Decision Document DD 2012-88: Determination of the Safety of Pioneer Hi-Bred Production Ltd.'s Canola (Brassica napus L.) Events 73496 and 61061

This Decision Document has been prepared to explain the regulatory decision reached under Directive 94-08 (Dir94-08), entitled "Assessment Criteria for Determining Environmental Safety of Plants with Novel Traits", its companion biology document BIO1994-09, "The Biology of Brassica napus L. (Canola/Rapeseed)", and Chapter 2.6 of the Regulatory Guidance: Feed Registration Procedures and Labelling Standards, entitled "Guidelines for the Assessment of Novel Feeds: Plant Sources".

The Canadian Food Inspection Agency (CFIA), specifically the Plant Biosafety Office of the Plant Health and Biosecurity Directorate, the Plant and Biotechnology Risk Assessment Unit of the Plant Health Science Directorate and the Animal Feed Division of the Animal Health Directorate, has evaluated information submitted by Pioneer Hi-Bred Production Ltd. This information is in regard to the herbicide tolerant canola events 73496 and 61061. The CFIA has determined that these plants with a novel trait (PNT) do not present altered environmental risk nor, as a novel feed, do they present livestock feed safety concerns when compared to currently commercialized canola varieties in Canada.

Taking into account these evaluations, unconfined release into the environment and use as livestock feed of canola events 73496 and 61061 is therefore authorized by the Plant Biosafety Office of the Plant Health and Biosecurity Directorate and the Animal Feed Division of the Animal Health Directorate as of May 3, 2012. Any canola lines derived from canola events 73496 and 61061 may also be released into the environment and used as livestock feed, provided that

1. no inter-specific crosses are performed,
2. the intended uses are similar,
3. it is known based on characterization that these plants do not display any additional novel traits and are substantially equivalent to currently grown canola in Canada, in terms of their potential environmental impact and livestock feed safety, and
4. the novel genes are expressed at a level similar to that of the authorized lines.

Canola events 73496 and 61061 are subject to the same phytosanitary import requirements as their unmodified counterparts. Canola events 72496 and 61061 are required to meet the requirements of other jurisdictions; including but not limited to, the Food & Drugs Act and the Pest Control Products Act.

Please note, that the livestock feed and environmental safety assessments of novel feeds and PNTs are critical steps in the potential commercialization of these plant types. Other requirements, such as the evaluation of food safety by Health Canada, have been addressed separately from this review.

This bulletin is published by the Canadian Food Inspection Agency. For further information, please contact the Plant Biosafety Office or the Animal Feed Division at:

Table of Contents

1. Brief Identification of the Modified Plant
2. Background Information
3. Description of the Novel Trait
   1. Development Method
   2. Tolerance to Glyphosate
   3. Stable Integration into the Plant Genome
4. Criteria for the Environmental Assessment
   1. Potential of Canola Events 73496 and 61061 to Become Weeds of Agriculture or be Invasive of Natural Habitats
   2. Potential for Gene Flow from Canola Events 73496 and 61061 to Sexually Compatible Plants Whose Hybrid Offspring May Become More Weedy or More Invasive
   3. Altered Plant Pest Potential of Canola Events 73496 and 61061
   4. Potential Impact of Canola Events 73496 and 61061 on Non-Target Organisms
   5. Potential Impact of Canola Events 73496 and 61061 on Biodiversity
5. Criteria for the Livestock Feed Assessment
   1. Potential Impact of Canola Events 73496 and 61061 on Livestock Nutrition
   2. Potential Impact of Canola Events 73496 and 61061 on Animal Health and Human Safety as it Relates to the Potential Transfer of Residues into Foods of Animal Origin, and Worker/Bystander Exposure to the Feed
6. New Information Requirements
7. Regulatory Decision

I. Brief Identification of the Modified Plant

II. Background Information

Canola events 73496 and 61061 were developed by Pioneer Hi-Bred Production Ltd. using recombinant DNA technology, resulting in the introduction of the gat4621 gene. The gat4621 gene encodes a glyphosate acetyltransferase enzyme, GAT4621, which is derived from Bacillus licheniformis enzymes. GAT4621 acetylates the herbicide glyphosate, rendering it non-phytotoxic and conferring glyphosate herbicide tolerance to the plant.

