DD2010-79: Determination of the Safety of Syngenta Seeds Canada Inc.'s Corn (Zea mays L.) Event MIR162

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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 BIO1994-11, "The Biology of Zea mays L. (Corn/Maize)", 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) has evaluated information submitted by Syngenta Seeds Canada Inc. in regard to the corn event MIR162, which is resistant to certain lepidopteran insects. The CFIA has determined that this plant with novel traits (PNT) does not present altered environmental risk nor, as a novel feed, does it present livestock feed safety concerns when compared to currently commercialized corn varieties in Canada.

Taking into account these evaluations, unconfined release into the environment and use as livestock feed of corn event MIR162 is therefore authorized by Plant Biosafety Office (PBO) of the Plant Health and Biosecurity Directorate and the Animal Feed Division of the Animal Health Directorate as of February 11, 2010. Any corn lines derived from event MIR162 may also be released into the environment and used as livestock feed, provided that (i) no inter-specific crosses are performed, (ii) the intended uses are similar, (iii) it is known based on characterization, that these plants do not display any additional novel traits and are substantially equivalent to currently grown corn varieties in Canada, in terms of their potential environmental impact and livestock feed safety, and (iv) the novel genes are expressed at a level similar to that of the authorized line. Before corn event MIR162 is cultivated in Canada as a single event, an Insect Resistance Management (IRM) plan must be submitted to and approved by the PBO.

Corn event MIR162 is subject to the same phytosanitary import requirements as its unmodified counterparts.

Please note, that the livestock feed and environmental safety 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.

Brief Identification of the Modified Plant
Background Information
Description of the Novel Traits
Criteria for the Environmental Assessment
Criteria for the Livestock Feed Assessment
1. Potential Impact of Corn Event MIR162 on Livestock Nutrition
2. Potential Impact of Corn Event MIR162 on Livestock and Workers/Bystanders
New Information Requirements
Regulatory Decision

I. Brief Identification of the Modified Plant

Designation of the Modified Plant:	Corn Event MIR162, OECD Unique Identifier SYN-IR162-4
Applicant:	Syngenta Seeds Canada Inc.
Plant Species:	Maize (Zea mays L.)
Novel Traits:	Resistance to lepidopteran pests including fall armyworm (Spodoptera frugiperda), true armyworm (Pseudaletia unipuncta), beet armyworm (Spodoptera exigua), corn earworm (Helicoverpa zea), black cutworm (Agrotis ipsilon), western bean cutworm (Striacosta albicosta)
Trait Introduction Method:	Agrobacterium-mediated transformation
Proposed Use of the Modified Plant:	Production of corn for human consumption (wet mill products, dry mill products and seed oil) and oil, meal, grain, silage and other byproducts for livestock feed. Corn event MIR162 is not intended to be cultivated as an individual event in Canada

II. Background Information

Syngenta Seeds Canada Inc. developed, through the use of recombinant DNA techniques, a corn event resistant to a number of lepidopteran pests of corn. The corn event, designated as MIR162, was developed to provide a method to control yield losses from insect feeding damage caused by lepidopteran larvae. In Canada, the target lepidopteran pests include the fall armyworm, true armyworm, corn earworm, western bean cutworm and black cutworm.

Corn event MIR162 was developed using recombinant DNA technology, resulting in the introduction of the modified Vip3Aa (Vip3Aa20) gene and the pmi marker gene. The Vip3Aa20 gene is derived from the Vip3Aa1 gene from Bacillus thuringiensis strain AB88 that encodes a vegetative insecticidal protein (Vip), with activity against a range of lepidopteran insects. The pmi gene from Escherichia coli encodes the phosphomannose isomerase (PMI) enzyme. Expression of the PMI enzyme allows the plant to utilize mannose as a carbon source. This trait is of no agronomical interest but was used to select transformed from non-transformed plants during the development phase of corn event MIR162.

