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

<p style="text-align: justify;">The primary use of commodity soybeans is for soybean meal that is consumed by animals. Raw soybeans contain several antinutritional factors (trypsin inhibitors, urease, and hemagglutinins). Heat treatment is the most common processing method used to minimize the activity of such factors. Soybean oil is the major soybean fraction consumed by humans. Soybean oil accounts for 80% of total United States consumption of oils and fats.</p>

<p style="text-align: justify;">Pioneer states that the 305423 soybean variety is intended to be used for the production of high oleic soybean oil. The oil is intended to be a highly stable vegetable oil suitable for frying applications without the need for hydrogenation which produces trans fatty acids and &quot;hydrogenated flavor.&quot; Pioneer states that it is aware of no food or feed uses of current soybean varieties for which the 305423 soybean variety would not be equally suitable.</p>

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

<p style="text-align: justify;">Pioneer assessed the composition of grain and forage from the 305423 soybean and a null segregant (non-transgenic isoline) control. Pioneer states that the null segregant plants are an appropriate control because they are almost genetically identical to the corresponding 305423 soybean plants with the exception that they do not carry the transgenic DNA. Both the transgenic and control soybeans were grown at six field locations in soybean-growing areas of North America using a randomized complete block design with three replicates at each location. Pioneer also analyzed grain and forage from four different commercial soybean varieties.</p>

<p style="text-align: justify;">Pioneer measured 52 components in grain and 5 in forage. Composition data obtained from the commercial varieties were used to calculate 99% tolerance intervals with 95% confidence for all measured components. To interpret the composition results for 305423 soybeans, Pioneer used the confidence intervals and established a combined literature range using data from published literature and databases on soybean composition. Grain samples were analyzed for proximates (protein, fat, and ash), acid detergent fiber (ADF), neutral detergent fiber (NDF), fatty acids, amino acids, isoflavones, and antinutrients. Compositional analysis of forage samples included proximates, ADF, and NDF.</p>

<p style="text-align: justify;"><b>Grain Analysis:</b> Analysis of the levels of 14 fatty acids confirmed the expected high oleic acid phenotype as shown by a substantial increase in the mean level of oleic acid to 76.5% in 305423 soybean as compared to a mean level of 21% in the control soybean. As expected, the increase in the level of oleic acid was accompanied by a decrease in the level of linoleic acid. The mean level of linoleic acid in the control soybean was 52.5% and that in the 305423 soybean was 3.6%. The level of linolenic acid also decreased since linolenic acid is formed in soybeans directly from linoleic acid. Levels of two minor fatty acids, heptadecanoic and heptadecenoic, increased in the 305423 soybean to 0.8% and 1.2% of the total fatty acids, respectively. This increase in the levels of heptadecanoic and heptadecenoic fatty acids is not unexpected because the GM-HRA enzyme most likely results in the increased concentration of 2-ketobutyrate, which is the substrate in the biosynthesis of hepta fatty acids in soybeans.</p>

<p style="text-align: justify;">Mean levels of protein and fiber in grain from the 305423 and control soybean were not statistically significantly different. While the mean level of fat was statistically significantly lower in the 305423 soybean than in the control using the unadjusted P-value, no statistical difference was detected using the false discovery rate (FDR)-adjusted P-value. The mean level of ash was statistically significantly lower in the 305423 soybean as compared to the control soybean. Mean levels of the proximates and fiber in grain from 305423 and control soybean lines were within the 99% tolerance intervals for the reference varieties and within the combined literature range.</p>

<p style="text-align: justify;">There were no statistically significant differences observed in the mean levels of amino acids between 305423 and control soybean grain with the exception of threonine and glutamic acid. The mean levels of these two amino acids were statistically significantly increased in 305423 soybean when the unadjusted P-values were used, but not when the FDR-adjusted P-values were used. All these levels were within the 99% tolerance intervals.</p>

<p style="text-align: justify;">Mean levels of nine isoflavones were quantified in grain samples. The mean levels for daidzin, malonylgenistin, and malonyldaidzin were statistically significantly increased in the 305423 versus the control soybean. Mean genestin levels were only shown to be statistically significantly increased when the unadjusted P-value was used. Mean values for all the measured isoflavones were within the 99% tolerance intervals. In both the control and 305423 soybean the mean levels of genistin, glycitin, malonylgenistin, malonylglycitin, and malonyldaidzin were above the combined literature range. Daidzin levels were only above the combined literature range in the 305423 soybean. No literature data were available for the level of glycitein. The values for the other two isoflavones (genistein and daidzein) were within the combined literature range.</p>