Pioneer Hi-Bred Production Ltd. has provided data on the identity of canola events 73496 and 61061, a detailed description of the transformation method, data and information on the gene insertion site, gene copy number and levels of gene expression in the plants and the role of the inserted gene and regulatory sequences. The novel protein was identified and characterized. Data were provided for the evaluation of the potential toxicity of the novel protein to livestock and non-target organisms and potential allergenicity of the novel protein to humans and to livestock. Data were provided for the evaluation of herbicide residues in the feed commodities derived from the crop, following herbicide application.

Canola events 73496 and 61061 have been field tested at ten test sites located within the major canola-producing regions of the United States and Canada in 2008 and 2009. The Canadian locations were representative of canola growing regions in Canada.

Agronomic characteristics of canola events 73496 and 61061 such as seed dormancy, seedling vigour, plant height, lodging, yield, days to flowering, flowering duration, days to maturity and susceptibilities to various canola pests and pathogens were compared to those of the unmodified control.

Nutritional components of canola events 73496 and 61061, such as proximates (protein, fat, fibre, ash, and carbohydrates), acid detergent fibre, neutral detergent fibre, amino acids, fatty acids, vitamins, minerals, glucosinolates, phytosterols, antinutrients, and secondary metabolites, were compared with those of the unmodified canola control.

The Plant Biosafety Office of the Plant Health and Biosecurity Directorate, in conjunction with the Plant and Biotechnology Risk Assessment (PBRA) Unit of the Plant Health Science Directorate, CFIA, have reviewed the above information, in light of the assessment criteria for determining environmental safety of PNTs, as described in the Directive 94-08 (Dir94-08), entitled "Assessment Criteria for Determining Environmental Safety of Plants With Novel Traits". The PBRA Unit has considered:

• the potential of canola events 73496 and 61061 to become weeds of agriculture or be invasive of natural habitats;
• the potential for gene flow from canola events 73496 and 61061 to sexually compatible plants whose hybrid offspring may become more weedy or more invasive;
• the potential for canola events 73496 and 61061 to become a plant pest;
• the potential impact of canola events 73496 and 61061 or their gene products on non-target species, including humans; and
• the potential impact of canola events 73496 and 61061 on biodiversity.

The Animal Feed Division of the Animal Health Directorate, CFIA, has also reviewed the above information with respect to the assessment criteria for determining the safety and efficacy of livestock feed, as described in Chapter 2.6 of the Regulatory Guidance: Feed Registration Procedures and Labelling Standards, entitled "Guidelines for the Assessment of Novel Feeds: Plant Sources".

The Animal Feed Division has considered both intended and unintended effects and similarities and differences between the modified plants and their counterparts relative to the safety and efficacy of feed ingredients derived from canola events 73496 and 61061 for their intended purpose; including:

• potential impact of canola events 73496 and 61061 on livestock nutrition; and
• potential impact of canola events 73496 and 61061 on animal health and human safety as it relates to the potential transfer of residues into foods of animal origin, and worker/bystander exposure to the feed.

The Animal Feed Division has also considered whether feeds derived from canola events 73496 and 61061 meet the feed definitions and labelling requirements as listed in Schedule IV of the Feeds Regulations.

Pioneer Hi-Bred Production Ltd. has provided the CFIA with a method for the detection and identification of canola events 73496 and 61061.

III. Description of the Novel Traits

1. Development Method

Canola events 73496 and 61061 were developed through microprojectile bombardment of embryonic microspores with a DNA fragment containing only the gat4621 gene and its regulatory elements. Transformants were selected based on tolerance to glyphosate. Canola events 73496 and 61061 were identified as successful transformants and were chosen for further development based on molecular analyses, herbicide efficacy, and agronomic evaluations.

