</Reprint><Start_Page>1274</Start_Page><End_Page>1278</End_Page><Periodical>Nature Biotechnology</Periodical><Volume>14</Volume><Web_URL_Link2>file://Y:\References\GM References_in RefMan<u>\Wehrmann et al_1996_bar and pat.pdf</u></Web_URL_Link2><ZZ_JournalFull><f name="System">Nature Biotechnology</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(Wehrmann et al. 1996), has now been considered in 19 FSANZ approvals (A372, A375, A380, A385, A386, A446, A481, A518, A533, A543, A589, A1028, A1040, A1046, A1073 A1080, A1081, A1087 and A1094) as well as being accepted in the literature as having neither toxicity nor allergenicity concerns PFJlZm1hbj48Q2l0ZT48QXV0aG9yPkRlbGFuZXk8L0F1dGhvcj48WWVhcj4yMDA4PC9ZZWFyPjxS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ADDIN REFMGR.CITE PFJlZm1hbj48Q2l0ZT48QXV0aG9yPkRlbGFuZXk8L0F1dGhvcj48WWVhcj4yMDA4PC9ZZWFyPjxS
ZWNOdW0+Mjg4PC9SZWNOdW0+PElEVGV4dD5FdmFsdWF0aW9uIG9mIHByb3RlaW4gc2FmZXR5IGlu
IHRoZSBjb250ZXh0IG9mIGFncmljdWx0dXJhbCBiaW90ZWNobm9sb2d5PC9JRFRleHQ+PFByZWZp
eD5zZWUgZS5nLiA8L1ByZWZpeD48TURMIFJlZl9UeXBlPSJKb3VybmFsIj48UmVmX1R5cGU+Sm91
cm5hbDwvUmVmX1R5cGU+PFJlZl9JRD4yODg8L1JlZl9JRD48VGl0bGVfUHJpbWFyeT5FdmFsdWF0
aW9uIG9mIHByb3RlaW4gc2FmZXR5IGluIHRoZSBjb250ZXh0IG9mIGFncmljdWx0dXJhbCBiaW90
ZWNobm9sb2d5PC9UaXRsZV9QcmltYXJ5PjxBdXRob3JzX1ByaW1hcnk+RGVsYW5leSxCLjwvQXV0
aG9yc19QcmltYXJ5PjxBdXRob3JzX1ByaW1hcnk+QXN0d29vZCxKLkQuPC9BdXRob3JzX1ByaW1h
cnk+PEF1dGhvcnNfUHJpbWFyeT5DdW5ueSxILjwvQXV0aG9yc19QcmltYXJ5PjxBdXRob3JzX1By
aW1hcnk+RWljaGVuIENvbm4sUi48L0F1dGhvcnNfUHJpbWFyeT48QXV0aG9yc19QcmltYXJ5Pkhl
cm91ZXQtR3VpY2hlbmV5LEMuPC9BdXRob3JzX1ByaW1hcnk+PEF1dGhvcnNfUHJpbWFyeT5NYWNJ
bnRvc2gsUy48L0F1dGhvcnNfUHJpbWFyeT48QXV0aG9yc19QcmltYXJ5Pk1leWVyLEwuUy48L0F1
dGhvcnNfUHJpbWFyeT48QXV0aG9yc19QcmltYXJ5PlByaXZhbGxlLEwuUy48L0F1dGhvcnNfUHJp
bWFyeT48QXV0aG9yc19QcmltYXJ5PkdhbyxZLjwvQXV0aG9yc19QcmltYXJ5PjxBdXRob3JzX1By
aW1hcnk+TWF0dHNzb24sSi48L0F1dGhvcnNfUHJpbWFyeT48QXV0aG9yc19QcmltYXJ5Pkxldmlu
ZSxNLjwvQXV0aG9yc19QcmltYXJ5PjxBdXRob3JzX1ByaW1hcnk+SUxTSTwvQXV0aG9yc19Qcmlt
YXJ5PjxEYXRlX1ByaW1hcnk+MjAwODwvRGF0ZV9QcmltYXJ5PjxLZXl3b3Jkcz5TYWZldHk8L0tl
eXdvcmRzPjxLZXl3b3Jkcz5Qcm90ZWluczwvS2V5d29yZHM+PEtleXdvcmRzPm1ldGhvZHM8L0tl
eXdvcmRzPjxLZXl3b3Jkcz5BbWlubyBBY2lkIFNlcXVlbmNlPC9LZXl3b3Jkcz48UmVwcmludD5J
biBGaWxlPC9SZXByaW50PjxTdGFydF9QYWdlPlM3MTwvU3RhcnRfUGFnZT48RW5kX1BhZ2U+Uzk3
PC9FbmRfUGFnZT48UGVyaW9kaWNhbD5Gb29kIGFuZCBDaGVtaWNhbCBUb3hpY29sb2d5PC9QZXJp
b2RpY2FsPjxWb2x1bWU+NDY8L1ZvbHVtZT48V2ViX1VSTF9MaW5rMz5maWxlOi8vWTpcUmVmZXJl
bmNlc1xHTSBSZWZlcmVuY2VzX2luIFJlZk1hbjx1PlxEZWxhbmV5IGV0IGFsXzIwMDhfUHJvdGVp
biBTYWZldHkucGRmPC91PjwvV2ViX1VSTF9MaW5rMz48WlpfSm91cm5hbEZ1bGw+PGYgbmFtZT0i
U3lzdGVtIj5Gb29kIGFuZCBDaGVtaWNhbCBUb3hpY29sb2d5PC9mPjwvWlpfSm91cm5hbEZ1bGw+
PFpaX1dvcmtmb3JtSUQ+MTwvWlpfV29ya2Zvcm1JRD48L01ETD48L0NpdGU+PENpdGU+PEF1dGhv
cj5Iw6lyb3VldDwvQXV0aG9yPjxZZWFyPjIwMDU8L1llYXI+PFJlY051bT4zOTU8L1JlY051bT48
SURUZXh0PlNhZmV0eSBldmFsdWF0aW9uIG9mIHRoZSBwaG9zcGhpbm90aHJpY2luIGFjZXR5bHRy
YW5zZmVyYXNlIHByb3RlaW5zIGVuY29kZWQgYnkgdGhlIHBhdCBhbmQgYmFyIHNlcXVlbmNlcyB0
aGF0IGNvbmZlciB0b2xlcmFuY2UgdG8gZ2x1Zm9zaW5hdGUtYW1tb25pdW0gaGVyYmljaWRlIGlu
IHRyYW5zZ2VuaWMgcGxhbnRzPC9JRFRleHQ+PE1ETCBSZWZfVHlwZT0iSm91cm5hbCI+PFJlZl9U
eXBlPkpvdXJuYWw8L1JlZl9UeXBlPjxSZWZfSUQ+Mzk1PC9SZWZfSUQ+PFRpdGxlX1ByaW1hcnk+
U2FmZXR5IGV2YWx1YXRpb24gb2YgdGhlIHBob3NwaGlub3RocmljaW4gYWNldHlsdHJhbnNmZXJh
c2UgcHJvdGVpbnMgZW5jb2RlZCBieSB0aGUgPGk+cGF0PC9pPiBhbmQgPGk+YmFyPC9pPiBzZXF1
ZW5jZXMgdGhhdCBjb25mZXIgdG9sZXJhbmNlIHRvIGdsdWZvc2luYXRlLWFtbW9uaXVtIGhlcmJp
Y2lkZSBpbiB0cmFuc2dlbmljIHBsYW50czwvVGl0bGVfUHJpbWFyeT48QXV0aG9yc19QcmltYXJ5
PkgmI3hFOTtyb3VldCxDLjwvQXV0aG9yc19QcmltYXJ5PjxBdXRob3JzX1ByaW1hcnk+RXNkYWls
ZSxELkouPC9BdXRob3JzX1ByaW1hcnk+PEF1dGhvcnNfUHJpbWFyeT5NYWxseW9uLEIuQS48L0F1
dGhvcnNfUHJpbWFyeT48QXV0aG9yc19QcmltYXJ5PkRlYnJ1eW5lLEUuPC9BdXRob3JzX1ByaW1h
cnk+PEF1dGhvcnNfUHJpbWFyeT5TY2h1bHosQS48L0F1dGhvcnNfUHJpbWFyeT48QXV0aG9yc19Q
cmltYXJ5PkN1cnJpZXIsVC48L0F1dGhvcnNfUHJpbWFyeT48QXV0aG9yc19QcmltYXJ5PkhlbmRy
aWNreCxLLjwvQXV0aG9yc19QcmltYXJ5PjxBdXRob3JzX1ByaW1hcnk+dmFuIGRlciBLbGlzLFIu
LUouPC9BdXRob3JzX1ByaW1hcnk+PEF1dGhvcnNfUHJpbWFyeT5Sb3VhbixELjwvQXV0aG9yc19Q
cmltYXJ5PjxEYXRlX1ByaW1hcnk+MjAwNTwvRGF0ZV9QcmltYXJ5PjxLZXl3b3Jkcz5TYWZldHk8
L0tleXdvcmRzPjxLZXl3b3Jkcz5Qcm90ZWluczwvS2V5d29yZHM+PEtleXdvcmRzPmdsdWZvc2lu
YXRlIGFtbW9uaXVtPC9LZXl3b3Jkcz48S2V5d29yZHM+UGxhbnRzPC9LZXl3b3Jkcz48UmVwcmlu
dD5JbiBGaWxlPC9SZXByaW50PjxTdGFydF9QYWdlPjEzNDwvU3RhcnRfUGFnZT48RW5kX1BhZ2U+
MTQ5PC9FbmRfUGFnZT48UGVyaW9kaWNhbD5SZWd1bGF0b3J5IFRveGljb2xvZ3kgYW5kIFBoYXJt
YWNvbG9neTwvUGVyaW9kaWNhbD48Vm9sdW1lPjQxPC9Wb2x1bWU+PFdlYl9VUkw+PHU+aHR0cDov
L3d3dy5zY2llbmNlZGlyZWN0LmNvbS9zY2llbmNlP19vYj1NSW1nJmFtcDtfaW1hZ2VrZXk9QjZX
UFQtNEY4VFcxQy0xLUMmYW1wO19jZGk9Njk5OSZhbXA7X3VzZXI9NjcxMjQ0NyZhbXA7X29yaWc9
c2VhcmNoJmFtcDtfY292ZXJEYXRlPTAzJTJGMDElMkYyMDA1JmFtcDtfc2s9OTk5NTg5OTk3JmFt
cDt2aWV3PWMmYW1wO3djaHA9ZEdMelZ0Yi16U2tXQSZhbXA7bWQ1PThlOTkwOTZkZmVkOGNiYzMy
NGQwMTVkODQ5MGVhMjU4JmFtcDtpZT0vc2RhcnRpY2xlLnBkZjwvdT48L1dlYl9VUkw+PFdlYl9V
UkxfTGluazI+ZmlsZTovL1k6XFJlZmVyZW5jZXNcR00gUmVmZXJlbmNlc19pbiBSZWZNYW48dT5c
SGVyb3VldCBldCBhbF8yMDA1X3NhZmV0eSBvZiBQQVQucGRmPC91PjwvV2ViX1VSTF9MaW5rMj48
WlpfSm91cm5hbEZ1bGw+PGYgbmFtZT0iU3lzdGVtIj5SZWd1bGF0b3J5IFRveGljb2xvZ3kgYW5k
IFBoYXJtYWNvbG9neTwvZj48L1paX0pvdXJuYWxGdWxsPjxaWl9Xb3JrZm9ybUlEPjE8L1paX1dv
cmtmb3JtSUQ+PC9NREw+PC9DaXRlPjwvUmVmbWFuPgB=
ADDIN EN.CITE.DATA (Hérouet et al. 2005; see e.g. Delaney et al. 2008).A summary of these previous characterisations is provided in Table 6 which shows the application in which the most recent detailed study or information was considered by FSANZ and is available on the FSANZ website. For the bioinformatic studies, which analyse sequence similarity to known protein toxins and allergens, where the Applicant provided searches (see updated studies listed below) using a more recent (and hence larger) database, the results did not alter the conclusions reached previously that Cry1F, Cry34Ab1, Cry35Ab1 and PAT are not allergenic or toxic.Table SEQ Table \* ARABIC 6: Summary of consideration of Cry1F, Cry34Ab1, Cry35Ab1 and PAT in previous FSANZ safety assessmentsConsiderationSub-sectionCry1F (link)Cry34Ab1 & Cry35Ab1 (link)PAT (link)Potential toxicityHistory of human consumptionA446 (FSANZ, 2003)A543 (FSANZ, 2005)A1087 ADDIN REFMGR.CITE <Refman><Cite><Author>FSANZ</Author><Year>2013</Year><RecNum>1556</RecNum><IDText>Application A1087 - Food derived from insect-protected soybean line DAS-81419-2</IDText><MDL Ref_Type="Report"><Ref_Type>Report</Ref_Type><Ref_ID>1556</Ref_ID><Title_Primary>Application A1087 - Food derived from insect-protected soybean line DAS-81419-2</Title_Primary><Authors_Primary>FSANZ</Authors_Primary><Date_Primary>2013</Date_Primary><Keywords>soybean</Keywords><Reprint>Not in File</Reprint><Publisher>Report prepared by Food Standards Australia New Zealand</Publisher><Web_URL><u>;(FSANZ 2013a)Amino acid sequence similarity to protein toxinsThis application – using search updated in June 2014This application – using search updated in July 2014This application – using search updated in June 2014Stability to heatA446 (FSANZ, 2003)A543 (FSANZ, 2005)A1080 ADDIN REFMGR.CITE <Refman><Cite><Author>FSANZ</Author><Year>2013</Year><RecNum>1557</RecNum><IDText>Application A1080 - Food derived from herbicide-tolerant cotton line MON88701</IDText><MDL Ref_Type="Report"><Ref_Type>Report</Ref_Type><Ref_ID>1557</Ref_ID><Title_Primary>Application A1080 - Food derived from herbicide-tolerant cotton line MON88701</Title_Primary><Authors_Primary>FSANZ</Authors_Primary><Date_Primary>2013</Date_Primary><Reprint>Not in File</Reprint><Publisher>Report prepared by Food Standards Australia New Zealand</Publisher><Web_URL><u>;(FSANZ 2013b)Acute oral toxicityA446 (FSANZ, 2003)A543 (FSANZ, 2005)A1080 ADDIN REFMGR.CITE <Refman><Cite><Author>FSANZ</Author><Year>2013</Year><RecNum>1557</RecNum><IDText>Application A1080 - Food derived from herbicide-tolerant cotton line MON88701</IDText><MDL Ref_Type="Report"><Ref_Type>Report</Ref_Type><Ref_ID>1557</Ref_ID><Title_Primary>Application A1080 - Food derived from herbicide-tolerant cotton line MON88701</Title_Primary><Authors_Primary>FSANZ</Authors_Primary><Date_Primary>2013</Date_Primary><Reprint>Not in File</Reprint><Publisher>Report prepared by Food Standards Australia New Zealand</Publisher><Web_URL><u>;(FSANZ 2013b)Potential allergenicityIn vitro digestibilityA446 (FSANZ, 2003)A543 (FSANZ, 2005)A1080 ADDIN REFMGR.CITE <Refman><Cite><Author>FSANZ</Author><Year>2013</Year><RecNum>1557</RecNum><IDText>Application A1080 - Food derived from herbicide-tolerant cotton line MON88701</IDText><MDL Ref_Type="Report"><Ref_Type>Report</Ref_Type><Ref_ID>1557</Ref_ID><Title_Primary>Application A1080 - Food derived from herbicide-tolerant cotton line