Syngenta Seeds Canada Inc. has provided data on the identity of corn event MIR162, 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 plant and the role of the inserted genes and regulatory sequences. The novel proteins were identified and characterized. Data was provided for the evaluation of the potential toxicity of the novel proteins to livestock and non-target organisms and potential allergenicity of the novel proteins to humans and to livestock.

Corn event MIR162 has been field tested in the United States and the data for trial years 2005 and 2006 were submitted.

Agronomic characteristics of corn event MIR162 such as seedling emergence, plant height, time to reproduction, lodging, susceptibilities to pathogens, and yield parameters were compared to those of unmodified corn counterparts.

Nutritional components of corn event MIR162, such as proximates, amino acids and fatty acids were compared with those of unmodified corn counterparts.

The Plant and Biotechnology Risk Assessment (PBRA) Unit of the Science Strategies Directorate, CFIA, has 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:

potential of corn event MIR162 to become a weed of agriculture or be invasive of natural habitats;
potential for gene flow from corn event MIR162 to wild relatives whose hybrid offspring may become more weedy or more invasive;
potential for corn event MIR162 to become a plant pest;
potential impact of corn event MIR162 or its gene products on non-target species, including humans; and
potential impact of corn event MIR162 on biodiversity.
potential for development of insect resistance to corn event MIR162

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:

potential impact of corn event MIR162 on livestock nutrition; and
potential impact of corn event MIR162 on livestock and workers/bystanders

Syngenta Seeds Canada Inc. has provided the CFIA with a method for the detection and identification of corn products containing the corn event MIR162.

III. Description of the Novel Traits

1. Resistance to Lepidopteran Insects

Vegetative insecticidal proteins are a class of insecticidal proteins that are produced during the vegetative growth of B. thuringiensis (Bt), a common gram-positive soil bacterium. Bt cultures continue to produce Vip proteins during the stationary phase of development and sporulation. The Vip3A proteins are active against a range of lepidopteran insects. The proteins are insecticidal to susceptible lepidopteran insects after ingestion and cleavage by proteases in the insect's gut, forming a protease-resistant active fragment that is the bio-active form of the protein. Insecticidal activity is believed to depend on the binding of the active fragment to specific receptors present on midgut epithelial cells of susceptible insects, forming pores which disrupt osmotic balance and eventually result in insect death. The vip3Aa20 gene introduced into corn event MIR162 derives from the native vip3Aa1 gene isolated from the Bt strain AB88. The vip3Aa20 coding sequence was optimized to accommodate the preferred codon usage for maize. Except for two amino-acid substitutions, the Vip3Aa20 protein expressed in corn event MIR162 event is identical to the native Vip3Aa1 protein. The two amino acid substitutions were demonstrated to have no apparent impact on the protein's structure and insecticidal activity, and the two proteins were considered equivalent.

The vip3Aa20 gene is expressed in corn event MIR162 using a promoter which confers constitutive expression of the Vip3Aa20 protein in the corn tissues. Tissue samples were collected at various growth stages from two field-grown MIR162 hybrids. Levels of Vip3Aa20 protein were evaluated by enzyme-linked immunosorbent assays (ELISAs). Average Vip3Aa20 concentrations across growth stages, expressed in micro-grams Vip3Aa20 protein per gram dry weight tissue (µg/g dwt), ranged from 14-148 µg/g dwt in leaves, 13-33 µg/g dwt in roots and 25-94 µg/g dwt in whole plants. Average concentrations measured in kernels at seed maturity and senescence ranged from 41-46 µg/g dwt and 34-35 µg/g, respectively. Average concentrations measured in pollen and silk at anthesis ranged from 43-52 µg/g dwt and 63-132 µg/g dwt, respectively.

The Vip3Aa20 protein was purified from leaves of corn event MIR162 and characterized. The identity of the purified protein was confirmed by Western immunoblot analysis, mass spectral analysis of tryptic peptides and insecticidal activity of corn event MIR162 leaf protein extracts against S. frugiperda.