<p style="text-align: justify;">Concentrations of several antinutrients were measured in soybean grain including non-digestible oligosaccharides stachyose and raffinose, lectins, phytic acid, and trypsin inhibitor. No statistically significant differences were observed between the 305423 and control soybean in mean values for raffinose, lectins, and phytic acid. The mean value for trypsin inhibitor was statistically significantly lower for 305423 soybean as compared to the control soybean. The reduction in the mean value of trypsin inhibitor in 305423 soybean was expected as Pioneer reported that the promoter used to drive expression of the <i>FAD2-1</i> gene, the Kunitz trypsin inhibitor promoter, has been shown to silence the KTi3 gene which encodes the Kunitz trypsin inhibitor. The mean value for stachyose was statistically significantly increased when the unadjusted P-value was used, but not when the FDR-adjusted P-value was used. Mean values for all the measured antinutrients were within the 99% tolerance intervals and within the combined literature ranges.</p>

<p style="text-align: justify;"><b>Forage Analysis:</b> Pioneer analyzed forage for protein, fat, ash, ADF, and NDF. No statistically significant differences were observed between the mean levels of these components in forage from the 305423 and control soybeans with the exception of the level of fat. The mean level of fat was statistically significantly decreased in 305423 using the unadjusted P-value, but not when using the FDR-adjusted P-value. All mean levels were within the 99% tolerance intervals and combined literature ranges, with the exception of NDF. The mean level of NDF in both the control and 305423 soybean was above the combined literature range.</p>

<p style="text-align: justify;"><b>Endogenous Allergens:</b> A study was conducted to assess whether the transformation process may have increased the overall allergenicity of 305423 soybean compared to conventional soybean. Using sera from clinically reactive soy allergic patients, Pioneer conducted IgE immunoblot and ELISA studies using protein extracts from 305423 soybean and conventional soybean. The SDS-PAGE Coomassie blue-stained protein profiles for 305423 and control soybean extracts appeared to be the same; they are similar in their IgE binding profile, and showed the same IgE binding capacity for 305423 and control soy extracts. It was concluded that the levels of endogenous allergens in and the allergic potential of 305423 soybean are comparable to those in nontransgenic control soybean.</p>

<p style="text-align: justify;"><b>Nutritional Quality</b></p>

<p style="text-align: justify;">A poultry feeding study over a period of 42 days has been carried out with diets containing 305423 soybean, non-GM control soybean with comparable genetic background and three commercial non-GM soybeans. The mortality, body weight gain, feed efficiency, organ yield, carcass yield, breast, thigh, wing and leg yield and abdominal fat of the chickens fed with 305423 soybean were compared to chickens fed non-GM control soybean diets. The results from this 42-day poultry study confirm the safety of 305423 soybean and that 305423 soybean is nutritionally equivalent to non-GM control soybean with a comparable genetic background and to commercial soybeans.</p>

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

<p style="text-align: justify;">The only newly expressed protein in 305423 soybean is the GM-HRA protein. Based on amino acid sequence similarity searches against the National Center for Biotechnology Information (NCBI) Protein dataset, there were no significant similarities with known protein toxins. Pioneer conducted an acute oral toxicity study in mice in which a single dose of 582 mg per kilogram of body weight (mg/kg bw) of GM-HRA 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. The result of this study shows that the GM-HRA protein does not cause acute toxicity.</p>

<p style="text-align: justify;">While soybean is one of the major food allergens, none of the identified allergens is a member of the ALS family and the ALS protein from soybean has not been characterized as a soy allergen. Comparisons of the amino acid sequence of the GM-HRA protein to the amino acid sequences of known allergens in the Food Allergy Research and Resource Program (FARRP, version 6.0) database using the FASTA34 sequence alignment program showed that none of the identified alignments met or exceeded the threshold of greater than or equal to 35% identity over 80 amino acids and no contiguous stretches of 8 or greater amino acids were shared between the GM-HRA protein and the proteins in the allergen database. Additionally, the GM-HRA protein is not glycosylated.</p>

<p style="text-align: justify;">The GM-HRA protein is rapidly (&lt; 30 seconds) hydrolyzed in simulated gastric fluid (SGF) to fragments of &lt; 3 kDa; and when subjected to simulated intestinal fluid (SIF), the GM-HRA protein, including the low weight molecular fragments seen in SGF, is completely and rapidly (&lt; 2 minutes) hydrolyzed.</p>

<p style="text-align: justify;">Based on this weight of evidence assessment, it was concluded that the GM-HRA protein is unlikely to be a toxin and is not a potential allergen.</p>