MON88701</Title_Primary><Authors_Primary>FSANZ</Authors_Primary><Date_Primary>2013</Date_Primary><Reprint>Not in File</Reprint><Publisher>Report prepared by Food Standards Australia New Zealand</Publisher><Web_URL><u>;(FSANZ 2013b)Amino acid sequence similarity to allergensThis application – using search updated in April 2014This application – using search updated in April 2014This application – using search updated in April 2014Updated bioinformatics studies submitted2014. Evaluation of the Amino Acid Sequence Similarity of the Cry1F Protein to the NCBI Protein Sequence Datasets. Study ID PHI-2008-240/074. Pioneer Hi-Bred International, Inc (unpublished).2014. Evaluation of the Amino Acid Sequence Similarity of the Cry34Ab1 and Cry35Ab1 Protein to the NCBI Protein Sequence Datasets. Study ID PHI-2008-243/074. Pioneer Hi-Bred International, Inc (unpublished).2014. Evaluation of the Amino Acid Sequence Similarity of the PAT Protein to the NCBI Protein Sequence Datasets. Study ID PHI-2008-242/074. Pioneer Hi-Bred International, Inc (unpublished).2014. Comparison of the Amino Acid Sequence Identity Between the Cry1F Protein and Known Protein Allergens (≥ 35% Identity over ≥ 80 Amino Acids and 8 Amino Acid Exact Match as Search Criteria). Study ID PHI-2008-236/074. Pioneer Hi-Bred International, Inc (unpublished).2014. Comparison of the Amino Acid Sequence Identity Between the Cry34Ab1 and Cry35Ab1 Proteins and Known and Putative Protein Allergens (≥ 35% Identity over ≥ 80 Amino Acids and 8 Amino Acid Exact Match as Search Criteria). Study ID PHI-2008-237/074. Pioneer Hi-Bred International, Inc (unpublished).2014. Comparison of the Amino Acid Sequence Identity Between the PAT Protein and Known and Putative Protein Allergens (≥ 35% Identity over ≥ 80 Amino Acids and 8 Amino Acid Exact Match as Search Criteria). Study ID PHI-2008-238/074. Pioneer Hi-Bred International, Inc (unpublished).4.1.6Bioinformatic analysis of additional ORFs created by the transformation procedure Study submitted:2015. Junction Reading Frame Analysis at the Insertion Site of maize Event DP-004114-3. Study ID PHI-R036-Y15. Pioneer Hi-Bred International, Inc (unpublished) A bioinformatics analysis was performed to assess the similarity to known allergens and toxins of the putative 99 amino acid polypeptide encoded by the sequence obtained from the ORF analysis (refer to Section 3.4.3). To evaluate the similarity to known allergens of proteins that might potentially be produced by translation of the ORF, an epitope search was carried out to identify any short sequences of amino acids that might represent an isolated shared allergenic epitope. This search compared the sequences with known allergens residing in the FARRP (Food Allergy Research and Resource Program) dataset within version 15 of AllergenOnline (University of Nebraska). The Fast Alignment Search Tool - All (FASTA) algorithm ADDIN REFMGR.CITE <Refman><Cite><Author>Pearson</Author><Year>1988</Year><RecNum>152</RecNum><IDText>Improved tools for biological sequence comparison</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>152</Ref_ID><Title_Primary>Improved tools for biological sequence comparison</Title_Primary><Authors_Primary>Pearson,W.R.</Authors_Primary><Authors_Primary>Lipman,D.J.</Authors_Primary><Date_Primary>1988/4</Date_Primary><Keywords>Amino Acid Sequence</Keywords><Keywords>Animals</Keywords><Keywords>Base Sequence</Keywords><Keywords>Comparative Study</Keywords><Keywords>Dna</Keywords><Keywords>genetics</Keywords><Keywords>Globins</Keywords><Keywords>Humans</Keywords><Keywords>Information Systems</Keywords><Keywords>Molecular Sequence Data</Keywords><Keywords>Peptides</Keywords><Keywords>Receptors,Antigen,T-Cell</Keywords><Keywords>Sequence Homology,Nucleic Acid</Keywords><Keywords>Software</Keywords><Keywords>Transforming Growth Factors</Keywords><Reprint>Not in File</Reprint><Start_Page>2444</Start_Page><End_Page>2448</End_Page><Periodical>Proceedings of the National Academy of Sciences</Periodical><Volume>85</Volume><Issue>8</Issue><Address>Department of Biochemistry, University of Virginia, Charlottesville 22908</Address><Web_URL>PM:3162770</Web_URL><Web_URL_Link1><u>file://F:\Standards Management\APPLICATIONS\A1001-A1050\A1018 High Oleic GM Soybean\A1018 Application\A1018 Literature References\Improved_tools_for_biological_sequence_comparison_-_pdf.PDF</u></Web_URL_Link1><Web_URL_Link2>file://Y:\References\GM References_in RefMan<u>\Pearson </u>and<u> Lipman_1988_tools for sequence comparison.pdf</u></Web_URL_Link2><ZZ_JournalFull><f name="System">Proceedings of the National Academy of Sciences</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(Pearson and Lipman 1988), version 35.4.4, was used to search the database. No alignments generated an E-score of ≤1e-4, no alignment met or exceeded the Codex Alimentarius ADDIN REFMGR.CITE <Refman><Cite><Author>Codex</Author><Year>2009</Year><RecNum>1656</RecNum><IDText>Foods derived from modern biotechnology, 2nd edition</IDText><MDL Ref_Type="Report"><Ref_Type>Report</Ref_Type><Ref_ID>1656</Ref_ID><Title_Primary>Foods derived from modern biotechnology, 2nd edition</Title_Primary><Authors_Primary>Codex</Authors_Primary><Date_Primary>2009</Date_Primary><Keywords>Biotechnology</Keywords><Reprint>Not in File</Reprint><Pub_Place>Rome</Pub_Place><Publisher>Codex Alimentarius Commission, Joint FAO/WHO Food Standards Programme, Food and Agriculture Organization of the United Nations</Publisher><Web_URL><u>;\References\GM References_in RefMan\Codex_2009_food derived from modern biotech.pdf</u></Web_URL_Link2><ZZ_WorkformID>24</ZZ_WorkformID></MDL></Cite></Refman>(Codex 2009) FASTA alignment threshold for potential allergenicity (35% identity over 80 amino acids) and no alignments of eight or more consecutive identical amino acids ADDIN REFMGR.CITE <Refman><Cite><Author>Metcalfe</Author><Year>1996</Year><RecNum>39</RecNum><IDText>Assessment of the allergenic potential of foods derived from genetically engineered crop plants</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>39</Ref_ID><Title_Primary>Assessment of the allergenic potential of foods derived from genetically engineered crop plants</Title_Primary><Authors_Primary>Metcalfe,D.D.</Authors_Primary><Authors_Primary>Astwood,J.D.</Authors_Primary><Authors_Primary>Townsend,R.</Authors_Primary><Authors_Primary>Sampson,H.A.</Authors_Primary><Authors_Primary>Taylor,S.L.</Authors_Primary><Authors_Primary>Fuchs,R.L.</Authors_Primary><Date_Primary>1996</Date_Primary><Keywords>allergenicity</Keywords><Keywords>Allergens</Keywords><Keywords>Amino Acid Sequence</Keywords><Keywords>Animals</Keywords><Keywords>chemistry</Keywords><Keywords>Drug Stability</Keywords><Keywords>Genetic Engineering</Keywords><Keywords>genetics</Keywords><Keywords>Human</Keywords><Keywords>immunology</Keywords><Keywords>Molecular Sequence Data</Keywords><Keywords>Plant Proteins</Keywords><Keywords>Plants</Keywords><Keywords>Plants,Edible</Keywords><Reprint>Not in File</Reprint><Start_Page>S165</Start_Page><End_Page>S186</End_Page><Periodical>Critical Reviews in Food Science and Nutrition</Periodical><Volume>36 Suppl</Volume><Address>ILSI Allergy and Immunology Institute, USA</Address><Web_URL>PM:8959382</Web_URL><Web_URL_Link2>file://Y:\References\GM References_in RefMan<u>\Metcalfe et al. 1996.pdf</u></Web_URL_Link2><ZZ_JournalFull><f name="System">Critical Reviews in Food Science and Nutrition</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(Metcalfe et al. 1996) were found. It was concluded that the ORF does not contain any cross-reactive Immunoglobulin E (IgE) binding epitopes with known allergens.The ORF amino acid sequence was also compared with non-redundant sequences present in the National Center for Biotechnology (NCBI) database which incorporated entries from several databases including GenBank, RefSeq, Swiss-Prot, Protein Information Resource, Protein Research Foundation and Worldwide Protein Data Bank database. The search used the BLASTP (Basic Local Alignment Search Tool Protein – version 2.2.26) ADDIN REFMGR.CITE <Refman><Cite><Author>Altschul</Author><Year>1997</Year><RecNum>174</RecNum><IDText>Gapped BLAST and PSI-BLAST: a new generation of protein database search programs</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>174</Ref_ID><Title_Primary>Gapped BLAST and PSI-BLAST: a new generation of protein database search programs</Title_Primary><Authors_Primary>Altschul,S.F.</Authors_Primary><Authors_Primary>Madden,T.L.</Authors_Primary><Authors_Primary>Schaffer,A.A.</Authors_Primary><Authors_Primary>Zhang,J.</Authors_Primary><Authors_Primary>Zhang,Z.</Authors_Primary><Authors_Primary>Miller,W.</Authors_Primary><Authors_Primary>Lipman,D.J.