The levels of Vip3Aa20 protein in corn event MIR162 tissues were too low to extract sufficient amounts for evaluation of environmental and feed safety. To obtain sufficient quantities of Vip3Aa20 protein for safety studies, it was necessary to express vip3Aa20 genes in an E. coli production system. The equivalency of the plant-produced protein to the E. coli- produced protein was evaluated by comparing their molecular weight, immunological reactivity, tryptic peptide mass map, insecticidal activity and glycosylation status. Based on the results, Vip3Aa20 proteins from both sources were found to be equivalent.

The potential mammalian toxicity and allergenicity of the Vip3Aa20 protein was evaluated. The Vip3Aa20 protein lacks sequence similarity to known mammalian allergens and toxins. No toxic effects were observed when the Vip3Aa20 protein was ingested by mice at a single oral dose of 1,250 mg/kg body weight or when the Vip3Aa1 protein, which was demonstrated to be equivalent to Vip3Aa20, was ingested by mice at a single oral dose of 2,700 mg/kg body weight. Incubation for 30 minutes at 65°C resulted in complete inactivation of the Vip3Aa20 protein. In vitro digestive fate studies have shown that the protein is rapidly degraded in simulated gastric fluid, unlike protein allergens which are normally resistant to digestion. The Vip3Aa20 protein expressed in corn event MIR162 is not glycosylated, unlike many known allergens. Therefore, based on the weight-of-evidence, the Vip3Aa20 protein expressed in corn event MIR162 is unlikely to be toxic or allergenic to mammals.

Soil degradation experiments were conducted to evaluate the degradation of the Vip3Aa20 protein in soil over time. Four live soils representing various textures and characteristics and one artificial soil were used. The experiments were conducted using leaves from a corn event expressing a Vip3Aa protein variant (Vip3Aa19) that differs from the Vip3Aa20 protein by a single amino-acid. As this single amino acid substitution has no impact on the properties and insecticidal activity of the Vip3Aa19 protein, the Vip3Aa19 protein represented an appropriate surrogate for the Vip3Aa20 protein expressed in corn event MIR162. The average time to degrade 50% of the initial soil concentration of the Vip3Aa19 protein ranged from 6.0 to 12.6 days across soil types. As very similar degradation rates are anticipated for the Vip3Aa20 protein, the Vip3Aa20 protein is not expected to persist in the soil.

2. Expression of Phosphomannose Isomerase

The pmi gene from Escherichia coli, which encodes the enzyme phosphomannose isomerase (PMI) was introduced in corn event MIR162. Expression of PMI was used as a selectable marker during the process of developing corn event MIR162. Maize cells producing PMI can utilize mannose as a primary carbon source, whereas cells lacking the pmi gene fail to proliferate in a mannose-based culture medium. Cells that can grow in the mannose-based culture medium should also be cells that carry the VIP3Aa20 gene.

The pmi gene expressed in corn event MIR162 is linked to a constitutive promoter. Tissue samples were collected at various growth stages from two field-grown MIR162 hybrids. Levels of PMI protein were evaluated by enzyme-linked immunosorbent assays (ELISA). Average PMI levels across growth stages, expressed in micro-grams PMI protein per gram dry weight tissue (µg/g dwt), ranged from <0.2-13 µg/g dwt in leaves, 0.8-5 µg/g dwt in roots and 2-9 µg/g dwt in whole plants. Average levels measured in kernels at seed maturity and senescence ranged from 1.6-2.2 µg/g dwt and 0.8-0.8 µg/g dwt, respectively. Average levels measured in pollen and silk at anthesis ranged from 4-6 µg/g dwt and 16-26 µg/g dwt, respectively.

The PMI protein expressed in corn event MIR162 was purified from leaves and characterized. The identity of the purified protein was confirmed by Western immunoblot analysis and enzymatic activity. The potential toxicity and allergenicity of the PMI protein produced in corn event MIR162 was evaluated by comparing it with a recombinant E. coli-produced PMI protein whose lack of toxicity and allergenicity had previously been established (see DD2007-68 and DD-2008-70). Based on similar molecular weight, immunoreactivity and enzymatic activity, both proteins were found to be equivalent, with no significant differences. Therefore, the PMI protein expressed in corn event MIR162 is unlikely to be a toxin or an allergen.

