<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. Typically, canola oil is used by itself as a salad oil or cooking oil, or blended with other vegetable oils in the manufacture of margarine, shortening, salad oil and cooking oils. Refined edible canola oil consists of purified triglycerides (96-97%) and does not contain any detectable protein, hence no amounts of PAT were detected in the refined oil of HCN28 canola. Furthermore, processing would destroy the enzymatic activity of the PAT protein. As such, there will be no human exposure to the introduced PAT protein as result of the consumption of refined oil from line HCN28.</p>

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

<p style="text-align: justify;">Compositional analyses of processed canola oil from HCN28 and current commercial canola cultivars were compared for fatty acids, glucosinolates, chlorophyll and phytosterol content. These comparisons indicated no statistically significant differences for the components analyzed. The use of refined oil from HCN28 would therefore have no significant impact on the nutritional quality of the food supply.</p>

<p style="text-align: justify;">Because canola meal is used as a livestock feed, additional compositional studies were conducted that demonstrated there were no significant differences in the moisture, oil, protein, crude fibre, ash, phytosterol and gross energy of HCN28, Innovator, and non-transgenic lines Excel, Legend and Cyclone. The <i>pat</i> gene did not result in any secondary effects in the seed composition or gross energy levels between glufosinate tolerant and non-transformed <i>B. napus</i> varieties.</p>

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

<p style="text-align: justify;">It was determined that there were no toxicity or allergenicity concerns with HCN28, since refined canola oil is the only product for human consumption and does not contain any detectable amount of protein. The absence of toxicity was further demonstrated by examining the amino aid sequence homology and the characteristics of the PAT protein.<br />
The potential toxicity of the PAT protein was evaluated in a 14-day repeated dose feeding study with rats. Subject groups consisting of male and female Wistar rats were fed a diet amended with either 5000 or 50,000 ppm purified PAT protein and monitored for dietary intake, weight gain, and observable adverse effects. There were no clinical signs noted and food consumption and body weights were unaffected by treatment. No treatment-related changes were seen in hematology or urinalysis parameters. Organ weight data, macroscopical and microscopical findings also did not distinguish treated groups from controls. Based on these results, there was no evidence of acute toxicity for the PAT protein.</p>

<p style="text-align: justify;">The potential of the PAT protein expressed in T45 transgenic canola to elicit allergic reactions was evaluated by comparing the nucleotide sequence of the <i>pat</i> gene from <i>S. viridochromogenes</i> with available nucleotide sequences contained within the GENBank database. Significant similarity was judged to be &gt;60% identity for &gt;100 base pairs. Using this criterion, the only significant similarity at the nucleotide sequence level was between the <i>pat</i> gene and a previously characterized PPT acetyltransferase from <i>S. viridochromogenes</i> (70.7% identity in 546 nucleotide overlap) and the analogous <i>bar</i> gene from <i>S. hygroscopicus</i> (65.8% identity in 546 nucleotide overlap). When comparing the deduced amino acid sequence of the PAT protein with other proteins in the database, the only noted significant similarity was with an rRNA methyltransferase also isolated from a Streptomyces species (68.2% identity in 132 amino acid overlap). The results of the GENBank database search failed to identify any homologies with known toxins or allergens that have been sequenced.</p>

<p style="text-align: justify;">Also, unlike known protein toxins and allergens, the PAT protein was extremely susceptible to proteolytic degradation. Crude PAT protein containing extracts from transgenic canola plants were compared with purified PPT-acetyltransferase with respect to acid and/or enzymatic hydrolysis under conditions simulating the mammalian stomach. Samples were incubated at 37&deg;C in stomach fluid from beagle dogs for up to 15 minutes, following which aliquots were removed, adjusted to pH 8.0, and tested for acetyltransferase activity. The enzymatic activity of purified PPT-acetyltransferase was completely lost within 1 minute of exposure to stomach fluid (pH 1.1), and within 10 minutes of exposure to fluid adjusted to pH 4.0. While the acetyltransferase activity of crude extracts from transgenic plants was lost equally quickly at pH 1.1, the rate of inactivation was slower at higher pH values.</p>