</Authors_Primary><Date_Primary>1997/9/1</Date_Primary><Keywords>Algorithms</Keywords><Keywords>Amino Acid Sequence</Keywords><Keywords>Animals</Keywords><Keywords>chemistry</Keywords><Keywords>Databases,Factual</Keywords><Keywords>Dna</Keywords><Keywords>Humans</Keywords><Keywords>Molecular Sequence Data</Keywords><Keywords>Proteins</Keywords><Keywords>Sequence Alignment</Keywords><Keywords>Software</Keywords><Reprint>In File</Reprint><Start_Page>3389</Start_Page><End_Page>3402</End_Page><Periodical>Nucleic Acids Research</Periodical><Volume>25</Volume><Issue>17</Issue><Address>National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA. altschul@ncbi.nlm.</Address><Web_URL><u>:\References\GM References_in RefMan<u>\Altschul et al_1997_gapped BLAST searches.mht</u></Web_URL_Link2><ZZ_JournalFull><f name="System">Nucleic Acids Research</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(Altschul et al. 1997) algorithm. No significant similarities of any sequence to any sequences in the databases (including those of known toxins) were found.It is concluded that, in the unlikely event the 99-amino acid potential ORF was expressed in line 4114, there is no significant similarity between the encoded sequence and any known protein toxins or allergens.4.1.7ConclusionCorn line 4114 is a molecular stack expressing four novel proteins, Cry1F, Cry34Ab1, Cry35Ab1 and PAT. These proteins have previously been assessed in two corn lines 1507 and 59122 where the coding regions and regulatory elements of the various gene cassettes are the same as in line 4114. Food from the breeding stack created from these two lines is also commercially available.For Cry34Ab1, Cry35Ab1 and PAT, mean levels were lowest in the pollen (9.2, 0.34 ?g/g dry weight and <LOQ respectively). The highest levels of these three proteins were in the leaf samples at either the R1 or R4 stages. In contrast, the level of Cry1F was lowest in leaf tissue at R6 (the stage at which grain is harvested) and highest in pollen (35 ?g/g dry weight). In the grain, levels of Cry1F, Cry34Ab1, Cry35Ab1 and PAT were 3.3, 24, 1.1 and <LOQ ?g/g dry weight respectively.Western blot analyses confirmed that the four novel proteins in line 4114 have the expected molecular weights and immunoreactivity and are equivalent to the corresponding proteins produced in the breeding stack 1507 x 59122.4.2Herbicide metabolitesAs part of the safety assessment it is important to establish whether the expression of a novel protein(s) is likely to result in the accumulation of any novel metabolites. If such substances are found to occur as a result of the genetic modification, then it is important to determine their potential toxicity.The glufosinate-tolerance trait is present in lines from 19 previous applications to FSANZ (see Section 4.1.5). The enzyme activity of PAT results in the acetylation of the free amino group of glufosinate to produce the non-herbicidal N-acetyl glufosinate. This is a well-known metabolite in glufosinate-tolerant plants and was previously considered in detail by FSANZ in cotton line LL25 (FSANZ 2006). There are no concerns that the spraying of line 4114 with glufosinate would result in the production of any novel metabolites that have not been previously assessed.5Compositional analysisThe main purpose of compositional analysis is to determine if, as a result of the genetic modification, any unexpected changes have occurred to the food. These changes could take the form of alterations in the composition of the plant and its tissues and thus its nutritional adequacy. Compositional analysis can also be important for evaluating the intended effect where there has been a deliberate change to the composition of the food.The classic approach to the compositional analysis of GM food is a targeted one. Rather than analysing every single constituent, which would be impractical, the aim is to analyse only those constituents most relevant to the safety of the food or that may have an impact on the whole diet. Important analytes therefore include the key nutrients, toxicants and anti-nutrients for the food in question. The key nutrients and anti-nutrients are those components in a particular food that may have a substantial impact in the overall diet. They may be major constituents (fats, proteins, carbohydrates or enzyme inhibitors such as anti-nutrients) or minor constituents (minerals, vitamins). Key toxicants are those toxicologically significant compounds known to be inherently present in an organism, such as compounds whose toxic potency and level may be significant to health (e.g. solanine in potatoes).5.1Key componentsFor corn there are a number of components that are considered to be important for compositional analysis ADDIN REFMGR.CITE <Refman><Cite><Author>OECD</Author><Year>2002</Year><RecNum>79</RecNum><IDText>Consensus document on compositional considerations for new varieties of maize (Zea mays): Key food and feed nutrients, anti-nutrients and secondary plant metabolites</IDText><MDL Ref_Type="Report"><Ref_Type>Report</Ref_Type><Ref_ID>79</Ref_ID><Title_Primary>Consensus document on compositional considerations for new varieties of maize (<i>Zea mays</i>): Key food and feed nutrients, anti-nutrients and secondary plant metabolites</Title_Primary><Authors_Primary>OECD</Authors_Primary><Date_Primary>2002</Date_Primary><Keywords>Zea mays</Keywords><Reprint>Not in File</Reprint><Pub_Place>Paris</Pub_Place><Publisher>Organisation for Economic Co-operation and Development</Publisher><Title_Series>Series on the safety of novel foods and feeds, No. 6</Title_Series><Web_URL><u>(2002)25&doclanguage=en</u></Web_URL><ZZ_WorkformID>24</ZZ_WorkformID></MDL></Cite></Refman>(OECD 2002). As a minimum, the key nutrients of corn grain appropriate for a comparative study include the proximates (crude protein, fat, ash, acid detergent fibre [ADF] and neutral detergent fibre [NDF]), amino acids and fatty acids. In addition, mineral and vitamin levels may be considered and international guidance also suggests that levels of the key anti-nutrients and secondary metabolites could be determined for new varieties of corn.Corn contains a number of anti-nutrients: phytic acid, raffinose, 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) and trypsin and chymotrypsin inhibitor. Only phytic acid and raffinose are considered to be biologically relevant (OECD 2002). DIMBOA is present at highly variable levels in corn hybrids and little evidence is available on either its toxicity or anti-nutritional effects, and corn contains only low levels of trypsin and chymotrypsin inhibitor, neither of which is considered nutritionally significant. Phytic acid is considered an important anti-nutrient for animals, especially non-ruminants, since it can significantly reduce the bioavailability of phosphorus in the plant. Feed formulators add the enzyme phytase to pig and poultry diets to improve the utilisation of phosphorus. Raffinose is a non-digestible oligosaccharide and is considered an anti-nutrient because of its gastrointestinal effects (flatulence).Secondary metabolites are defined as those natural products which do not function directly in the primary biochemical activities which support the growth, development and reproduction of the organism in which they occur. Secondary plant metabolites are neither nutrients nor anti-nutrients but are sometimes analysed as further indicators of the absence of unintended effects of the genetic modification on metabolism (OECD 2002). Characteristic metabolites in corn are furfural and the phenolic acids, ferulic acid and p-coumaric acid. There are no generally recognised anti-nutrients in corn at levels considered to be harmful, but for the purposes of comparative assessment, the OECD has recommended considering analytical data for the content of the anti-nutrients phytic acid and raffinose, and the secondary metabolites furfural, ferulic acid, p-coumaric acid.5.2Study design and conduct for key componentsStudy submitted:2011. Agronomic Characteristics and Nutrient Composition of a Maize Line Containing Event DP-??4114-3: US and Canada Test Sites. Study ID PHI-2010-058/021. Pioneer Hi-Bred International, Inc (unpublished).The line 4114 hybrid generation F1*5 (refer to Figure 4) was used for compositional analysis because it represents a commercial hybrid form of line 4114 that would be most applicable to food and feed use. Ideally, the comparator in compositional analyses should be the near isogenic parental line grown under identical conditions ADDIN REFMGR.CITE <Refman><Cite><Author>OECD</Author><Year>2002</Year><RecNum>79</RecNum><IDText>Consensus document on compositional considerations for new varieties of maize (Zea mays): Key food and feed nutrients, anti-nutrients and secondary