2. Tolerance to Glyphosate

The herbicide glyphosate blocks the shikimic acid pathway required for biosynthesis of the aromatic amino acids phenylalanine, tyrosine, and tryptophan in plants, leading to growth suppression or death of the plants. The gat4621 gene encodes a glyphosate acetyltransferase enzyme, which inactivates glyphosate by N-acetylation. The GAT4621 enzyme is derived from three B. licheniformis glyphosate acetyltransferases, to which it bears 75–78 percent amino acid sequence identity. Introduction of the gat4621 gene into canola during the transformation process to develop canola events 73496 and 61061 confers field level tolerance to glyphosate.

The GAT4621 protein has been subject to a previous CFIA safety assessment study in Pioneer Hi-Bred Production Ltd.'s Corn Event 98140 (DD2009-78).

GAT4621 protein expression in canola events 73496 and 61061 is driven by a constitutive promoter. Samples of canola tissue were collected from plants from six field trials in the United States and Canada. Whole plant samples were collected at stages from five true leaves unfolding to full flowering, root samples were collected at full flowering, and seed samples were collected at senescence. GAT4621 protein expression in micro-grams of protein per gram of dry weight tissue (µg/g dwt) was evaluated by enzyme-linked immunosorbent assay (ELISA). Average expression levels for canola event 73496 are as follows: 6.9 µg/g dwt in the whole plant at the BBCH15 stage, 5.3 µg/g dwt in the whole plant at the BBCH33 stage, 5.2 µg/g dwt in the whole plant at the BBCH65 stage, 6.6 µg/g dwt in root, and 6.2 µg/g dwt in seed. Average expression levels for canola event 61061 are as follows: 12 µg/g dwt in the whole plant at the BBCH15 stage, 9.0 µg/g dwt in the whole plant at the BBCH33 stage, 7.9 µg/g dwt in the whole plant at the BBCH 65 stage, 5.2 µg/g dwt in root, and 5.1 µg/g dwt in seed.

To obtain sufficient quantities of GAT4621 protein for evaluation of environmental and feed safety, it was necessary to express the gat4621 gene in a microbial production system. Equivalency was demonstrated between canola events 73496 and 61061-produced GAT4621 protein and a microbial-produced GAT4621 protein that had been used in studies previously submitted for Pioneer Hi-Bred Production Ltd.'s corn event 98140, and these studies were accepted to support the safety of canola events 73496 and 61061. This demonstration of equivalency was based on a comparison of their molecular weights, immunoreactivities, N-terminal amino acid sequences, and tryptic peptide mass maps.

Demonstration of protein equivalence between microbial and canola events 73496 and 61061-produced GAT4621 proteins allows use of the existing data to confirm the safety of the GAT4621 protein in canola events 73496 and 61061. Previous assessments have shown that the GAT4621 protein did not cause any adverse effects in mice at a level of 1900 mg/kg body weight, and that the GAT4621 protein, unlike many allergens, is readily degraded in simulated mammalian gastric and intestinal fluids. The GAT4621 protein expressed in canola events 73496 and 61061 is similar to glyphosate acetyltransferases in B. licheniformis, to which animals and humans are regularly exposed. In addition, Pioneer Hi-Bred Production Ltd. provided an updated bioinformatic evaluation of the GAT4621 protein, which confirmed the lack of relevant similarities between the GAT4621 protein sequence and sequences of known allergens and toxins. Therefore, based on the weight of evidence, the GAT4621 protein expressed in canola events 73496 and 61061 is unlikely to be toxic or allergenic to mammals.

3. Stable Integration into the Plant Genome

Molecular characterization by Southern blot analysis demonstrated that canola events 73496 and 61061 each contain one intact copy of the gat4621 gene cassette at a single site in the canola genome. No additional elements, including intact or partial DNA fragments of the gat4621 gene cassette or backbone sequences from the vector, linked or unlinked to the intact insert, were detected in canola events 73496 and 61061.