The PMI protein has no agronomic significance.

3. Development Method

The vip3Aa20 and pmi genes were introduced in corn event MIR162 via Agrobacterium-mediated transformation of immature maize embryos derived from a proprietary maize line. Embryos capable of further regeneration were transferred to cell culture medium containing mannose. Corn event MIR162 was identified as a successful transformant and was chosen for further development as a cultivated line.

4. Stable Integration into the Plant Genome

Molecular characterization by Southern blot analysis demonstrated that corn event MIR162 contains one intact copy of the vip3Aa20 and pmi gene cassettes inserted at a single site in the corn genome. No additional elements, including intact or partial DNA fragments of the vip3Aa20 or pmi cassette or backbone sequences from the plasmid vector, linked or unlinked to the intact insert, were detected in corn event MIR162. Sequencing of the introduced DNA confirmed the organization of the genetic elements and revealed that the right and left borders of the insert were truncated along with very short fragments of non-functional plant genomic sequence. These deletions have no effect on the functionality of the DNA insert or the plant and this phenomenon has been previously observed with Agrobacterium transformation. In addition, two single nucleotide changes were noted within the coding sequence of the vip3Aa20 gene. Only one of the nucleotide changes resulted in an amino acid substitution. This amino acid substitution has no effect on the insecticidal activity of the Vip3Aa20 protein since it is not within the portion of the protein that is active in the insect gut.

The stability of the insert within corn event MIR162 was verified by Southern blot analysis over three backcross generations. The inheritance pattern of the vip3Aa20 and pmi genes within three segregating generations of corn event MIR162 showed that both genes segregate according to Mendelian rules of inheritance for a single genetic locus. Concentrations of VIP3Aa20 and PMI proteins in corn event MIR162 leaf tissues measured across three backcross generations indicate stability of VIP3Aa20 and PMI protein expression across generations.

IV. Criteria for the Environmental Assessment

1. Potential of Corn Event MIR162 to Become a Weed of Agriculture or be Invasive of Natural Habitats

The biology of corn, described in the CFIA Biology Document BIO1994-11, shows that unmodified plants of this species are not invasive of unmanaged habitats in Canada. Corn does not possess the potential to become weedy due to the lack of seed dormancy, the non-shattering nature of corn cobs and the poor competitive ability of seedlings. According to the information provided by Syngenta Seeds Canada Inc., corn event MIR162 was determined to be similar to unmodified corn in this respect.

MIR162 corn hybrids were tested in the United States corn belt at 6 locations in 2005 and at 10 locations in 2006. A total of 19 agronomic traits were evaluated. These agronomic traits covered a broad range of characteristics that encompass the entire life cycle of the maize plant and included data assessing seedling emergence, vegetative vigor, time to maturity and seed production. For the majority of agronomic traits, no statistically significant differences between MIR162 hybrids and their non-transformed isogenic counterparts were observed. Although instances of statistically significant differences between MIR162 and control hybrids were observed for some traits, there were no consistent trends in the data across locations, MIR162 hybrids or years that would indicate that any of these differences were due to the genetic modification.

In addition to the agronomic characteristics, susceptibility of corn event MIR162 to various corn pests and pathogens was evaluated. The results showed no competitive advantage was conferred to corn event MIR162 by expression of vip3Aa20 and pmi proteins, other than that conferred resistance to a number of lepidopteran insect pests of corn. As feeding damage by lepidopteran larvae is not known to be a major factor restricting the establishment or distribution of corn in Canada, the introduction of this novel trait does not make MIR162 corn weedy or invasive of natural habits.

The above considerations led the CFIA to conclude that corn event MIR162 has no increased weediness or invasiveness potential compared to currently commercialized corn varieties.

