<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 CP4 EPSPS or GOX proteins were detected in the refined oil of GT73 canola. The genetic modification of GT73 canola will not result in any change in the consumption pattern for this product. As the introduced gene products were not detectable in the refined oil produced from transgenic canola, there is little or no anticipated human exposure to these proteins</p>

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

<p style="text-align: justify;">The analysis of nutrients from transgenic GT73 canola and non-transgenic canola did not reveal any significant differences in the levels of crude protein, crude fat, crude fibre, ash and gross energy in either whole seed or processed meal. The fatty acid composition of oils and levels of the shikimate pathway aromatic amino acids phenylalanine, tyrosine and tryptophan extracted from both transgenic GT73 and non-transgenic canola was statistically the same and, in the case of fatty acids, within the normal range for canola oil. These results collectively demonstrated that the introduction of the novel genes in GT73 should not result in any secondary effects impacting on composition or nutritional quality. Furthermore, the transgenic line GT73 meets the standards for canola oil in Canada of containing less than 2% erucic acid and less than 30 micromoles/g glucosinolates in the oil-free meal. It was determined that the consumption of refined oil from GT73 would have no significant impact on the nutritional quality of the food supply in the United States and Canada.</p>

<p style="text-align: justify;">Several animal feeding studies were conducted to demonstrate the wholesomeness of canola meal. Canola meal is used only for animal feed and is not considered a human food fraction. The feeding studies were conducted to establish the nutritional adequacy of canola meal for animal feeds and represented a worst case scenario in terms of canola consumption by humans. These studies included a four-week rat study on processed and unprocessed meal, a ten-week trout study on processed meal and a five-day quail (Northern Bobwhite) study on unprocessed meal. The studies all supported the safety of meal from glyphosate-tolerant canola as a component in animal feed.</p>

<p style="text-align: justify;">The rat feeding study examined six week-old Sprague-Dewley rats (10/sex/group) fed either 0, 5 or 15% w/w ground (unprocessed) and processed (toasted and defatted) glyphosate-tolerant canola (GT73 and GT200) and &#39;Westar&#39; canola meal and a diet control (commercial rodent chow with no added canola meal). The study determined that rats fed on diets with unprocessed canola seed had relative liver and kidney weights 5 - 20% greater compared to the diet controls. However, there were no differences in absolute or relative organ weights between the glyphosate-tolerant canola and parental line groups. When fed processed canola meal, there were no meaningful differences observed in body weights and body weight gains between groups fed processed glyphosate-tolerant canola meal, parental line canola meal and a control diet. Liver weights, however, increased approximately 12-16% for both sexes fed 15% GT73 meal. The increase in liver weight was attributed to a higher level of alkyl glucosinolate toxicants in the glyphosate-tolerant canola line GT73 which was 4 g/kg compared to 1.8 g/kg for the parental line. The higher level of glucosinolates present in glyphosate-tolerant canola was not attributed to the genetic modification.</p>

<p style="text-align: justify;">The quail feeding study examined thirty northern bobwhite (<i>Colinus virginianus</i>) chicks fed diets for five days and observed for a further three days. Treatment groups were fed a control diet, glyphosate-tolerant canola (both line GT73 and GT200) or the parental line &#39;Westar&#39;, where canola was incorporated at a rate of 20% of the total weight of the diet. The study did not find any significant effects on body weight or feed consumption between birds in the control or treatment groups. There were no mortalities or overt signs of toxicity in either treatment or control groups.<br />
Similarly a trout feeding study examined triplicate groups of 15 fish rainbow trout (<i>Oncorhynchus mykiss</i>) fed canola meal at 0, 5, 10, 15 or 20% weight of the dry diet for 10 weeks (<i>i.e.</i>, 45 fish/treatment).There was statistical overlap in weight gain of fish fed each dietary treatment and no differences were detected between glyphosate-tolerant canola (both line GT73 and GT200) diets and controls. Fish fed the glyphosate-tolerant canola did not exhibit any adverse effects.</p>

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

<p style="text-align: justify;">It was determined that there were no toxicity or allergenicity concerns with GT73, 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 physiochemical characteristics of the CP4 EPSPS and GOX proteins, and acute toxicity study in mice.</p>

<p style="text-align: justify;">The deduced amino acid sequences of both the CP4 EPSPS and GOX proteins were compared to the amino acid sequences of 1935 known protein toxins. No significant similarities were found other than would be expected given that certain functional domains are generally conserved between proteins.</p>

<p style="text-align: justify;">The CP4 EPSPS enzyme belongs to the same family of EPSPS enzymes that occur in plants, fungi and bacteria. The EPSPS proteins are naturally present in foods derived from plants and microbes and have no history of being toxic or allergenic. Similarly, the <i>gox</i> gene was sourced from a common soil bacterium, which has no history of pathogenicity. The GOX enzyme degrades glyphosate and experiments demonstrated that the enzyme displayed a narrow substrate specificity, and appeared not to have any secondary effects on other plant specific pathways.</p>

<p style="text-align: justify;">The acute oral toxicity of CP4 EPSPS, GOX and GOXv247 proteins, was studied in groups of ten CD-1 mice/sex in order to directly assess the potential for toxicity associated with these proteins. Based on previously assessed studies, there were no adverse effects or mortalities noted in mice administered CP4 EPSPS protein by gavage at doses up to 572 mg/kg. Similarly, no adverse effects were observed in mice administered the GOX protein by gavage at doses up to 100 and 104 mg/kg for GOX and GOXv247 respectively. The doses were well above the level of expression of the proteins found in glyphosate-tolerant canola plants, estimated to be a 1300-fold increase of CP4 EPSPS and 5000-fold increase of GOX. Clinical observations observed up to 9 days after dosing showed no signs of toxicity (<i>i.e.</i>, no adverse effects for either protein on body weight, food consumption, survival, or gross pathology).</p>

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

<p style="text-align: justify;">The possibility of exposure to CP4 EPSPS and GOX is significantly limited by the combined facts that these proteins are present at low levels in leaf tissue and are absent in refined oil. The low potential for allergenicity was further demonstrated by examining the amino aid sequence homology and a study on the digestibility of the CP4 EPSPS and GOX proteins.</p>

<p style="text-align: justify;">The amino acid sequence of the CP4 EPSPS and GOX proteins were compared to the amino acid sequences of 219 known allergens present in public domain databases (<i>e.g.</i>, GenBank, EMBL, Swissprot, PIR). No significant similarities (<i>i.e.</i> a sequence of more than 8 consecutive amino acids) were found with any of these known allergens.<br />
The study of the digestibility of both proteins in model digestion systems was done <i>in vitro</i> using simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) as mammalian digestion models. The exposure of CP4 EPSPS and GOX proteins to SGF and SIF was conducted over a series of timed incubations at 37&deg;C. The products of the digestion were analyzed using gel electrophoresis, Western blot analysis and enzymatic activity assays. From the simulated digestion experiments and Western blot analyses, the CP4 EPSPS protein had a half-life of less than 15 seconds in the gastric system and 10 minutes in the intestinal system. The GOX protein had a half-life of less than 30 seconds in the intestinal system as determined by Western blot analyses. In summary, both the CP4 EPSPS and GOX proteins were digested by proteases present in the mammalian digestive system, suggesting that they would not survive peptic and tryptic digestion or the acidic conditions of the human digestive system.</p>