The stability of the inserted DNA in canola events 73496 and 61061 was demonstrated by Southern blot analysis across five generations. Analysis of the inheritance pattern of the gat4621 gene and the herbicide tolerance phenotype within multiple segregating generations of canola events 73496 and 61061 confirmed the stability of the inserted DNA and the stability of GAT4621 protein expression. The results of the analysis were consistent with the finding of a single site of insertion that segregates according to the Mendelian laws of genetics.

IV. Criteria for the Environmental Assessment

1. Potential of Canola Events 73496 and 61061 to Become Weeds of Agriculture or be Invasive of Natural Habitats

Canola (Brassica napus) possesses some of the characteristics that are common to weeds and invasive plants. It is an annual crop that may persist in unmanaged ecosystems without human intervention. There have been reports of B. napus becoming a weed of agriculture in North America and other parts of the world, however it has not become an abundant or problematic weed in Canada, despite being cultivated in Canada for many years. B. napus plants can grow as volunteers in cultivated fields in the seasons following a B. napus crop, but they are usually eliminated by soil cultivation or the use of herbicides.

The CFIA has evaluated data submitted by Pioneer Hi-Bred Production Ltd. on the agronomic characteristics and biology of canola events 73496 and 61061. These characteristics were: seed germination, seedling vigour, early growth, early population, days to flowering, flowering duration, days to maturity, plant height, lodging, pod-shattering, final population, disease incidence, insect damage and grain yield. Canola events 73496 and 61061 were not significantly different from the control lines and/or were within the range of expression of these traits in other conventional B. napus varieties for all measured characteristics. No differences were observed between the control lines and canola events 73496 and 61061 at any of the sites for Sclerotinia and powdery mildew. Monitoring of trial locations did not indicate any changes in seed dormancy of canola events 73496 and 61061 in comparison to other B. napus varieties, nor is the glyphosate tolerance trait expected to contribute to increased seed dormancy.

No competitive advantage was conferred to plants of canola events 73496 or 61061, other than that conferred by tolerance to glyphosate, as the reproductive or growth characteristics of canola events 73496 and 61061 were comparable to those of unmodified B. napus varieties. Tolerance to glyphosate provides a competitive advantage only when this herbicide is used, and will not, in and of itself, make a glyphosate-tolerant plant weedier or more invasive of natural habitats. Glyphosate tolerance will not cause canola events 73496 or 61061 to become weedier or more invasive in unmanaged habitats than unmodified B. napus. Glyphosate-tolerant volunteers will not be controlled in subsequent crops if glyphosate is used as the only weed control tool. However, control of glyphosate-tolerant B. napus as a volunteer weed in other crops or in fallow ground can readily be achieved by the use of other classes of herbicides, or by mechanical means.

The novel trait has no intended or observed effects on weediness or invasiveness. The CFIA has therefore concluded that canola events 73496 and 61061 have no altered weed or invasiveness potential in Canada when compared to conventional B. napus varieties.

The CFIA considered the changes in usual agronomic practices that may arise from volunteer plants with novel herbicide tolerances. Similarly, the CFIA considered the potential that continued application of the same herbicide in subsequent rotations may lead to increased selection pressure for herbicide resistant weed populations. In order to address these issues, an herbicide stewardship plan which includes integrated pest management strategies should be implemented. These plans may include a recommendation to rotate or combine weed control products with alternate modes of action and to employ other weed control practices.

Pioneer Hi-Bred Production Ltd. has submitted an herbicide tolerance stewardship plan (Stewardship of Herbicide Tolerant 73496 and 61061 Canola) to the CFIA, which was determined to be satisfactory when evaluated by the PBRA Unit.