2. Potential for Gene Flow from Corn Event MIR162 to Wild Relatives Whose Hybrid Offspring May Become More Weedy or More Invasive

The biology of corn, as described in CFIA Biology Document BIO1994-11, indicates that there are no wild relatives in Canada that can hybridize with Zea mays.

The CFIA has therefore concluded that gene flow from corn event MIR162 to wild relatives is not possible in Canada.

3. Altered Plant Pest Potential of Corn Event MIR162

Corn is not considered a plant pest in Canada. The expression of the Vip3Aa20 and PMI proteins in corn event MIR162 are not expected to have effects on susceptibility to corn pathogens. Field evaluation of MIR162 corn hybrids did not show any increase or decrease in susceptibility to plant diseases, including gray leaf spot disease, compared to unmodified corn counterparts. Non-target studies on corn insect pests such as Western corn rootworm and Western flower thrips indicate that the Vip3Aa20 and PMI proteins do not change the susceptibility of corn to non-target pests.

The unconfined environmental release of MIR162 without a proper management plan could potentially lead to the development of resistance to the insecticidal proteins by its target pests. Since corn event MIR162 is not intended to be cultivated as an individual event in Canada, there will not be an effect on the development of insect resistance to the insecticidal protein Vip3Aa20 by target pests in this country. An insect resistance management stewardship plan specific to this product is not required at this time. Before corn event MIR162 is cultivated in Canada as a single event, an IRM plan must be submitted to and approved by the PBO.

Thus, if used as intended, corn event MIR162 will not display any altered plant pest potential compared to currently commercialized corn varieties.

4. Potential Impact of Corn Event MIR162 on Non-Target Organisms

The lack of toxicity of the phosphomannose isomerase PMI on non-target organisms has been established previously (see DD2007-68 and DD-2008-70). Syngenta Seeds Canada Inc. has provided information on the receptor-mediated mechanism of action of Vip3A proteins which indicates that Bacillus thuringiensis Vip3A proteins are not toxic to species outside of the insect order Lepidoptera. They have provided information on the selective toxicity of Vip3Aa20, as well as information that demonstrates a lack of toxicity to endangered species and species of concern such as the Karner blue butterfly (Lycaeides melissa samuelis) and the Monarch butterfly (Danaus plexippus), together with data and scientific rationale to demonstrate nontoxicity of Vip3A proteins to mammals, birds, and fish.

The available literature indicates that the toxicity spectrum of Vip3Aa proteins is limited to certain lepidopteran species. Syngenta Seeds Canada Inc. has reported results from studies on the insecticidal activity of Vip3Aa proteins on a range of insect species from eight orders, including 23 lepidopteran species (Agrostis ipsilon, Helicoverpa zea, Helicoverpa armigera, Helicoverpa punctigera, Heliothis virescens, Spodoptera exigua, Spodoptera frugiperda, Spodoptera litura, Striacosta albicosta, Trichloplusia ni, Phthorimaea operculella, Manduca sexta, Chilo partellus, Ostrinia nubilalis, Plutella xylostella, Danaus plexippus, Pieris brassicae, Bombyx mori, Earias vittella, Ephestia kuehniella, Diatrea grandiosella, Diatraea saccharalis, and Homoeosoma electellum), 6 Coleopteran species (Coleomegilla maculata, Diabrotica virgifera virgifera, Leptinotarsa decemlineata, Anthonomus grandis, Aleochara bilineata, and Coccinella septempunctata), 3 Dipteran species (Culex pipiens, Culex quinquefasciatus, and Drosophila melanogaster), 1 Hemipteran species (Orius insidiosus), 1 Isotomid species (Folsomia candida), 1 Neuropteran species (Chrysoperla carnea), 1 Haplotaxid species (Eisenia foetida), and 1 Thysanoptera species (Frankliniella occidentalis). The results confirmed that the activity of Vip3Aa proteins is restricted to certain lepidopteran species. The susceptible lepidopteran species include, among others, certain corn pests such as the fall armyworm, corn earworm, true armyworm, black cutworm, but not the European corn borer.

