<p style="text-align: justify;"><b>The Introduced DNA</b></p>

<p style="text-align: justify;">Southern blot analyses and nucleotide sequencing demonstrated that MIR162 maize contains a single intact T-DNA insert in the nuclear maize genome. Southern blot analyses further demonstrated that the T-DNA insert contains: i) single copies of a <i>vip3Aa</i> gene and a <i>pmi</i> gene; ii) two copies of the ZmUbiInt promoter; iii) one copy of the NOS terminator; and iv) no backbone sequences from transformation plasmid pNOV1300. Nucleotide sequencing of the T-DNA insert in MIR162 maize revealed two codon changes within the <i>vip3Aa</i> coding sequence relative to the intended <i>vip3Aa</i> sequence; one of these was a silent mutation and the other codon change resulted in an amino acid substitution. The <i>vip3Aa</i> gene variant present in MIR162 maize has been designated <i>vip3Aa20</i>. Nucleotide sequencing additionally determined that the MIR162 maize T-DNA insert did not locate within any known <i>Z. mays</i> gene. Further, no novel open reading frames were created that spanned either the 5&rsquo; or 3&rsquo; junctions between the T-DNA and <i>Z. mays</i> genomic sequences. These genetic characterization data demonstrate that, apart from the well-characterized change that resulted in a single altered amino acid in the <i>vip3Aa</i> coding sequence, there were no unintended changes in the MIR162 maize genome as a result of the T-DNA insertion. Observations of <i>vip3Aa20</i> and <i>pmi</i> segregation ratios over several generations of MIR162 maize were consistent with the genes being linked at a single locus in the maize genome, and indicate stable inheritance of the transgenes. These data also indicated that no novel proteins, other than Vip3Aa20 and PMI, would be produced in MIR162 maize.</p>

<p style="text-align: justify;"><b>Expressed Material</b></p>

<p style="text-align: justify;">The levels of expression of the Vip3Aa20 and PMI proteins in maize plants derived from event MIR162 were determined in several plant tissues and whole plants at four growth stages (V9-V12, anthesis, kernel maturity and senescence) in two field maize hybrids that were hemizygous for both transgenes. Kernels from MIR162 maize are the most likely tissue to comprise foodstuffs, either as grain or grain by-products. The average Vip3Aa20 concentration measured in kernels from MIR162 maize (43.56 &micro;g Vip3Aa20/g dry weight at senescence) represents less than 0.004% of the total protein. The average PMI concentration measured in kernels from MIR162 maize (1.93 &micro;g PMI/g dry weight at senescence) represents less than 0.0002% of the total protein (these calculations are based on maize grain or kernels containing 10% total protein by weight). Given the low levels of Vip3Aa20 and PMI in MIR162 kernels, dietary exposure potential can be considered minimal. Since no health hazards were identified for either the Vip3Aa20 or PMI proteins (summarized below), specific dietary exposure estimates are not necessary to support a conclusion regarding the safety of MIR162 maize.</p>