Pioneer Hi-Bred Production Ltd. will make this stewardship plan readily available to growers and agriculture extension personnel, in both private and public sectors, to promote careful management practices for canola events 73496 and 61061 and other glyphosate-tolerant B. napus. These practices include the use of alternate control tools as appropriate to achieve complete volunteer control of glyphosate-tolerant B. napus. Pioneer Hi-Bred Production Ltd. will provide an efficient mechanism for growers to report agronomic problems to the company, which will facilitate the ongoing monitoring of glyphosate-tolerant B. napus. Pioneer Hi-Bred Production Ltd. will monitor grower compliance to determine the effectiveness of the stewardship plan and make any changes to the plan as appropriate.

This information led the CFIA to conclude that canola events 73496 and 61061 have no altered weed or invasiveness potential compared to canola varieties currently grown in Canada.

2. Potential for Gene Flow from Canola Events 73496 and 61061 to Sexually Compatible Plants Whose Hybrid Offspring May Become More Weedy or More Invasive

Successful interspecific and intergeneric crosses between B. napus and some related species have been reported in the scientific literature (see biology document BIO1994-09 for more information). However, many of these crosses have required extensive human intervention and the rates of natural hybridization between B. napus and weedy relatives resulting in fertile offspring appear to be very low. Sinapis arvensis is considered the worst of the weedy relatives of B. napus in Western Canada. Hybrids between both species can be produced under field conditions, however at very low frequency. Additionally, backcrossing of the hybrids to S. arvensis failed to produce viable progeny. Therefore, the likelihood of introgression of traits from B. napus to S. arvensis appears to be very low. Raphanus raphanistrum, another economically damaging weed, has been shown to hybridize with B. napus at low frequency. However, introgression has never been demonstrated and can be considered unlikely. In crosses with other wild related species (e.g. Erucastrum gallicum), no viable hybrid seed was produced. Thus, there have been no reports to date despite the widespread adoption and cultivation of glyphosate-tolerant canola.

Stable gene transfer from B. napus is most likely with Brassica crops, such as B. juncea and B. rapa. Any hybrids resulting from outcrossing between B. rapa and canola events 73496 and 61061 could be controlled by herbicides other than glyphosate or by mechanical means. Any unintended presence of glyphosate tolerance in non-glyphosate tolerant varieties of B. napus can be controlled by standard crop management practices.

If glyphosate-tolerant individuals arose through interspecific or intergeneric hybridization, the novel trait would confer no competitive advantage to these plants unless challenged by glyphosate. This would only occur in managed ecosystems where glyphosate is used for weed control. As with glyphosate-tolerant canola events 73496 and 61061, these herbicide-tolerant individuals, should they arise, could be controlled using mechanical means or herbicides other than glyphosate. Hybrids, if they developed, could potentially result in the loss of glyphosate as a tool to control these species. This, however, can be avoided by the use of sound crop management practices.

This information led the CFIA to conclude that gene flow from canola events 73496 and 61061 to related species in Canada is possible but would not result in increased weediness or invasiveness of the resulting progeny.

3. Altered Plant Pest Potential of Canola Events 73496 and 61061

B. napus is not a plant pest in Canada, and the intended effect of the novel trait (tolerance to glyphosate) is unrelated to plant pest potential. Based on the incidence of Sclerotinia and powdery mildew in the field trials, the agronomic characteristics of canola events 73496 and 61061 were shown to be within the normal range of conventional B. napus varieties. This indicates that it is unlikely there are any major unintended effects in canola events 73496 or 61061 that could alter their plant pest potential relative to that of other B. napus varieties.

The CFIA therefore concludes that canola events 73496 and 61061 do not display any altered pest potential compared to canola varieties currently grown in Canada.

4. Potential Impact of Canola Events 73496 and 61061 on Non-Target Organisms

The glyphosate-tolerance trait introduced into canola events 73496 and 61061 is not expected to have an impact on non-target organisms. The GAT4621 protein expressed in canola events 73496 and 61061 is identical to the GAT4621 protein produced in corn event 98140, which has been reviewed and authorized in Canada (DD2009-78). As the environmental safety of the GAT4621 protein has previously been established, no negative impacts resulting from exposure of organisms to the GAT4621 protein expressed in canola events 73496 and 61061 are expected.