Syngenta Seeds Canada Inc. has submitted data from dietary toxicity studies on the effect of the Vip3Aa protein on non-target terrestrial invertebrate indicator species, including the honeybee (Apis mellifera), insidious flower bug (Orius insidiosus), seven-spot ladybird beetle (Coccinella septempunctata), pink spotted ladybird beetle (Coleomegilla maculata), green lacewing (Chrysoperla carnea), rove beetle (Aleochara bilineata), collembola (Folsomia candida), and earthworm (Eisenia foetida). Test organisms were exposed to Vip3Aa20 protein, or to a variant (Vip3Aa19 protein) which differs from the Vip3Aa20 protein by a single amino acid substitution. As this amino-acid substitution has no impact on the properties and insecticidal activity of the Vip3Aa19 protein, the Vip3Aa19 protein was used as an appropriate Vip3Aa20 surrogate for some of the bioassays. In all cases, the Vip3Aa protein was demonstrated to be safe to these non-lepidopteran indicator species at doses exceeding the estimated environmental concentration of Vip3Aa20 protein in the diet of non-target invertebrates feeding on corn event MIR162 tissues or exposed to Vip3Aa20 protein through their predation on insects feeding on corn event MIR162 tissue.

Daphne magna (water flea) was used as an indicator species to assess the effects of Vip3Aa20 protein on aquatic invertebrates. No adverse effects on the water flea were observed in the presence of corn event MIR162 pollen at a concentration of 120 mg/L, or 1,485 times the expected environmental concentration. Taking into consideration the narrow spectrum of activity of the Vip3Aa20 protein, which is restricted to certain lepidopteran species, this observation adds to the weight of evidence that no hazard to aquatic invertebrates is anticipated from exposure to MIR162 corn.

As the activity of Vip3A proteins is restricted to certain lepidopteran species, the endangered species assessment focused on larvae of the order Lepidoptera.

The habitat of the Karner Blue butterfly (Lycaeides melissa samuelis) has the potential to occur in proximity of corn fields; however, the Karner Blue butterfly is listed by the Committee on the Status of Endangered Wildlife as extirpated in Canada (Please refer to http://www.cosewic.gc.ca/index.htm for more information). Even if Karner Blue butterfly populations were recovered in Canada, Karner Blue larvae would have low likelihood of exposure to corn pollen because corn pollen shed typically occurs after Karner Blue larvae have finished feeding. The lepidopteran species currently listed by the Committee on the Status of Endangered Wildlife in Canada as being a threatened or endangered species are not expected to be found in the proximity of corn fields. Therefore the potential exposure of threatened or endangered lepidopteran species in Canada to the Vip3Aa20 protein expressed in corn event MIR162 will be minimal.

Among the butterfly species of special concern in Canada, the Monarch (Danaus plexippus) is the only species that may be exposed to significant amounts of the Vip3Aa20 protein, as Monarch larvae feeding on milkweed plants may be exposed to corn pollen drifting from adjacent MIR162 corn plants. However, the Vip3A protein is not lethal to Monarch larvae when ingested at a concentration of 1,000 ng/cm² (Lee et al., 2003), a concentration equivalent to 57,500 corn event MIR162 pollen grains/cm². This dose exceeds the highest corn pollen concentration Monarch larvae may be exposed to in the field (up to 1,400 pollen grains/cm², Pleasants et al., 2001). Additionally, activated Vip3A protein fails to induce pores in the midgut of Monarch larvae (Lee et al., 2003). Taking into account these results, corn event MIR162 is unlikely to pose any risk to the Monarch butterfly.

