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

<p style="text-align: justify;">The human consumption of canola products is limited to the refined oil. Because virtually no protein is present in the oil extracted from the plants, the potential for human exposure is exceedingly low. Furthermore, the amounts of PAT protein present in seed-meal fed to animals would be too low to cause concern. Additionally, the barnase RNAse and its inhibitor encoded by barnase and barstar genes, respectively, were not detected in dry seeds. As the introduced gene products were not detectable in the refined oil produced from transgenic canola, there will be no human exposure to these proteins based on normal consumption patterns.</p>

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

<p style="text-align: justify;">The composition of refined canola oil from MS8 x RF3 hybrid canola was compared to that for refined oil from non-transgenic canola. Some statistical differences in fatty acid composition were noted in the comparison, however, the fatty acids for the transgenic lines, including the erucic acid content of the oil, were within the normal range for canola oil fatty acids. Processing according to protocols mimicking industrial practices (including tempering, flaking, cooking, pressing, desolventizing oil and meal, oil blending, degumming, oil refining, water washing, bleaching, hydrogenation and deodorizing) further demonstrated that the composition and physical characteristics of the oil from MS8 x RF3 hybrids and control canola varieties were equivalent. The use of refined oil from MS8 x RF3 canola hybrids would therefore have no significant impact on the nutritional quality of the food supply. Similarly, the glucosinolate content of seed meal derived from transgenic canola hybrids was the same as that from non-transgenic control cultivars.</p>

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

<p style="text-align: justify;">Since only the processed oil from transgenic MS8, RF3, or hybrids derived therefrom (MS8 x RF3), are available for human consumption, and the processing removes proteinaceous material, there were no additional toxicity or allergenicity concerns regarding this product. This was further assessed by searching for amino acid sequence homologies with known protein toxins and allergens, and by examining the physiochemical characteristics of the introduced RNAse (barnase), RNAse inhibitor (barstar), and PAT (<i>bar</i>) proteins.</p>

<p style="text-align: justify;">A comparative analysis using the FASTDB algorithm to search three amino acid sequence databases determined that amino acid sequence for each novel protein did not show significant homology with other proteins present in the databases, with the exception of other related compounds. For example, barnase RNAse was similar to ribonucleases from other bacilliform bacteria and PAT was similar to other phosphinothricin acetyltransferases originating from different organisms. No homologies with potential toxins or allergens were observed.</p>

<p style="text-align: justify;">The RNAse, encoded by the barnase gene, was a small single-domain protein, containing no disulfide bonds, metal-ion cofactors or other non-peptide components. When heated, it unfolded completely into an inactive form.</p>

<p style="text-align: justify;">No toxic or allergic effects were expected from PAT, as acetyltransferases are ubiquitous in nature, do not possess proteolytic or heat stability and are highly substrate specific. PAT has a extremely high substrate specificity for L-PPT and dimethylphosphinothricin (DMPT), and experimental data clearly showed that neither L-PPT&#39;s analog L-glutamic acid, D-PPT, nor any other amino acid can be acetylated by the PAT enzyme.</p>