Composition analyses showed that the levels of key nutrients, anti-nutrients and secondary metabolites in grain of canola events 73496 and 61061 are comparable to those in conventional canola varieties. The elevated concentrations of some acetylated amino acids in tissues of canola events 73496 and 61061 are unlikely to negatively impact organisms interacting with canola, as conventional canola already contains the five acetylated amino acids found in canola events 73496 and 61061 without known adverse effects on interacting organisms. In addition, published studies showed no adverse effects in rats following acute gavage with high doses (2,000 mg/kg body weight) of these acetylated amino acids. Therefore, it is very unlikely that the genetic transformation may have caused unintended changes to the composition of tissues of canola events 73496 and 61061 that would negatively impact organisms interacting with these canola events.

Field evaluations of canola events 73496 and 61061 did not show any increased resistance to damage caused by pest insects and pathogens compared to commercial canola varieties (see Section 3 on Altered Plant Pest Potential of Canola Events 73496 and 61061).

The CFIA has therefore determined that the unconfined release of canola events 73496 and 61061 will not result in altered impacts on non-target organisms, including humans, compared to canola varieties currently grown in Canada.

5. Potential Impact of Canola Events 73496 and 61061 on Biodiversity

Canola events 73496 and 61061 are not expected to nor have they shown any negative effects on non-target organisms and do not present altered weediness, invasiveness or plant pest potential. The novel trait is not expected to expand the range of cultivation of B. napus in Canada. No changes in current agronomic practices for B. napus are expected, including the use of glyphosate for weed control. Pioneer Hi-Bred Production Ltd.'s herbicide tolerance stewardship plan for canola events 73496 and 61061 contains recommendations for delaying the development of glyphosate resistance in weeds.

The introduction of these glyphosate-tolerant B. napus lines is not likely to affect typical crop rotation practices.

The CFIA has concluded that the novel gene and its corresponding trait does not confer to canola events 73496 and 61061 any characteristic that would result in unintended environmental effects following unconfined release. The CFIA has therefore concluded that the potential impact on biodiversity of canola events 73496 and 61061 is unlikely to be different from that of canola varieties currently grown in Canada.

V. Criteria for the Livestock Feed Assessment

The Animal Feed Division considered nutrient and anti-nutrient profiles; the safety of feed ingredients derived from canola events 73496 and 61061, including the presence of gene products, residues, and metabolites in terms of animal health and human safety as it relates to the potential transfer of residues into foods of animal origin, and worker/bystander exposure to the feed; and whether feeds derived from canola events 73496 and 61061 meet the definitions and requirements of feeds as listed in Schedule IV of the Feeds Regulations.