plant metabolites</IDText><MDL Ref_Type="Report"><Ref_Type>Report</Ref_Type><Ref_ID>79</Ref_ID><Title_Primary>Consensus document on compositional considerations for new varieties of maize (<i>Zea mays</i>): Key food and feed nutrients, anti-nutrients and secondary plant metabolites</Title_Primary><Authors_Primary>OECD</Authors_Primary><Date_Primary>2002</Date_Primary><Keywords>Zea mays</Keywords><Reprint>Not in File</Reprint><Pub_Place>Paris</Pub_Place><Publisher>Organisation for Economic Co-operation and Development</Publisher><Title_Series>Series on the safety of novel foods and feeds, No. 6</Title_Series><Web_URL><u>(2002)25&doclanguage=en</u></Web_URL><ZZ_WorkformID>24</ZZ_WorkformID></MDL></Cite></Refman>(OECD 2002). In the case of line 4114, the control was the hybrid produced by crossing PHNARxPHTFE since this represents the closest non-GM genetic line to F1*5 for the purposes of comparison. The test and control lines were grown from verified seed lots at six field sites in the US and Canada during the 2010 growing season. Three of these sites were the same as those used for the protein expression analysis (Section 4.1.3). There were four replicated blocks at each site planted in a randomised complete-block design. Maintenance fertilizer and pesticides were applied to all lines as needed in order to maintain a relatively weed-free and insect-free environment. In addition, a sub-set of line 4114 plants was sprayed at the V4 and V7 growth stages with glufosinate herbicide (Ignite? 268 SL) at application rates of 0.44 – 0.47 and 0.43 – 0.46 kg ai/ha respectively. No symptoms of plant injury were observed in these glufosinate-treated plants. Compositional data from a total of four proprietary non-GM lines grown at six locations in 2003 and four lines grown at six locations in 2007 was collected. Procedures for planting, harvesting and processing, of these reference lines were similar to those employed for the trials containing line 4114 and the PHNARxPHTFE control. These data were used to aid in the determination of the normal variation found in corn analyte levels.Grain was harvested at physiological maturity (R6) and samples (each sample comprised grain from five pooled ears) were analysed for proximates, fibre (acid detergent fibre – ADF; neutral detergent fibre – NDF; total dietary fibre), fatty acids, amino acids, minerals, vitamins, anti-nutrients and secondary metabolites. Key analyte levels (proximates, fibre, calcium and phosphorus) for forage (harvested at R4) were also obtained but are not reported here; it is noted, however, that in the combined site analysis none of the analyte levels in line 4114 differed significantly from those of the control. Methods of composition analysis were based on internationally recognised procedures (e.g. those of the Association of Official Analytical Chemists), methods specified by the manufacturer of the equipment used for analysis, or other published methods. 5.3Analyses of key components in grainThe analysis of line 4114 is only for glufosinate-treated plants. In total, 81 analyte levels were measured and carbohydrate was calculated rather than being measured i.e. there was a total of 82 analytes considered. Moisture values were measured for conversion of components to dry weight, but were not statistically analysed. A total of 26 analytes had more than 80% of the observations below the assay limit of quantitation (LOQ) and were excluded from the statistical analysis. The data for 56 analytes were therefore analysed. Data were transformed into Statistical Analysis Software (SAS) data sets and analysed using SAS version 9.2. A least squares mean (LSM) value was generated (combined across all sites) and used for each analyte comparison, and minimum and maximum values were also noted for each analyte (summarised in Tables 7 – 13). A mixed model Analysis of Variance was used for over-all analysis. The significance of an overall treatment effect was estimated using an F-test, while paired contrasts were made using t-tests. Probability values were adjusted using False Discovery Rate (FDR) procedures to improve discrimination of true differences ADDIN REFMGR.CITE <Refman><Cite><Author>Benjamini</Author><Year>1995</Year><RecNum>184</RecNum><IDText>Controlling the false discovery rate: a practical and powerful approach to multiple testing</IDText><MDL Ref_Type="Journal (Full)"><Ref_Type>Journal (Full)</Ref_Type><Ref_ID>184</Ref_ID><Title_Primary>Controlling the false discovery rate: a practical and powerful approach to multiple testing</Title_Primary><Authors_Primary>Benjamini,Y.</Authors_Primary><Authors_Primary>Hochberg,Y.</Authors_Primary><Date_Primary>1995</Date_Primary><Reprint>In File</Reprint><Start_Page>289</Start_Page><End_Page>300</End_Page><Periodical>Journal of the Royal Statistical Society Series B-Methodological</Periodical><Volume>57</Volume><Issue>1</Issue><ISSN_ISBN>0035-9246</ISSN_ISBN><Web_URL_Link1><u>file://F:\Standards Management\APPLICATIONS\A1001-A1050\A1018 High Oleic GM Soybean\A1018 Application\A1018 Literature References\Controlling_the_False_Discovery_Rate__a_Practical_and_Powerf.PDF</u></Web_URL_Link1><Web_URL_Link2>file://Y:\References\GM References_in RefMan<u>\Benjamin </u>and<u> Hochberg_1995_false discovery rate.pdf</u></Web_URL_Link2><ZZ_JournalFull><f name="System">Journal of the Royal Statistical Society Series B-Methodological</f></ZZ_JournalFull><ZZ_WorkformID>32</ZZ_WorkformID></MDL></Cite></Refman>(Benjamini and Hochberg 1995). In assessing the significance of any difference between the mean analyte value for line 4114 and the control, an FDR-adjusted P-value of 0.05 was used. This means that approximately 5% of statistically significant differences are expected to occur due to chance alone.Any statistically significant differences between line 4114 and the hybrid control have been compared to the 99% tolerance interval compiled from the results for each analyte of the eight non-GM reference lines combined across all sites/times, to assess whether the differences are likely to be biologically meaningful. These tolerance intervals contain, with 95% confidence, 99% of the values contained in the population of commercial lines. Additionally, the results for line 4114 and the hybrid control have been compared to a combined literature range for each analyte, compiled from published literature for commercially available corn. It is noted, however, that information in the published literature is limited and is unlikely to provide a broad reflection of the natural diversity that occurs within corn PFJlZm1hbj48Q2l0ZT48QXV0aG9yPkhhcnJpZ2FuPC9BdXRob3I+PFllYXI+MjAxMDwvWWVhcj48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ADDIN REFMGR.CITE PFJlZm1hbj48Q2l0ZT48QXV0aG9yPkhhcnJpZ2FuPC9BdXRob3I+PFllYXI+MjAxMDwvWWVhcj48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ADDIN EN.CITE.DATA (Harrigan et al. 2010; Ridley et al. 2011; Zhou et al. 2011). Therefore, even if means fall outside the published range, this does not necessarily mean the value falls outside the natural variation.5.3.1Proximates and fibreResults of the proximate and fibre analysis are shown in Table 7. There are no significant differences between the means of line 4114 and the control for any analyte and all means are also within both the tolerance interval and the literature range.Table SEQ Table \* ARABIC 7: Mean (range) percentage dry weight (%dw) of proximates and fibre in grain from glufosinate-treated line 4114 and the hybrid (PHNAR x PHTFE) control.AnalyteLine 4114(%dw)PHNARxPHTFE (%dw) adj P-valueTolerance interval (%dw)Combined literaturerange (%dw)Ash1.43 (1.31 – 1.58)1.33 (1.12 – 1.52)20.05390.531, 2.160.62 – 6.28Crude Protein10.2 (7.64 – 11.6)10.2 (8.92 – 11.4)0.9436.59, 13.56.15 – 17.26Crude Fat4.65 (4.32 – 5.86)4.81 (4.28 – 5.73)0.3321.45, 5.751.74 – 5.82Carbohydrate183.8 (82.3 – 86.4)83.7 (82.5 – 84.9)0.87480.3, 89.777.4 – 89.5ADF4.05 (3.27 – 4.76)3.83 (3.24 – 4.48)0.2191.43, 5.731.82 – 11.34NDF10.7 (9.42 – 12.1)10.4 (9.39 – 11.3)0.3635.75, 20.65.59 – 22.64Crude Fibre2.70 (2.39 – 3.09)2.57 (1.36 – 3.38)0.6720.941, 3.730.49 – 5.501 Carbohydrate calculated as 100% - (protein %dw+ fat %dw + ash %dw)5.3.2Fatty AcidsThe levels of 29 fatty acids were measured. Of these, the following had ≥80% of observations below the LOQ and were therefore excluded from analysis - C8:0 caprylic, C10:0 capric, C12:0 lauric, C14:0 myristic, C14:1 myristoleic, C15:0 pentadecanoic, C15:1 pentadecenoic, C16:1 palmitoleic, C17:0 heptadecanoic, C17:1 heptadecenoic, C17:2 heptadecadienoic, C18:2 (9,15) isomer of linoleic, C18:3 gamma linolenic, C19:0 nonadecanoic, C20:2 eicosadienoic, C20:3 eicosatrienoic, C20:4 arachidonic, C21:0 heneicosanoic, C22:0 behenic, C22:1 erucic