A1106-AppR-SD1 - Food and Agriculture Organization



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 &amp; 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&amp;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 &amp; Medical Microbiology</Periodical><Volume>18</Volume><ZZ_JournalFull><f name="System">FEMS Immunology &amp; 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 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> T&#xFC;494 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>K&#xFC;tzner,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>Tr&#xFC;per,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.&apos;t</Keywords><Keywords>Support,U.&apos;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.&apos;t</Keywords><Keywords>Support,U.&apos;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. 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ADDIN 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&apos; 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&apos; 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.&apos;t</Keywords><Keywords>Support,U.&apos;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.&apos;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. 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ADDIN 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&apos; 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&apos; 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.&apos;t</Keywords><Keywords>Support,U.&apos;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> T&#xFC;494 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 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>H&#xF6;fte,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>Sober&#xF3;n,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). 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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). 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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 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&amp;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&amp;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 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

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