1. Potential Impact of Canola Events 73496 and 61061 on Livestock Nutrition

Nutritional Composition

The compositional equivalence of canola events 73496 and 61061 (sprayed with glyphosate) to an unmodified control canola was examined at six replicated field sites in Canada and US during the 2009 growing season. Seed samples were analysed for proximates, acid detergent fibre (ADF), neutral detergent fibre (NDF), amino acids, fatty acids, vitamins, minerals, glucosinolates, phytosterols, secondary metabolites (N-acetylated amino acids) and anti-nutrients (phytic acid, sinapine, and tannins). Whole plants and processed products (meal, oil) were also analysed for secondary metabolites. Statistical analyses were performed on the composition data to determine if there were statistically significant differences (P<0.05) between canola events 73496 and 61061 and unmodified control canola. No statistically significant differences were observed between canola events 73496 and 61061 and the unmodified control canola for proximates, ADF, and NDF. There were small, but statistically significant differences between the canola events 73496 and 61061 and the unmodified control canola for concentrations of oleic and linoleic acids. Additionally, stearic, linolenic, arachidic, behenic and lignoceric acid concentrations were statistically significantly different for canola event 61061 compared to the unmodified control canola. However, all means were within the tolerance interval of conventional canola varieties and therefore the differences were not considered biologically relevant. No statistically significant differences were observed between canola events 73496 or 61061 and the unmodified control canola for any amino acid analysed. Small, but statistically significant differences were observed between canola event 73496 and the unmodified control canola for magnesium, vitamin B2, delta- and total tocopherols, and between canola event 61061 and the unmodified control canola for magnesium, phosphorus, pantothenic acid, alpha-, delta-, gamma-, and total tocopherols. All mean concentrations were within the tolerance intervals and therefore these differences were considered not biologically meaningful. The total glucosinolate concentration in canola events 73496 and 61061 seed were 5.66 and 5.50  µmoles/g dry weight, respectively, and these were not statistically different from the concentration in unmodified control canola (5.77 µmoles/g dry weight). All means were within the tolerance interval of conventional canola varieties. No statistically significant differences were found between canola event 73496 and the unmodified control canola for soluble and insoluble tannins, phytic acid and sinapine, while the concentration of insoluble tannins was statistically lower in canola event 61061 compared to the unmodified control canola. Cholesterol levels in canola events 73496 and 61061 were statistically significantly higher than the unmodified control canola levels, however the means were within the tolerance interval for conventional canola varieties. All other phytosterols were not different for both canola events 73496 and 61061 and the unmodified control canola.

The concentration of N-acetylaspartate (NAA) and N-acetylglutamate (NAG) in seeds and whole plants from canola events 73496 and 61061 were significantly higher relative to the corresponding concentrations in the unmodified control canola. All NAA and NAG values for both canola events 73496 and 61061 were above the upper limit of the respective tolerance intervals. Pioneer Hi-Bred Production Ltd. provided evidence to show that due to the low livestock exposure to these metabolites as well as the rapid rate of deacetylation of NAA and NAG into free aspartate and glutamate during metabolism, the increased concentration of NAA and NAA in canola events 73496 and 61061 poses no adverse effects to livestock as feed. No statistically significant difference was observed between both transgenic lines and control canola seed for N-acetylglycine (NAGly). Statistically significant differences were observed between canola event 73496 and unmodified control canola for N-acetylserine (NAS) and N-acetylthreonine (NAT); while statistically significant differences were only found between canola event 61061 and unmodified control canola for NAT. All NAGly, NAS and NAT values in seed for both transgenic canola lines were within their respective tolerance intervals. No significant differences were observed between canola events 73496 and 61061 and unmodified control whole plants for NAS. The concentration of NAGly and NAT in whole plant samples derived from canola events 73496 and 61061 were elevated compared to those of the unmodified control and were outside the tolerance intervals. The levels of NAA and NAG were the most abundant in canola events 73496 and 61061 meal (hulled and de-hulled) and these were elevated compared to unmodified control. It is not expected that this increase is likely to result in adverse effects based on scientific evidence and livestock exposure estimates generated by Pioneer Hi-Bred Production Ltd. There were no detected levels of N-acetylated amino acids in the canola oil analysed.

Conclusion

The evidence provided by Pioneer Hi-Bred Production Ltd. supports the conclusion that the nutritional composition of canola events 73496 and 61061 are comparable to conventional canola varieties except for the elevated levels of N-acetylglutamate (NAG) and N-acetylaspartate (NAA). The data support the conclusion that feeds produced from the two transgenic canola lines are expected to not pose a risk to livestock with the increased levels of NAA and NAG in the novel crops.

2. Potential Impact of Canola Events 73496 and 61061 on Animal Health and Human Safety as it Relates to the Potential Transfer of Residues into Foods of Animal Origin, and Worker/Bystander Exposure to the Feed

Canola events 73496 and 61061 are tolerant to glyphosate as a result of the insertion of the gat4621 gene encoding the GAT4621 protein. The assessment of canola events 73496 and 61061 evaluated the impact of the following potential hazards relative to the safety of feed ingredients derived from these events:

• The presence of the novel GAT4621 protein;
• The chemical pesticide residue profile.