and C23:0 tricosanoic. Results for the remaining eight fatty acids are given in Table 8 and can be summarised as follows:There was no significant difference between the means of line 4114 and the control for palmitic, stearic, oleic, linoleic, linolenic, arachidic, and lignoceric acids.The mean level of eicosenoic acid was significantly higher in grain of line 4114 compared with grain from the control but fell within both the tolerance interval and the combined literature range.The mean level of oleic acid was significantly lower in grain of line 4114 compared with grain from the control but fell within both the tolerance interval and the combined literature range.Table SEQ Table \* ARABIC 8: Mean (range) percentage composition, relative to total fat, of major fatty acids in grain from glufosinate-treated line 4114 and the hybrid (PHNAR x PHTFE) controlAnalyteLine 41141(%total)PHNARxPHTFE (%total)Adj P-valueTolerance interval (%total)Combined literaturerange (%total)Palmitic acid (C16:0)13.9 (13.2 – 14.7)14.1 (13.2 – 15.6)0.6725.51, 18.47.94 – 20.71Stearic acid (C18:0)1.55 (1.40 – 1.93)1.6 (1.45 – 2.07)0.3510.566, 4.671.02 – 3.40Oleic acid (C18:1)22.0 (20.0 – 23.9)22.6 (20.7 – 25.8)0.035410.4, 65.617.4 – 40.2Linoleic acid (C18:2)60.9 (58.0 – 64.2)60.1 (54.9 – 62.9)0.15730.4, 81.736.2 – 66.5Linolenic acid (C18:3)0.844 (0.432 – 1.23)0.787 (0.410 – 1.22)0.5300, 3.340.57 – 2.25Arachidic acid (C20:0)0.384 (0.330 – 0.462)0.392 (0.343 – 0.505)0.2190.159, 0.8490.28 – 0.965Eicosenoic acid (C20:1)0.258 (0.231 – 0.282)0.235 (0.204 – 0.255)0.02060.213, 0.370.17 – 1.917Lignoceric (C24:0)0.264 (0.222 - 0.346)0.240 (0.185 – 0.318)0.6090, 0.6750.14 – 0.231 orange shading represents line 4114 mean that is significantly higher than the control mean; mauve shading represents line 4114 mean that is significantly lower than the control mean5.3.3Amino acidsLevels of 18 amino acids were measured. Since asparagine and glutamine are converted to aspartate and glutamate respectively during the analysis, levels for aspartate include both aspartate and asparagine, while glutamate levels include both glutamate and glutamine. The results in Table 9 show there was no significant difference between the control and line 4114 for any of the analyte means. All means also fell within both the tolerance interval and the literature range.Table SEQ Table \* ARABIC 9: Mean (range) %dw of amino acids in grain from glufosinate-treated line 4114 and the hybrid (PHNAR x PHTFE) controlAnalyteLine 41141(%dw)PHNARxPHTFE (%dw)Adj P-valueTolerance interval (%dw)Combined literaturerange (%dw)Alanine0.738 (0.521 - 0.890)0.739 (0.604 – 0.84)0.9800.491, 1.090.439 – 1.393Arginine0.430 (0.363 – 0.516)0.434 (0.362 – 0.492)0.8740.253, 0.5510.119 – 0.639Aspartate0.691 (0.524 – 0.791)0.680 (0.562 – 0.751)0.8170.442, 0.9470.335 – 1.208Cystine0.188 (0.132 – 0.241)0.193 (0.157 – 0.253)0.8740.136, 0.4180.125 – 0.514Glutamate1.92 (1.36 – 2.31)1.93 (1.59 – 2.24)0.9431.17, 2.880.965 – 3.536Glycine0.371 (0.303 – 0.439)0.372 (0.318 – 0.442)0.9320.249, 0.4850.184 – 0.539Histidine0.295 (0.222 – 0.365)0.293 (0.257 – 0.342)0.8740.18, 0.3620.137 – 0.434Isoleucine0.341 (0.237 – 0.417)0.339 (0.288 – 0.388)0.8740.143, 0.5870.179 – 0.692Leucine1.27 (0.813 – 1.61)1.26 (1.02 – 1.49)0.8740.659, 1.950.642 – 2.492Lysine0.286 (0.223 – 0.334)0.293 (0.224 – 0.349)0.5560.112, 0.5510.172 – 0.668Methionine0.179 (0.127 – 0.225)0.179 (0.144 – 0.231)0.9820.072, 0.490.124 – 0.468Phenylalanine0.535 (0.353 – 0.679)0.523 (0.443 – 0.611)0.8050.298, 0.6930.244 – 0.930Proline0.905 (0.642 – 1.10)0.899 (0.763 – 1.01)0.8740.454, 1.640.462 – 1.632Serine0.506 (0.364 – 0.620)0.5 (0.421 – 0.554)0.8740.266, 0.6830.235 – 0.769Threonine0.364 (0.272 – 0.426)0.361 (0.318 – 0.402)0.8740.176, 0.5780.224 – 0.666Tryptophan0.0656 (0.0530 – 0.0740)0.0634 (0.0367 – 0.0726)0.5510.0087, 0.1270.0271 – 0.215Tyrosine0.248 (0.175 – 0.332)0.263 (0.199 – 0.333)0.4800.0707, 0.5050.103 – 0.642Valine0.462 (0.346 – 0.550)0.460 (0.393 – 0.507)0.8970.159, 0.7490.266 – 0.8555.3.4MineralsThe levels of nine minerals in grain from line 4114 and the hybrid control were measured. For copper, ≥ 80% of the data points were below the LOQ and no statistical analysis was done. Results for the remaining analytes are given in Table 10 and can be summarised as follows:There was no significant difference between the means of line 4114 and the control for calcium, iron, magnesium, manganese, sodium and zinc.The mean levels of phosphorus and potassium were significantly higher in grain of line 4114 compared with grain from the control but fell within both the tolerance interval and the combined literature range.Table SEQ Table \* ARABIC 10: Mean (range) levels of minerals in the grain of glufosinate-treated line 4114 and the hybrid (PHNAR x PHTFE) controlAnalyteUnitLine 41141PHNARxPHTFEAdj P-value)Tolerance intervalCombined literaturerangeCalciummg/kg dw33.5 (26.1 – 52.7) 32.5 (27 – 39.9)0.57512.7, 90.212.7 – 208.4Ironmg/kg dw17.5 (15.0 – 33.4)16.8 (14.6 – 22.8)0.5998.57 – 26.910.4 – 49.1Magnesium%dw0.134 (0.109 – 0.168)0.133 (0.103 – 0.155)0.8740.038 – 0.190.0594 – 0.194Manganesemg/kg dw6.27 (4.94 – 9.70)6.21 (4.92 – 9.58)0.8742.5, 12.21.69 – 14.30Phosphorus%dw0.311 (0.283 – 0.362)0.289 (0.258 – 0.334)0.02060.127 – 0.4720.147 – 0.533Potassium%dw0.421 (0.381 – 0.514)0.387 (0.343 – 0.441)0.02080.194, 0.6870.181 – 0.603Sodiummg/kg dw0.732 (<LOQ – 2.84)0.582 (<LOQ – 4.89)0.5750 – 20.70.2 - 732Zincmg/kg dw17.5 (15.0 – 20.1)17.4 (15.0 – 20.3)0.87410.4, 27.16.5 – 37.21 orange shading represents line 4114 means that are significantly higher than the control means.5.3.5Vitamins Levels of 12 vitamins were measured. For riboflavin, β-tocopherol and δ-tocopherol because more than 80% of the readings were below the LOQ, no analysis was done.Results for the remaining nine vitamins are given in Table 11 and show the mean level of Vitamin B1 in line 4114 was significantly higher than the mean in the control. However, both means fell within both the tolerance interval and the literature range. There were no significant differences between the means of line 4114 and the control for any other analytes.Table SEQ Table \* ARABIC 11: Mean (range) weight (mg/k g dw) of vitamins in grain from glufosinate-treated line 4114 and the hybrid (PHNAR x PHTFE) controlAnalyteLine 4114(mg/kg dw)1PHNARxPHTFE (mg/kg dw)Adj P-value)Tolerance interval (mg/kg dw)Combined literaturerange (mg/kg dw)Vitamin A (β-carotene)18.9 (12.0 – 33.6)16.4 (7.27 – 26.2)0.3510, 68.30.19 – 46.81Vitamin B1 (Thiamine HCl)2.59 (<LOQ – 3.61)1.97 (<LOQ – 2.92)0.04790.414, 6.641.26 – 40.00Vitamin B3 (Niacin)13.7 (10.5 – 16.7)13.9 (10.6 – 18)0.8360, 51.710.37 – 46.94Vitamin B5 (Pantothenic acid)6.06 (3.94 – 27.0)4.92 (<LOQ – 10.9)0.3223.02, 8.203.50 – 14.0Vitamin B6 (Pyridoxine HCl)4.83 (1.81 – 8.14)4.68 (2.35 – 6.88)0.8741.83, 11.13.68 – 11.32Vitamin B9 (Folic acid)0.943 (0.138 – 1.48)0.872 (0.181 – 1.91)0.8240, 2.30.147 – 1.464Vitamin E (α-tocopherol)6.50 (3.02 – 13.0)5.47 (2.09 – 13.3)0.3032.18, 28.21.5 – 68.7?-tocopheroal4.05 (2.88 – 5.54)3.38 (2.30 – 5.04)0.06620, 39.96.46 – 61.0Total tocopherols11.0 (7.09 – 17.9)9.23 (4.77 – 17.0)0.1950, 53.68.69 - 1331 orange shading represents line 4114 mean that is significantly higher than the control mean.5.3.6Anti-nutrientsLevels of three key anti-nutrients were measured. Results in Table 12 show that none of the means differed significantly between line 4114 and the control. All means fell within both the tolerance interval and the literature range.Table SEQ Table \* ARABIC 12: Mean (range) of anti-nutrients in grain from glufosinate-treated line 4114 and the hybrid (PHNAR x PHTFE) controlAnalyteLine 41141PHNARxPHTFE Adj P-valueTolerance interval Combined literaturerangePhytic acid (%dw)1.04 (0.796 – 1.36)0.998 (0.842 – 1.17)0.5750.418, 1.410.111 – 1.570Raffinose %dw)0.0928 (<LOQ – 0.227)0.0823 (<LOQ – 0.210)0.6720, 0.3980.020 – 0.320Trypsin inhibitor (TIU/mg)2.39 (1.60 – 3.05)2.67 (1.45 – 4.68)0.5301.60, 4.891.09 – 7.185.3.7Secondary metabolitesThe levels of four secondary metabolites were measured but furfural was below the level of quantitation in line 4114, the control and all of the reference hybrids. For the three remaining metabolites p-coumaric acid, ferulic acid and inositol (see Table 13) there was no significant difference between the control and line 4114 for either of the means.Table SEQ Table \* ARABIC 13: Mean %dwt (range) of three secondary metabolites in grain from glufosinate-treated line 4114 and the hybrid (PHNAR x PHTFE) controlAnalyteLine 4114(%dw)PHNARxPHTFE (%dw)Adj P-valueTolerance interval (%dw)Combined literaturerange (%dw)p-coumaric acid0.0190 (0.0152 – 0.0255)0.0183 (0.0120 – 0.0276)0.5300.000341, 0.03870.003 – 0.0576Ferulic