<p style="text-align: justify;"><b>Toxicity and Allergenicity:</b></p>

<p style="text-align: justify;">Amino acid sequence alignment analysis, using the FASTA algorithm, revealed 23.9% identity between cDHDPS and <i>Mercurialis annua</i> profilin allergen over a 92 amino acid window. However, the length of the overlap is relatively short when compared to the full length (303 amino acid) cDHDPS protein and the longest stretch of the contiguous amino acid identities consisted of three amino acids. Based on this result, it is considered that cross-reactivity between cDHDPS and <i>M. annua</i> profilin allergen is highly unlikely. No other immunologically relevant sequences were detected when cDHDPS amino acid sequence was compared to the allergen sequences in AD4 database and it was concluded that cDHDPS protein is unlikely to share structurally and immunologically relevant sequence similarities with known allergens.</p>

<p style="text-align: justify;">In <i>in vitro</i> digestion studies, greater than 96% of the cDHDPS protein was digested in simulated gastric fluid within 30 seconds supporting the conclusion that cDHDPS has a low allergenic potential.</p>

<p style="text-align: justify;">An acute high dose oral toxicity study in mice using <i>E. coli</i>-derived cDHDPS protein involving a single gavage dose of 800 mg of cDHDPS/kg body weight did not result in any mortalities or adverse clinical reactions in mice.</p>

<p style="text-align: justify;"><b>Dietary Exposure:</b></p>

<p style="text-align: justify;">The levels of cDHDPS protein in LY038 were evaluated in tissue samples collected from plants grown at five field sites in the U.S., using an ELISA assay with goat polyclonal antibody specific for cDHDPS. The negative segregant LY038(-) was used as the negative control and <i>E. coli</i>-derived cDHDPS as the reference standard. The mean cDHDPS protein levels in LY038 grain, forage in the early dent stage, whole plant, forage root, root, and pollen tissues were 26.0, 0.94, 0.081, 0.069, 1.5, and 0.78 &micro;g/g dry weight, respectively. cDHDPS in leaf tissue was measured at four time points throughout the growing season and was not detected at the assay detection limit of 0.013 &micro;g/g fresh weight. These results indicate that cDHDPS is expressed primarily in the grain tissue as expected from the use of the Glb1 promoter. The estimated potential maximum daily intake of the cDHDPS protein for broilers, young pigs, and finishing pigs was calculated to be 1482, 962, 598 &micro;g/kg BW/day, respectively.</p>

<p style="text-align: justify;">Humans consume relatively little whole kernel or processed maize, compared to maize-based food ingredients. Maize is a raw material for the manufacture of starch, the majority of which is converted to a variety of sweetener and fermentation products, including high fructose syrup and ethanol. Maize oil is commercially processed from the germ. As the target use of LY038 is for animal feed, dietary exposure to humans of the cDHDPS protein will be significantly lower than the estimated levels calculated above for animals fed with this maize.</p>

<p style="text-align: justify;"><b>Lysine Catabolites:</b></p>

<p style="text-align: justify;">The grain of LY038 maize contains elevated levels of lysine and its catabolites saccharopine and ?-aminoadipic acid. Animals are continually exposed to saccharopine and ?-aminoadipic acid in the normal course of endogenous lysine metabolism and these compounds are degraded and ultimately become substrates for the tricarboxylic acid cycle, entering as acetoacetyl-CoA. Even if farm animals consumed large amounts of LY038 maize grain, the saccharopine would be completely degraded because of the excess capacity of saccharopine dehydrogenase in their liver and it would neither cause a safety problem to animals, nor would it accumulate in the tissue to pose a problem to humans consuming meat. Articles in the literature report studies in pigs, rats, and broiler chickens showing no adverse effects when animals were exposed to saccharopine or ?-aminoadipic acid. Furthermore, substantial amounts of ?-aminoadipic acid and saccharopine are found in foods commonly consumed by people. For example, ?-aminoadipic acid is found in lentils, garden peas, broccoli, cauliflower, green beans and lettuce and saccharopine is normally found in asparagus, edible mushrooms, lettuce, lentils and brie cheese.</p>

<p style="text-align: justify;"><b>Nutritional and Compositional Data:</b></p>

<p style="text-align: justify;">The composition of forage and grain from maize LY038 was evaluated using forage and grain from a negative segregant LY038(-), which did not contain the <i>cordapA</i> gene, as the control material. LY038 and LY038(-) maize lines were grown during the 2002 field season at five replicated fields in a randomized complete block design with three replicates per block. In addition, 20 conventional maize hybrids (reference material) were grown, four per site, to determine the amount of variation in nutrient composition that might be expected at each site. Forage and grain samples were collected from all plots and analyzed for nutritional components, anti-nutrients and secondary metabolites. In addition, six lysine-related secondary metabolites from lysine biosynthetic and catabolic pathways, as well as free (not incorporated into protein) and total lysine, were analyzed in LY038, LY038(-), and the conventional maize hybrid grain samples.</p>

<p style="text-align: justify;">Forage was harvested at the early dent stage. Compositional analyses of the forage revealed no statistically significant differences in the mean levels of all measured components except for phosphorus. The level of phosphorus was lower in LY038 than in LY038(-). However, the levels of all components, including phosphorus, fell within ranges detailed in the literature and within the 99% tolerance interval calculated for the conventional maize hybrids grown as controls.</p>

<p style="text-align: justify;">Grain samples were harvested at maturity (R6 growth stage) and analyzed for 75 components. Analytical data showed that, as intended, the levels of free and total lysine were higher in the LY038 grain than in the control LY038 (-) grain. However, the range of total lysine levels in LY038 was within the range reported for field maize. As expected, the levels of lysine catabolites, (saccharopine and ?-aminoadipic acid) were also elevated in the LY038 grain.</p>

<p style="text-align: justify;">Other statistically significant compositional differences were either within the 99 % tolerance level and/or within historical ranges for maize based on the field studies or ranges reported in the literature. No statistically significant differences were observed between LY038 and LY038(-) in the levels of antinutrients (phytic acid and raffinose) and secondary metabolites (ferulic acid and p-coumaric acid).</p>

<p style="text-align: justify;"><b>Bioefficacy and Safety of LY038 in Broiler Chickens:</b></p>

<p style="text-align: justify;">Maize-based diets for poultry and swine are typically deficient in the amino acid lysine and these diets are commonly supplemented with crystalline lysine to meet the animal&#39;s amino acid requirements. LY038 was developed to provide maize grain with consistently higher lysine content. To assess the nutritional value of the increased level of lysine in LY038, and the wholesomeness of LY038 when used as animal feed, a 42 day feeding study was conducted on growing broiler chickens, the primary intended market for LY038. A statistically significant increase in growth rate and feed efficiency was observed in broilers fed LY038 versus birds fed LY038(-) or four conventional varieties not supplemented with lysine. Chick mortality was low and random without any relationship to treatment. No unexpected effects on bird health were observed with the feeding of LY038 grain.</p>