Novel GAT4621 protein

The GAT4621 protein shares no significant biologically relevant sequence homology with any known toxins or allergens and lacks a mode of action that suggests that it is intrinsically toxic. This protein is also heat labile and rapidly degraded under conditions similar to those encountered in the gastrointestinal tract. The GAT4621 protein was previously assessed for Pioneer Hi-Bred Production Ltd.'s Corn Event 98140; and it has been determined, based on the established equivalence, that the safety data may be used to support the safety of the GAT4621from canola events 73496 and 61061. No signs of toxicity were demonstrated in the original single-dose oral toxicity studies in mice using purified microbial GAT4621 protein at doses up to 1900 mg/kg-bw. These factors support the lack of intrinsic toxicity of the GAT4621 protein.

Chemical pesticide residue profile

The safety of herbicide residues and metabolites in canola events 73496 and 61061, following the application of glyphosate, was also evaluated as part of the feed safety assessment. It was concluded, that potential glyphosate and residues of glyphosate metabolites (investigated as total residues of glyphosate + N-acetyl glyphosate + aminomethylphosphonic acid (AMPA) + N-acetyl AMPA) would not be a safety concern for livestock or for humans, when comparing the estimated exposure to the established legal residue limits in Canada and the US.

Conclusion

Feed ingredients derived from canola events 73496 and 61061, are considered to meet present ingredient definitions for canola and as such are approved for use as livestock feed in Canada.

VI. New Information Requirements

If at any time Pioneer Hi-Bred Production Ltd. becomes aware of any information regarding risk to the environment, including risk to human or animal health, which could result from release of canola events 73496 and 61061 in Canada or elsewhere, Pioneer Hi-Bred Production Ltd. will immediately provide such information to the CFIA. On the basis of such new information, the CFIA will re-evaluate the potential impact of canola events 73496 and 61061 on the environment, livestock and human health, and may re-evaluate its decision with respect to the livestock feed use and environmental release authorizations of canola events 73496 and 61061.

VII. Regulatory Decision

Based on the review of the data and information submitted by Pioneer Hi-Bred Production Ltd. and other relevant information, the CFIA has determined that canola events 73496 and 61061 do not present altered environmental risk when compared to currently commercialized canola varieties in Canada.

Based on the review of submitted data and information by Pioneer Hi-Bred Production Ltd., including comparisons of canola events 73496 and 61061 with the unmodified control, the Animal Feed Division of the Animal Health Directorate, CFIA, has concluded that the novel gene and its corresponding trait will not confer to canola events 73496 and 61061 any characteristic that would raise any concerns regarding the safety or nutritional composition of canola events 73496 and 61061. Canola oil, seed and meal are currently listed in Schedule IV of the Feeds Regulations and are, therefore approved for use in livestock feeds in Canada. Canola events 73496 and 61061 have been found to be as safe as and as nutritious as traditional canola varieties. Canola events 73496 and 61061 and its products are considered to meet present ingredient definitions and are approved for use as livestock feed ingredients in Canada.

Unconfined release into the environment and use as livestock feed of canola events 73496 and 61061 is therefore authorized by the Plant Biosafety Office of the Plant Health and Biosecurity Directorate and the Animal Feed Division of the Animal Health Directorate as of May 3, 2012. Any canola lines derived from canola events 73496 and 61061 may also be released into the environment and used as livestock feed, provided that

1. no inter-specific crosses are performed,
2. the intended uses are similar,
3. it is known based on characterization that these plants do not display any additional novel traits and are substantially equivalent to currently grown canola in Canada, in terms of their potential environmental impact and livestock feed safety,
4. and the novel genes are expressed at a level similar to that of the authorized lines.

Canola events 73496 and 61061 are subject to the same phytosanitary import requirements as their unmodified counterpart. Canola events 73496 and 61061 are required to meet the requirements of other jurisdictions; including but not limited to, the Food & Drugs Act, and the Pest Control Products Act.