acid0.275 (0.237 – 0.329)0.255 (0.199 – 0.299)0.2190.0553, 0.03090.0200 – 0.0389Inositol0.0224 (0.0151 – 0.0325)0.0256 (0.0197 – 0.0341)0.1320, 0.04610.0089 – 0.3775.3.8Summary of analysis of key componentsStatistically significant differences in the analyte levels found between grain of line 4114 and the PHNAR x PHTFE control are summarised in Table 14. Table SEQ Table \* ARABIC 14: Summary of analyte levels found in grain of glufosinate-treated line 4114 that are significantly (P < 0.05) different from those found in grain of the hybrid (PHNAR x PHTFE) controlAnalyteUnitLine 4114 mean1Hybrid control meandiff between line411 & hybrid control meansdiff between max and min in hybrid controlLine 4114 within tolerance interval?line 4114 within literature range?Ash% dw1.441.330.110.40yesyesOleic acid% total fatty acid22.022.60.63.9yesyesEicosenoic acid% total fatty acid0.2650.2350.0300.051yesyesPhosphorus% dw0.3110.2890.0220.076yesyesPotassium% dw0.4160.3870.0290.098yesyesVitamin B1mg/kg dw2.591.970.62> 1.12yesyes1 orange shading represents line 4114 means that are significantly higher than the hybrid control means; mauve shading represents line 4114 mean that is significantly lower than the control mean5.4Conclusion from compositional analysisDetailed compositional analyses were done to establish the nutritional adequacy of grain from line 4114 and to characterise any unintended compositional changes. Analyses were done of proximates, fibre, minerals, amino acids, fatty acids, vitamins, secondary metabolites and anti-nutrients. The levels were compared to levels in a) an appropriate non-GM hybrid line, PHNAR x PHTFE, b) a tolerance interval compiled from results taken for eight non-GM hybrid lines grown in similar field trials in different seasons, and c) levels recorded in the literature. Only five of the 56 analytes reported in Tables 7 - 13 deviated in level from the control in a statistically significant manner. However, the mean levels of all of these analytes fell within both the tolerance interval and the historical range from the literature. It is further noted that the differences between the line 4114 and control means for each analyte in Table 14 were less than the variation found within the control. It can therefore be concluded that grain from line 4114 is compositionally equivalent to grain from conventional corn varieties.6Nutritional impactIn assessing the safety of a GM food, a key factor is the need to establish that the food is nutritionally adequate and will support typical growth and well-being. In most cases, this can be achieved through an understanding of the genetic modification and its consequences, together with an extensive compositional analysis of the food.If the compositional analysis indicates biologically significant changes to the levels of certain nutrients in the GM food, additional nutritional assessment should be undertaken to assess the consequences of the changes and determine whether nutrient intakes are likely to be altered by the introduction of such foods into the food supply. Where a GM food has been shown to be compositionally equivalent to conventional varieties, the evidence to date indicates that feeding studies using target livestock species will add little to the safety assessment and generally are not warranted PFJlZm1hbj48Q2l0ZT48QXV0aG9yPkJhcnRob2xvbWFldXM8L0F1dGhvcj48WWVhcj4yMDEzPC9Z
ZWFyPjxSZWNOdW0+MTU0MzwvUmVjTnVtPjxJRFRleHQ+VGhlIHVzZSBvZiB3aG9sZSBmb29kIGFu
aW1hbCBzdHVkaWVzIGluIHRoZSBzYWZldHkgYXNzZXNzbWVudCBvZiBnZW5ldGljYWxseSBtb2Rp
ZmllZCBjcm9wczogTGltaXRhdGlvbnMgYW5kIHJlY29tbWVuZGF0aW9uczwvSURUZXh0PjxNREwg
UmVmX1R5cGU9IkpvdXJuYWwgKEZ1bGwpIj48UmVmX1R5cGU+Sm91cm5hbCAoRnVsbCk8L1JlZl9U
eXBlPjxSZWZfSUQ+MTU0MzwvUmVmX0lEPjxUaXRsZV9QcmltYXJ5PlRoZSB1c2Ugb2Ygd2hvbGUg
Zm9vZCBhbmltYWwgc3R1ZGllcyBpbiB0aGUgc2FmZXR5IGFzc2Vzc21lbnQgb2YgZ2VuZXRpY2Fs
bHkgbW9kaWZpZWQgY3JvcHM6IExpbWl0YXRpb25zIGFuZCByZWNvbW1lbmRhdGlvbnM8L1RpdGxl
X1ByaW1hcnk+PEF1dGhvcnNfUHJpbWFyeT5CYXJ0aG9sb21hZXVzLEEuPC9BdXRob3JzX1ByaW1h
cnk+PEF1dGhvcnNfUHJpbWFyeT5QYXJyb3R0LFcuPC9BdXRob3JzX1ByaW1hcnk+PEF1dGhvcnNf
UHJpbWFyeT5Cb25keSxHLjwvQXV0aG9yc19QcmltYXJ5PjxBdXRob3JzX1ByaW1hcnk+V2Fsa2Vy
LEsuPC9BdXRob3JzX1ByaW1hcnk+PERhdGVfUHJpbWFyeT4yMDEzPC9EYXRlX1ByaW1hcnk+PEtl
eXdvcmRzPlNhZmV0eTwvS2V5d29yZHM+PEtleXdvcmRzPlNhZmV0eSBhc3Nlc3NtZW50PC9LZXl3
b3Jkcz48S2V5d29yZHM+dG94aWNpdHk8L0tleXdvcmRzPjxLZXl3b3Jkcz5hbmFseXNpczwvS2V5
d29yZHM+PEtleXdvcmRzPmdlbmV0aWNzPC9LZXl3b3Jkcz48S2V5d29yZHM+UGxhbnRzPC9LZXl3
b3Jkcz48S2V5d29yZHM+R2VuZXRpYyBFbmdpbmVlcmluZzwvS2V5d29yZHM+PFJlcHJpbnQ+Tm90
IGluIEZpbGU8L1JlcHJpbnQ+PFN0YXJ0X1BhZ2U+MTwvU3RhcnRfUGFnZT48RW5kX1BhZ2U+MjQ8
L0VuZF9QYWdlPjxQZXJpb2RpY2FsPkNyaXRpY2FsIFJldmlld3MgaW4gVG94aWNvbG9neTwvUGVy
aW9kaWNhbD48Vm9sdW1lPjQzPC9Wb2x1bWU+PElzc3VlPlMyPC9Jc3N1ZT48TWlzY18yPjxmIG5h
bWU9IkFkdlBTRlQtTCI+RE9JOiAxMC4zMTA5LzEwNDA4NDQ0LjIwMTMuODQyOTU1PC9mPjwvTWlz
Y18yPjxXZWJfVVJMX0xpbmsyPmZpbGU6Ly9ZOlxSZWZlcmVuY2VzXEdNIFJlZmVyZW5jZXNfaW4g
UmVmTWFuPHU+XEJhcnRob2xvbWFldXMgZXQgYWxfMjAxM191c2Ugb2Ygd2hvbGUgZm9vZCBzdHVk
aWVzIGluIEdNIHNhZmV0eSBhc3Nlc3NtZW50LnBkZjwvdT48L1dlYl9VUkxfTGluazI+PFpaX0pv
dXJuYWxGdWxsPjxmIG5hbWU9IlN5c3RlbSI+Q3JpdGljYWwgUmV2aWV3cyBpbiBUb3hpY29sb2d5
PC9mPjwvWlpfSm91cm5hbEZ1bGw+PFpaX1dvcmtmb3JtSUQ+MzI8L1paX1dvcmtmb3JtSUQ+PC9N
REw+PC9DaXRlPjxDaXRlPjxBdXRob3I+SGVybWFuPC9BdXRob3I+PFllYXI+MjAxNDwvWWVhcj48
UmVjTnVtPjE2MDc8L1JlY051bT48SURUZXh0PkRvIHdob2xlLWZvb2QgYW5pbWFsIGZlZWRpbmcg
c3R1ZGllcyBoYXZlIGFueSB2YWx1ZSBpbiB0aGUgc2FmZXR5IGFzc2Vzc21lbnQgb2YgR00gY3Jv
cHM/PC9JRFRleHQ+PE1ETCBSZWZfVHlwZT0iSm91cm5hbCAoRnVsbCkiPjxSZWZfVHlwZT5Kb3Vy
bmFsIChGdWxsKTwvUmVmX1R5cGU+PFJlZl9JRD4xNjA3PC9SZWZfSUQ+PFRpdGxlX1ByaW1hcnk+
PGYgbmFtZT0iQWR2R3VsbGl2LVIiPkRvIHdob2xlLWZvb2QgYW5pbWFsIGZlZWRpbmcgc3R1ZGll
cyBoYXZlIGFueSB2YWx1ZSBpbiB0aGUgc2FmZXR5IGFzc2Vzc21lbnQgb2YgR00gY3JvcHM/PC9m
PjwvVGl0bGVfUHJpbWFyeT48QXV0aG9yc19QcmltYXJ5Pkhlcm1hbixSLkEuPC9BdXRob3JzX1By
aW1hcnk+PEF1dGhvcnNfUHJpbWFyeT5Fa21heSxSLjwvQXV0aG9yc19QcmltYXJ5PjxEYXRlX1By
aW1hcnk+MjAxNDwvRGF0ZV9QcmltYXJ5PjxLZXl3b3Jkcz5TYWZldHk8L0tleXdvcmRzPjxLZXl3
b3Jkcz5TYWZldHkgYXNzZXNzbWVudDwvS2V5d29yZHM+PEtleXdvcmRzPlByb3RlaW5zPC9LZXl3
b3Jkcz48S2V5d29yZHM+YWR2ZXJzZSBlZmZlY3RzPC9LZXl3b3Jkcz48S2V5d29yZHM+SHVtYW48
L0tleXdvcmRzPjxLZXl3b3Jkcz5EaWV0PC9LZXl3b3Jkcz48UmVwcmludD5Ob3QgaW4gRmlsZTwv
UmVwcmludD48U3RhcnRfUGFnZT4xNzE8L1N0YXJ0X1BhZ2U+PEVuZF9QYWdlPjE3NDwvRW5kX1Bh
Z2U+PFBlcmlvZGljYWw+UmVndWxhdG9yeSBUb3hpY29sb2d5IGFuZCBQaGFybWFjb2xvZ3k8L1Bl
cmlvZGljYWw+PFZvbHVtZT42ODwvVm9sdW1lPjxNaXNjXzI+PGYgbmFtZT0iQWR2R3VsbGl2LVIi
Pmh0dHA6Ly9keC5kb2kub3JnLzEwLjEwMTYvai55cnRwaC4yMDEzLjA3LjAwMzwvZj48L01pc2Nf
Mj48V2ViX1VSTF9MaW5rMj5maWxlOi8vWTpcUmVmZXJlbmNlc1xHTSBSZWZlcmVuY2VzX2luIFJl
Zk1hbjx1PlxIZXJtYW4gPC91PmFuZDx1PiBFa21heV8yMDE0X3dob2xlIGZvb2Qgc3R1ZGllcy5w
ZGY8L3U+PC9XZWJfVVJMX0xpbmsyPjxaWl9Kb3VybmFsRnVsbD48ZiBuYW1lPSJTeXN0ZW0iPlJl
Z3VsYXRvcnkgVG94aWNvbG9neSBhbmQgUGhhcm1hY29sb2d5PC9mPjwvWlpfSm91cm5hbEZ1bGw+
PFpaX1dvcmtmb3JtSUQ+MzI8L1paX1dvcmtmb3JtSUQ+PC9NREw+PC9DaXRlPjxDaXRlPjxBdXRo
b3I+T0VDRDwvQXV0aG9yPjxZZWFyPjIwMDM8L1llYXI+PFJlY051bT42Nzc8L1JlY051bT48SURU
ZXh0PkNvbnNpZGVyYXRpb25zIGZvciB0aGUgc2FmZXR5IGFzc2Vzc21lbnQgb2YgYW5pbWFsIGZl
ZWRzdHVmZnMgZGVyaXZlZCBmcm9tIGdlbmV0aWNhbGx5IG1vZGlmaWVkIHBsYW50czwvSURUZXh0
PjxQcmVmaXg+c2VlIGUuZy4gPC9QcmVmaXg+PE1ETCBSZWZfVHlwZT0iUmVwb3J0Ij48UmVmX1R5
cGU+UmVwb3J0PC9SZWZfVHlwZT48UmVmX0lEPjY3NzwvUmVmX0lEPjxUaXRsZV9QcmltYXJ5PkNv
bnNpZGVyYXRpb25zIGZvciB0aGUgc2FmZXR5IGFzc2Vzc21lbnQgb2YgYW5pbWFsIGZlZWRzdHVm
ZnMgZGVyaXZlZCBmcm9tIGdlbmV0aWNhbGx5IG1vZGlmaWVkIHBsYW50czwvVGl0bGVfUHJpbWFy
eT48QXV0aG9yc19QcmltYXJ5Pk9FQ0Q8L0F1dGhvcnNfUHJpbWFyeT48RGF0ZV9QcmltYXJ5PjIw
MDM8L0RhdGVfUHJpbWFyeT48S2V5d29yZHM+U2FmZXR5PC9LZXl3b3Jkcz48S2V5d29yZHM+U2Fm
ZXR5IGFzc2Vzc21lbnQ8L0tleXdvcmRzPjxLZXl3b3Jkcz5QbGFudHM8L0tleXdvcmRzPjxSZXBy
aW50Pk5vdCBpbiBGaWxlPC9SZXByaW50PjxWb2x1bWU+RU5WL0pNL01PTk8oMjAwMykxMDwvVm9s
dW1lPjxQdWJfUGxhY2U+UGFyaXM8L1B1Yl9QbGFjZT48UHVibGlzaGVyPk9yZ2FuaXNhdGlvbiBm
b3IgRWNvbm9taWMgQ28tb3BlcmF0aW9uIGFuZCBEZXZlbG9wbWVudDwvUHVibGlzaGVyPjxXZWJf
VVJMPjx1Pmh0dHA6Ly93d3cub2VjZC5vcmcvb2ZmaWNpYWxkb2N1bWVudHMvZGlzcGxheWRvY3Vt
ZW50cGRmP2NvdGU9RU5WL0pNL01PTk8oMjAwMykxMCZhbXA7ZG9jbGFuZ3VhZ2U9ZW48L3U+PC9X
ZWJfVVJMPjxXZWJfVVJMX0xpbmsyPmZpbGU6Ly9ZOlxSZWZlcmVuY2VzXEdNIFJlZmVyZW5jZXNf
aW4gUmVmTWFuPHU+XE9FQ0RfMjAwM19hbmltYWwgZmVlZHN0dWZmcy5wZGY8L3U+PC9XZWJfVVJM
X0xpbmsyPjxaWl9Xb3JrZm9ybUlEPjI0PC9aWl9Xb3JrZm9ybUlEPjwvTURMPjwvQ2l0ZT48L1Jl
Zm1hbj5=
ADDIN REFMGR.CITE PFJlZm1hbj48Q2l0ZT48QXV0aG9yPkJhcnRob2xvbWFldXM8L0F1dGhvcj48WWVhcj4yMDEzPC9Z
ZWFyPjxSZWNOdW0+MTU0MzwvUmVjTnVtPjxJRFRleHQ+VGhlIHVzZSBvZiB3aG9sZSBmb29kIGFu
aW1hbCBzdHVkaWVzIGluIHRoZSBzYWZldHkgYXNzZXNzbWVudCBvZiBnZW5ldGljYWxseSBtb2Rp
ZmllZCBjcm9wczogTGltaXRhdGlvbnMgYW5kIHJlY29tbWVuZGF0aW9uczwvSURUZXh0PjxNREwg
UmVmX1R5cGU9IkpvdXJuYWwgKEZ1bGwpIj48UmVmX1R5cGU+Sm91cm5hbCAoRnVsbCk8L1JlZl9U
