<p style="text-align: justify;"><b>Food and Feed Use</b></p>

<p style="text-align: justify;">Maize grain and its processed fractions are consumed as human food and animal feed. The majority of maize is used as animal feed, and a small percentage is harvested as forage and made into silage and fed to ruminants. The remainder of maize is exported, processed into food products, or converted to ethanol. Maize can be processed by wet and dry milling processes to convert the grain into food, feed, and fuel products.</p>

<p style="text-align: justify;"><b>Compositional Analysis</b></p>

<p style="text-align: justify;">The composition of forage and grain from event 98140 maize and a near isogenic, nontransgenic control maize was analyzed to assess whether there had been any unintentional compositional changes as a result of the genetic modification. Forage samples were analyzed for proximates (protein, fat, and ash), amino acids, free amino acids, acetylated amino acids [N-acetylaspartate (NAA) and N-acetylglutamate (NAG)], acid detergent fiber (ADF), neutral detergent fiber (NDF), calcium and phosphorus. Grain samples were analyzed for proximates, ADF, NDF, fatty acids, total amino acids, acetylated amino acids [NAA and NAG, N-acetylthreonine (NAThr), N-acetylserine (NASer), N-acetylglycine (NAGly)], free amino acids, minerals, vitamins, antinutrients, and secondary plant metabolites.</p>

<p style="text-align: justify;"><b>Forage Analysis:</b> No statistically significant differences between event 98140 and the nontransgenic control samples were observed in the mean levels of proximates, minerals, carbohydrates, ADF, NDF, total and free amino acids. The mean levels were also within the 99% tolerance intervals and combined literature ranges. Literature values and statistical tolerance intervals were not available for the amino acid content of maize forage, however, most of the means for the specific amino acids fell within or close to the literature ranges for maize silage published by OECD (2002). As expected, the mean levels for NAA and NAG were statistically significantly higher (p &lt; 0.05) for the 98140 samples compared to the non-transgenic control samples. No literature data were found regarding the level of the two acetylated amino acids in maize forage. In order to address the biological significance of the elevated levels of the two acetylated amino acids, their effect on the free amino acid pool was investigated. The free amino acids in maize forage were measured in both samples of nontransgenic control maize and samples of event 98140 maize, and no significant differences were detected in the mean levels of any of the free amino acids. NAG and NAA make up less than 1.2 % of the total amino acids in event 98140 forage samples and the protein levels and free amino acid pool were comparable to the non-transgenic control samples. The low levels of acetylation of aspartate and glutamate in the forage of event 98140 did not affect amino acid incorporation into proteins or the level of the free amino acid pool.</p>

<p style="text-align: justify;"><b>Grain Analysis:</b> No statistically significant differences between event 98140 and the non- transgenic control maize grain samples were observed in the mean levels of protein, ash, NDF, ADF, minerals, vitamins, antinutrients (raffinose, phytic acid, and trypsin inhibitor), secondary plant metabolites (furfural, p-coumaric acid, and ferulic acid), and fatty acids. All mean levels were within the 99% tolerance intervals and the combined literature ranges. No statistically significant differences between the 98140 and the non-transgenic control samples were observed in the mean levels of total amino acids in the grain, with the exception of the mean level of tryptophan, which was slightly higher in 98140 samples compared to control samples but still within the 99% tolerance interval and/or literature range.</p>

<p style="text-align: justify;">To clarify whether low levels of acetylation of amino acids affected the composition of the free amino acid pool in event 98140 grain, levels of individual free amino acids were measured. Two non-amino acid compounds (ethanolamine and ammonia) were measured along with the amino acids because they are recognized as analytes by the method used. There were no statistically significant differences in the mean levels of the free amino acids between 98140 and the non-transgenic control samples. Furthermore, all mean levels were within the 99% tolerance intervals; hence the free amino acid pools in event 98140 grain and non-transgenic control grain samples were comparable.</p>

<p style="text-align: justify;">Levels of acetylated amino acids were also compared between event 98140 and non-transgenic control grain samples. The mean levels of NAA and NAG in 98140 grain samples were 0.0403 % dw and 0.0079 % dw, respectively, compared to 0.00009 % dw and 0.00005 % dw, respectively, for control samples. It was calculated that NAA and NAG make up less than 0.05 % dw of maize grain and less than 0.5% of the total amino acids in event 98140 grain. Although the levels of NAThr, NASer, and NAGly were statistically significantly higher in 98140 grain samples than in the non-transgenic control samples, they were more than 100 fold lower than the levels of NAA and NAG. For example, the mean levels of NAThr, NASer, and NAGly in 98140 grain samples were less than 0.0003% on a dw basis.</p>

<p style="text-align: justify;">In addition to being found in conventional corn grain and forage, NAA and NAG are components of commonly consumed food such as eggs, chicken, turkey, and beef. An exposure assessment for humans to compare the estimates of dietary intake of NAA and NAG with and without exposure from event 98140 maize was conducted and it was concluded that commercialization of event 98140 maize may increase the dietary exposure to NAA and NAG in the U.S. population above current levels of exposure. However, based on the relatively low level of dietary exposure as well as the presence of deacetylases (aminoacylases that deacetylate N-acetylated amino acids) in the human body, it was concluded that no safety issues are expected as a result of the estimated increased consumption of acetylated amino acids in the human diet due to commercialization of 98140 maize.</p>

<p style="text-align: justify;">The biological effects of increased levels of NAA and NAG were also assessed in a 42-day broiler chicken feeding trial. No statistically significant differences were noted in mortality, weight gain, feed efficiency, and organ and carcass yield variables between broilers consuming diets produced with 98140 maize and those consuming diets produced with the non-transgenic control maize. No safety issues are expected as a result of the estimated increase in exposure to NAA and NAG in the broiler diet. Dietary exposure estimates for NAA and NAG in other livestock animal species were also calculated, and these were less than for the broiler chicken feeding trial.</p>

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

<p style="text-align: justify;">The GAT4621 and ZM-HRA proteins were evaluated for potential allergenicity and toxicity using a weight of evidence approach that examined: amino acid sequence similarities with known allergens and toxins, digestibility in model pepsin (simulated gastric fluid) and pancreatin (simulated intestinal fluid) systems, post-translational modification, and characteristics of the donor organism. Neither protein displayed significant amino acid sequence similarity with known allergens or toxins, both proteins were rapidly and completely digested in SGF within 30 seconds, and neither protein is glycosylated.</p>

<p style="text-align: justify;">The potential toxicity of GAT4621 protein was also assessed in an acute oral toxicity study in mice. A single dose of 1640 milligrams per kilogram of body weight (mg/kg bw) of <i>E. coli</i>-produced and purified GAT4621 protein was administered by oral gavage to five male and five female mice. No clinical symptoms of toxicity, body weight loss, gross organ lesions, or mortality were observed. It was concluded that GAT4621 protein is not acutely toxic. A similar study was conducted using the ZM-HRA protein with mice (5 male and 5 female) receiving a single oral dose of 1236 mg/kg bw of <i>E. coli</i>-produced and purified ZM-HRA protein. No clinical symptoms of toxicity, body weight loss, gross organ lesions, or mortality were observed, indicating that the ZM-HRA protein is not acutely toxic.</p>