eXBlPjxSZWZfSUQ+MTU0MzwvUmVmX0lEPjxUaXRsZV9QcmltYXJ5PlRoZSB1c2Ugb2Ygd2hvbGUg
Zm9vZCBhbmltYWwgc3R1ZGllcyBpbiB0aGUgc2FmZXR5IGFzc2Vzc21lbnQgb2YgZ2VuZXRpY2Fs
bHkgbW9kaWZpZWQgY3JvcHM6IExpbWl0YXRpb25zIGFuZCByZWNvbW1lbmRhdGlvbnM8L1RpdGxl
X1ByaW1hcnk+PEF1dGhvcnNfUHJpbWFyeT5CYXJ0aG9sb21hZXVzLEEuPC9BdXRob3JzX1ByaW1h
cnk+PEF1dGhvcnNfUHJpbWFyeT5QYXJyb3R0LFcuPC9BdXRob3JzX1ByaW1hcnk+PEF1dGhvcnNf
UHJpbWFyeT5Cb25keSxHLjwvQXV0aG9yc19QcmltYXJ5PjxBdXRob3JzX1ByaW1hcnk+V2Fsa2Vy
LEsuPC9BdXRob3JzX1ByaW1hcnk+PERhdGVfUHJpbWFyeT4yMDEzPC9EYXRlX1ByaW1hcnk+PEtl
eXdvcmRzPlNhZmV0eTwvS2V5d29yZHM+PEtleXdvcmRzPlNhZmV0eSBhc3Nlc3NtZW50PC9LZXl3
b3Jkcz48S2V5d29yZHM+dG94aWNpdHk8L0tleXdvcmRzPjxLZXl3b3Jkcz5hbmFseXNpczwvS2V5
d29yZHM+PEtleXdvcmRzPmdlbmV0aWNzPC9LZXl3b3Jkcz48S2V5d29yZHM+UGxhbnRzPC9LZXl3
b3Jkcz48S2V5d29yZHM+R2VuZXRpYyBFbmdpbmVlcmluZzwvS2V5d29yZHM+PFJlcHJpbnQ+Tm90
IGluIEZpbGU8L1JlcHJpbnQ+PFN0YXJ0X1BhZ2U+MTwvU3RhcnRfUGFnZT48RW5kX1BhZ2U+MjQ8
L0VuZF9QYWdlPjxQZXJpb2RpY2FsPkNyaXRpY2FsIFJldmlld3MgaW4gVG94aWNvbG9neTwvUGVy
aW9kaWNhbD48Vm9sdW1lPjQzPC9Wb2x1bWU+PElzc3VlPlMyPC9Jc3N1ZT48TWlzY18yPjxmIG5h
bWU9IkFkdlBTRlQtTCI+RE9JOiAxMC4zMTA5LzEwNDA4NDQ0LjIwMTMuODQyOTU1PC9mPjwvTWlz
Y18yPjxXZWJfVVJMX0xpbmsyPmZpbGU6Ly9ZOlxSZWZlcmVuY2VzXEdNIFJlZmVyZW5jZXNfaW4g
UmVmTWFuPHU+XEJhcnRob2xvbWFldXMgZXQgYWxfMjAxM191c2Ugb2Ygd2hvbGUgZm9vZCBzdHVk
aWVzIGluIEdNIHNhZmV0eSBhc3Nlc3NtZW50LnBkZjwvdT48L1dlYl9VUkxfTGluazI+PFpaX0pv
dXJuYWxGdWxsPjxmIG5hbWU9IlN5c3RlbSI+Q3JpdGljYWwgUmV2aWV3cyBpbiBUb3hpY29sb2d5
PC9mPjwvWlpfSm91cm5hbEZ1bGw+PFpaX1dvcmtmb3JtSUQ+MzI8L1paX1dvcmtmb3JtSUQ+PC9N
REw+PC9DaXRlPjxDaXRlPjxBdXRob3I+SGVybWFuPC9BdXRob3I+PFllYXI+MjAxNDwvWWVhcj48
UmVjTnVtPjE2MDc8L1JlY051bT48SURUZXh0PkRvIHdob2xlLWZvb2QgYW5pbWFsIGZlZWRpbmcg
c3R1ZGllcyBoYXZlIGFueSB2YWx1ZSBpbiB0aGUgc2FmZXR5IGFzc2Vzc21lbnQgb2YgR00gY3Jv
cHM/PC9JRFRleHQ+PE1ETCBSZWZfVHlwZT0iSm91cm5hbCAoRnVsbCkiPjxSZWZfVHlwZT5Kb3Vy
bmFsIChGdWxsKTwvUmVmX1R5cGU+PFJlZl9JRD4xNjA3PC9SZWZfSUQ+PFRpdGxlX1ByaW1hcnk+
PGYgbmFtZT0iQWR2R3VsbGl2LVIiPkRvIHdob2xlLWZvb2QgYW5pbWFsIGZlZWRpbmcgc3R1ZGll
cyBoYXZlIGFueSB2YWx1ZSBpbiB0aGUgc2FmZXR5IGFzc2Vzc21lbnQgb2YgR00gY3JvcHM/PC9m
PjwvVGl0bGVfUHJpbWFyeT48QXV0aG9yc19QcmltYXJ5Pkhlcm1hbixSLkEuPC9BdXRob3JzX1By
aW1hcnk+PEF1dGhvcnNfUHJpbWFyeT5Fa21heSxSLjwvQXV0aG9yc19QcmltYXJ5PjxEYXRlX1By
aW1hcnk+MjAxNDwvRGF0ZV9QcmltYXJ5PjxLZXl3b3Jkcz5TYWZldHk8L0tleXdvcmRzPjxLZXl3
b3Jkcz5TYWZldHkgYXNzZXNzbWVudDwvS2V5d29yZHM+PEtleXdvcmRzPlByb3RlaW5zPC9LZXl3
b3Jkcz48S2V5d29yZHM+YWR2ZXJzZSBlZmZlY3RzPC9LZXl3b3Jkcz48S2V5d29yZHM+SHVtYW48
L0tleXdvcmRzPjxLZXl3b3Jkcz5EaWV0PC9LZXl3b3Jkcz48UmVwcmludD5Ob3QgaW4gRmlsZTwv
UmVwcmludD48U3RhcnRfUGFnZT4xNzE8L1N0YXJ0X1BhZ2U+PEVuZF9QYWdlPjE3NDwvRW5kX1Bh
Z2U+PFBlcmlvZGljYWw+UmVndWxhdG9yeSBUb3hpY29sb2d5IGFuZCBQaGFybWFjb2xvZ3k8L1Bl
cmlvZGljYWw+PFZvbHVtZT42ODwvVm9sdW1lPjxNaXNjXzI+PGYgbmFtZT0iQWR2R3VsbGl2LVIi
Pmh0dHA6Ly9keC5kb2kub3JnLzEwLjEwMTYvai55cnRwaC4yMDEzLjA3LjAwMzwvZj48L01pc2Nf
Mj48V2ViX1VSTF9MaW5rMj5maWxlOi8vWTpcUmVmZXJlbmNlc1xHTSBSZWZlcmVuY2VzX2luIFJl
Zk1hbjx1PlxIZXJtYW4gPC91PmFuZDx1PiBFa21heV8yMDE0X3dob2xlIGZvb2Qgc3R1ZGllcy5w
ZGY8L3U+PC9XZWJfVVJMX0xpbmsyPjxaWl9Kb3VybmFsRnVsbD48ZiBuYW1lPSJTeXN0ZW0iPlJl
Z3VsYXRvcnkgVG94aWNvbG9neSBhbmQgUGhhcm1hY29sb2d5PC9mPjwvWlpfSm91cm5hbEZ1bGw+
PFpaX1dvcmtmb3JtSUQ+MzI8L1paX1dvcmtmb3JtSUQ+PC9NREw+PC9DaXRlPjxDaXRlPjxBdXRo
b3I+T0VDRDwvQXV0aG9yPjxZZWFyPjIwMDM8L1llYXI+PFJlY051bT42Nzc8L1JlY051bT48SURU
ZXh0PkNvbnNpZGVyYXRpb25zIGZvciB0aGUgc2FmZXR5IGFzc2Vzc21lbnQgb2YgYW5pbWFsIGZl
ZWRzdHVmZnMgZGVyaXZlZCBmcm9tIGdlbmV0aWNhbGx5IG1vZGlmaWVkIHBsYW50czwvSURUZXh0
PjxQcmVmaXg+c2VlIGUuZy4gPC9QcmVmaXg+PE1ETCBSZWZfVHlwZT0iUmVwb3J0Ij48UmVmX1R5
cGU+UmVwb3J0PC9SZWZfVHlwZT48UmVmX0lEPjY3NzwvUmVmX0lEPjxUaXRsZV9QcmltYXJ5PkNv
bnNpZGVyYXRpb25zIGZvciB0aGUgc2FmZXR5IGFzc2Vzc21lbnQgb2YgYW5pbWFsIGZlZWRzdHVm
ZnMgZGVyaXZlZCBmcm9tIGdlbmV0aWNhbGx5IG1vZGlmaWVkIHBsYW50czwvVGl0bGVfUHJpbWFy
eT48QXV0aG9yc19QcmltYXJ5Pk9FQ0Q8L0F1dGhvcnNfUHJpbWFyeT48RGF0ZV9QcmltYXJ5PjIw
MDM8L0RhdGVfUHJpbWFyeT48S2V5d29yZHM+U2FmZXR5PC9LZXl3b3Jkcz48S2V5d29yZHM+U2Fm
ZXR5IGFzc2Vzc21lbnQ8L0tleXdvcmRzPjxLZXl3b3Jkcz5QbGFudHM8L0tleXdvcmRzPjxSZXBy
aW50Pk5vdCBpbiBGaWxlPC9SZXByaW50PjxWb2x1bWU+RU5WL0pNL01PTk8oMjAwMykxMDwvVm9s
dW1lPjxQdWJfUGxhY2U+UGFyaXM8L1B1Yl9QbGFjZT48UHVibGlzaGVyPk9yZ2FuaXNhdGlvbiBm
b3IgRWNvbm9taWMgQ28tb3BlcmF0aW9uIGFuZCBEZXZlbG9wbWVudDwvUHVibGlzaGVyPjxXZWJf
VVJMPjx1Pmh0dHA6Ly93d3cub2VjZC5vcmcvb2ZmaWNpYWxkb2N1bWVudHMvZGlzcGxheWRvY3Vt
ZW50cGRmP2NvdGU9RU5WL0pNL01PTk8oMjAwMykxMCZhbXA7ZG9jbGFuZ3VhZ2U9ZW48L3U+PC9X
ZWJfVVJMPjxXZWJfVVJMX0xpbmsyPmZpbGU6Ly9ZOlxSZWZlcmVuY2VzXEdNIFJlZmVyZW5jZXNf
aW4gUmVmTWFuPHU+XE9FQ0RfMjAwM19hbmltYWwgZmVlZHN0dWZmcy5wZGY8L3U+PC9XZWJfVVJM
X0xpbmsyPjxaWl9Xb3JrZm9ybUlEPjI0PC9aWl9Xb3JrZm9ybUlEPjwvTURMPjwvQ2l0ZT48L1Jl
Zm1hbj5=
ADDIN EN.CITE.DATA (see e.g. OECD 2003; Bartholomaeus et al. 2013; Herman and Ekmay 2014). Line 4114 is the result of a genetic modification designed to confer protection against corn rootworm and tolerance to the herbicide glufosinate with no intention to significantly alter nutritional parameters in the food. In addition, the extensive compositional analyses of grain that have been undertaken to demonstrate the nutritional adequacy of line 4114 indicate it is equivalent in composition to conventional corn cultivars. The introduction of food from line 4114 into the food supply is therefore expected to have little nutritional impact and as such no additional studies, including animal feeding studies, are required.References ADDIN REFMGR.REFLIST Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25(17):3389–3402An G, Mitra A, Choi HK, Costa MA, An K, Thornburg RW, Ryan CA (1989) Functional analysis of the 3' control region of the potato wound-inducible proteinase inhibitor II gene. Plant Cell 1(1):115–122Aronson AI, Shai Y (2001) Why Bacillus thuringiensis insecticidal toxins are so effective: unique features of their mode of action. FEMS Microbiology Letters 195:1–8Barker RF, Idler KB, Thompson D.V., Kemp JD (1983) Nucleotide sequence of the T-DNA region from the Agrobacterium tumefaciens octopine Ti plasmid pTi15955. Plant Molecular Biology 2:335–350Bartholomaeus A, Parrott W, Bondy G, Walker K (2013) The use of whole food animal studies in the safety assessment of genetically modified crops: Limitations and recommendations. Critical Reviews in Toxicology 43(S2):1–24Baum JA, Chu C-R, Rupar M, Brown GR, Donovan WP, Huesing JE, Ilagan O, Malvar TM, Pleau M, Walters M, Vaughn T (2004) Binary toxins from Bacillus thuringiensis active against the western corn rootworm, Diabrotica virgifera virgifera LeConte. Applied and Environmental Microbiology 70(8):4889–4898Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate: a practical and powerful approach to multiple testing. Journal of the Royal Statistical Society Series B-Methodological 57(1):289–300Bradbury JF (1986) Guide to plant pathogenic bacteria. Cambridge News Ltd, AberystwythBravo A, Gill SS, Soberón M (2007) Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. Toxicon 49:423–435Broderick NA, Raffa KF, Handelsman J (2006) Midgut bacteria required for Bacillus thuringiensis insecticidal activity. Proceedings of the National Academy of Sciences 103:15196–15199CFIA (1994) The Biology of Zea mays L. (Corn/Maize). BIO1994-11. Canadian Food Inspection Agency, Ottawa. JA, Jelen A, Pearce GM, Jany CS, Johnson TB, Gawron-Burke C (1991) Isolation and characterisation of a novel insecticidal crystal protein gene from Bacillus thuringiensis subsp. aizawai. Journal of Bacteriology 173(13):3966–3976Christensen AH, Sharrock RA, Quail PH (1992) Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation. Plant Molecular Biology 18(4):675–689Codex (2009) Foods derived from modern biotechnology, 2nd edition. Codex Alimentarius Commission, Joint FAO/WHO Food Standards Programme, Food and Agriculture Organization of the United Nations, Rome. (2006) Corn oil. 5 ed, Corn Refiners Association, Washington D.C.. Damgaard PH, Granum PE, Bresciani J, Torregrossa MV, Eilenberg J, Valentino L (1997) Characterization of Bacillus thuringiensis isolated from infections in burn wounds. FEMS Immunology & Medical Microbiology 18:47–53de Maagd RA, Bravo A, Crickmore N (2001) How Bacillus thuringiensis has evolved specific toxins to colonize the insect world. Trends in Genetics 17:193–199Delaney B, Astwood JD, Cunny H, Eichen Conn R, Herouet-Guicheney C, MacIntosh S, Meyer LS, Privalle LS, Gao Y, Mattsson J, Levine M, ILSI (2008) Evaluation of protein safety in the context of agricultural biotechnology. Food and Chemical Toxicology 46:S71–S97Diehn, S., Lu, A. L., Nowatzki, T. M., Nubel, D. S., Pascual, M. A., Register III, J. C., Scelonge, C. J., Young, J. K., Crowgey, E. L. (2011) Maize event DP-004114-3 and methods for detection thereof. Patent US 2011/0154523 A1. RT, Stockhoff BA, Stamp L, Schnepf HE, Schwab GE, Knuth M, Russell J, Cardineau GA, Narva KE (2002) Novel Bacillus thuringiensis binary insecticidal crystal proteins active on western corn rootworm, Diabrotica virgifera virgifera Le Conte. Applied and Environmental Microbiology 68(3):1137–1145FAOSTAT (2014) Online database of the Food and Agriculture Organization of the United Nations. A, Guilley H, Jonard G, Richards K, Hirth L (1980) Nucleotide sequence of Cauliflower mosiac virus DNA. Cell 21(1):285–294FSANZ (2003) Application A446: Food derived from Insect-Protected and Glufosinate Ammonium-Tolerant Corn Line 1507. Report prepared by Food Standards Australia New Zealand. (2005) Application A543: Food derived from Insect-Protected, Glufosinate Ammonium-Tolerant Corn Line DAS-59122-7. Report prepared by Food Standards Australia New Zealand. (2013a) Application A1087 - Food derived from insect-protected soybean line DAS-81419-2. Report prepared by Food Standards Australia New Zealand. (2013b) Application A1080 - Food derived from herbicide-tolerant cotton line MON88701. Report prepared by Food Standards Australia New Zealand. Pool (2012) Sugar consumption in Australia. A statistical update. Green Pool Commodity Specialists, Brisbane. GG, Glenn KC, Ridley WP (2010) Assessing the natural variability in crop composition. Regulatory Toxicology and Pharmacology 58:S13–S20; doi:10.1016/j.yrtph.2010.08.023Herman RA, Ekmay R (2014) Do whole-food animal feeding studies have any value in the safety assessment of GM crops? Regulatory Toxicology and Pharmacology 68:171–174Herman RA, Scherer PN, Young DL, Mihaliak CA, Meade T, Woodsworth AT, Stockhoff BA, Narva KE (2002) Binary insecticidal crystal protein from Bacillus thuringiensis, strain PS149B1: effects of individual protein components and mixtures in laboratory bioassays . Journal of Economic Entomology 95(3):635–639Hernandez E, Ramisse F, Ducoureau J-P, Cruel T, Cavallo J-D (1998) Bacillus thuringiensis subsp. konkukian (serotype H34) superinfection: case report and experimental evidence of pathogenicity in immunosuppressed mice. Journal of Clinical Microbiology 36:2138–2139Hérouet C, Esdaile DJ, Mallyon BA, Debruyne E, Schulz A, Currier T, Hendrickx K, van der Klis R-J, Rouan D (2005) Safety evaluation of the phosphinothricin acetyltransferase proteins encoded by the pat and bar sequences that confer tolerance to glufosinate-ammonium herbicide in transgenic plants. Regulatory Toxicology and Pharmacology 41:134–149Hertig C, Rebmann G, Bull J, Mauch F, Dudler R (1991) Sequence and tissue-specific expression of a putative peroxidase gene from wheat (Triticum aestivum L.). Plant Molecular Biology 16(1):171–174Hofmann C, Vanderbruggen H, H?fte H, Van Rie J, Jansens S, Van Mellaert H (1988) Specificity of Bacillus thuringiensis d-endotoxins is correlated with the presence of high-affinity binding sites in the brush border membrane of target inscet midguts. Proceedings of the National Academy of Sciences 85:7844–7848H?fte H, Whiteley HR (1989) Insecticidal crystal proteins of Bacillus thuringiensis. Microbiological Reviews 53:242–255ILSI (2014) International Life Sciences Institute Crop Composition Database Version 4.0. SG, Goodbrand RB, Ahmed R, Kasatiya S (1995) Bacillus cereus and Bacillus thuringiensis isolated in a gastroeneritis outbreak investigation. Letters in Applied Microbiology 21:103–105Keil M, Sanchez-Serrano J, Schell J, Willmitzer L (1986) Primary structure of a proteinase inhibitor II gene from potato (Solanum tuberosum). Nucleic Acids Research 14(14):5641–5650Kim S-I, Veena, Gelvin SB (2007) Genome-wide analysis of Agrobacterium T-DNA integration sites in the Arabidopsis genome generated under non-selective conditions. The Plant Journal 51:779–791Koch MS, Ward JM, Levine SL, Baum JA, Vicini JL, Hammond BG (2015) The food and environmental safety of Bt crops. Frontiers in Plant Science 6:283Kützner HJ (1981) The Family Streptomycetaceae. In: Starr MP, Stolp H, Trüper HG, Ballows A, and Schlegel HG (eds) The Prokaryotes: A Handbook on Habitats, Isolation and Identification of Bacteria. Springer Verlag, Berlin, p. 2028–2090Masson L, Schwab G, Mazza A, Brousseau R, Potvin L, Schwartz JL (2004) A novel Bacillus thuringiensis (PS149B1) containing a Cry34Ab1/Cry35Ab1 binary toxin specific for the western corn rootworm Diabrotica virgifera virgifera LeConte forms ion channels in lipid membranes. Biochemistry 43(38):12349–12357Metcalfe DD, Astwood JD, Townsend R, Sampson HA, Taylor SL, Fuchs RL (1996) Assessment of the allergenic potential of foods derived from genetically engineered crop plants. Critical Reviews in Food Science and Nutrition 36 Suppl:S165–S186Moellenbeck DJ, Peters ML, Bing JW, Rouse JR, Higgins LS, Sims L, Nevshemal T, Marshall L, Ellis RT, Bystrak PG, Lang BA, Stewart JL, Kouba K, Sondag V, Gustafson V, Nour K, Xu D, Swenson J, Zhang J, Czapla T, Schwab G, Jayne S, Stockhoff BA, Narva K, Schnepf HE, Stelman SJ, Poutre C, Koziel M, Duck N (2001) Insecticidal proteins from Bacillus thuringiensis protect corn from corn rootworms. Nature Biotechnology 19(7):668–672NPTN (2000) Bacillus thuringiensis (Technical Fact Sheet)., National Pesticide Telecommunications Network, Oregon State University & U.S. Environmental Protection Agency. JT, Nagy F, Chua N-H (1985) Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313:810–812OECD (2002) Consensus document on compositional considerations for new varieties of maize (Zea mays): Key food and feed nutrients, anti-nutrients and secondary plant metabolites. Organisation for Economic Co-operation and Development, Paris.(2002)25&doclanguage=enOECD (2003) Considerations for the safety assessment of animal feedstuffs derived from genetically modified plants. ENV/JM/MONO(2003)10. Organisation for Economic Co-operation and Development, Paris.(2003)10&doclanguage=enOECD (2007) Consensus document on safety information on transgenic plants expressing Bacillus thuringiensis - derived insect control proteins. ENV/JM/MONO(2007)14. Organisation for Economic Co-operation and Development.(2007)14&docLanguage=EnOGTR (2008) The biology of Zea mays L. ssp mays (maize or corn). Prepared by the Office of the Gene Technology Regulator, Australia. WR, Lipman DJ (1988) Improved tools for biological sequence comparison. Proceedings of the National Academy of Sciences 85(8):2444–2448Pietrzak M, Shillito RD, Hohn T, Potrykus I (1986) Expression in plants of two bacterial antibiotic resistance genes after protoplast transformation with a new plant expression vector. Nucleic Acids Research 14:5857–5868Ransom J, Endres GJ (2014) Corn growth and management quick guide. A-1173, North Dakota State University Extension Service. WP, Harrigan GG, Breeze ML, Nemeth MA, Sidhu RS, Glenn KC (2011) Evaluation of compositional equivalence for multitrait biotechnology crops. Journal of Agricultural and Food Chemistry 59:5865–5876Siebert MW, Babock JM, Nolting S, Santos AC, Adamczyk JJ, Neese PA, King JE, Jenkins JN, McCarty J, Lorenz GM, Fromme DD, Lassiter RB (2014) Efficacy of Cry1F insecticidal protein in maize and cotton for control of fall armyworm (Lepidoptera: Noctuidae). Florida Entomologist 91(4):555–565Wang, N., Heetland, L. L., Solawetz, W. E., Zhao, Z.-Y. (2008) Highly transformable elite inbred line - PHWWE. Patent US 2008/0072344 A1Wehrmann A, Van Vliet A, Opsomer C, Botterman J, Schulz A (1996) The similarities of bar and pat gene products make them equally applicable for plant engineers. Nature Biotechnology 14:1274–1278White PJ, Pollak LM (1995) Corn as a food source in the United States: Part II. Processes, products, composition, and nutritive values. In: Cereal Foods World 40. p. 756–762WHO. Microbial pest control agent Bacillus thuringiensis. (217). 1999. Geneva, World Health Organization. Environmental Health Criteria. Wohlleben W, Arnold W, Broer I, Hillemann D, Strauch E, Punier A (1988) Nucleotide sequence of the phosphinothricin N-acetyltransferase gene from Streptomyces viridochromogenes Tü494 and its expression in Nicotiana tabacum. Gene 70:25–37Zhao Z, Gu W, Cai T, Tagliani L, Hondred D, Bond D, Schroeder S, Rudert M, Pierce D (2001) High throughput genetic transformation mediated by Agrobacterium tumefaciens in maize. Molecular Breeding 8:323–333Zhou J, Harrigan GG, Berman KH, Webb EG, Klusmeyer TH, Nemeth MA (2011) Stability in the composition equivalence of grain from insect-protected maize and seed from glyphosate-tolerant soybean to conventional counterparts over multiple seasons, locations, and breeding germplasms. Journal of Agricultural and Food Chemistry 59:8822–8828 ................
................
In order to avoid copyright disputes, this page is only a partial summary.
To fulfill the demand for quickly locating and searching documents.
It is intelligent file search solution for home and business.