Syngenta Seeds Canada Inc. has also submitted data from dietary toxicity studies on the effect of the Vip3Aa protein on non-target vertebrates including the mouse, the bobwhite quail and the catfish. These were selected as representative indicator species for potential mammalian, bird, and fish toxicity, respectively. The Vip3Aa protein variants used for the catfish and bobwhite quail studies had amino acid sequences that differed from that of the Vip3Aa20 protein by one or two amino acids, respectively. These amino acid substitutions have no impact on the properties and insecticidal activity of the proteins. No harmful effects were detected when mice were exposed to a single oral dose of 1,250 mg Vip3Aa20/kg body weight, represent 174 times the daily dose of Vip3Aa20 protein for mammals feeding on corn event MIR162 grain. No harmful effects were detected when quail were exposed to a single oral dose of 2,000 mg Vip3Aa/kg body weight, representing 76 times the daily dose of Vip3Aa20 protein for birds feeding on corn event MIR162 grain. No harmful effects were detected when juvenile catfish were fed for 30 days using a fish diet formulated to represent 4.8 times the corn event MIR162 grain concentration that would be found in a standard fish diet.

The Vip3Aa20 and PMI proteins are unlikely to be allergens (see Section III.1).

Composition analyses showed that the levels of key nutrients and anti-nutrients in corn event MIR162 grain and forage are comparable to those in commercial corn varieties.

Based on the above, the CFIA has determined that, compared to current commercial corn varieties, the unconfined release of corn event MIR162 will not result in altered impacts on non-target organisms, including humans.

5. Potential Impact of Corn Event MIR162 on Biodiversity

Corn event MIR162 has no novel phenotypic characteristics that would extend its range beyond the current geographic range of corn production in Canada. Since corn has no wild relatives with which it can outcross in Canada, there will be no transfer of the novel traits to other species in unmanaged environments. In addition the novel traits were determined to pose minimal risks to non-target organisms.

At present, the application of chemical insecticide treatments is a common practice to control some lepidopteran pests of corn in Canada, such as corn earworm, black cutworm, and armyworms. Corn event MIR162 provides an alternative method to existing methods of control of these pests. Therefore, the reduction in local pest species as a result of the release of corn event MIR162 does not present a significant change from existing agricultural practices.

The CFIA has therefore concluded that the potential impact on biodiversity of corn event MIR162 is equivalent to that of currently commercialized corn varieties.

6. Potential for Development of Insect Resistance to Corn Event MIR162

Since event MIR162 is not intended to be cultivated as an individual event in Canada, an insect resistance management stewardship plan specific to this product is not required at this time. Before corn event MIR162 is cultivated in Canada as a single event, an IRM plan must be submitted to and approved by the PBO.

V. Criteria for the Livestock Feed Assessment

1. Potential Impact of Corn Event MIR162 on Livestock Nutrition

Nutritional Composition:

Replicated trials of corn event MIR162 and non- transgenic controls were conducted at six locations in the US in 2005 to generate nutritional composition data. Forage and grain samples were analyzed for proximate, acid detergent fibre (ADF), neutral detergent fibre (NDF), total dietary fibre (TDF) and minerals. Amino acids, fatty acids, vitamins and secondary metabolites (furfural, ferulic acid, p-coumaric acid and phytosterols) were also analyzed in grain samples. No statistically significant differences were observed between corn event MIR162 and control for crude protein, fat, ADF and TDF. Ash, starch and NDF content in corn event MIR162 grain were significantly different from the control; however the means were within literature values. Statistically significant differences were observed between corn event MIR162 grain and the non-transgenic control for calcium, iron, phosphorus, vitamins A, B6 and E, however all means were within the literature ranges. No statistically significant differences were observed between corn event MIR162 and control grain for all amino acids analyzed. Statistically significant differences were observed between MIR162 and control for linoleic (C18:2) and linolenic (C18:3), however both means were within the literature ranges. Statistically significant differences were observed between corn event MIR162 and the control corn for ferulic and p-coumaric acids, but both means were within the literature ranges. Although statistically significant differences were observed between corn event MIR162 and control grain for ß-sitosterol (a phytosterol), the levels were within commercial ranges.

Broiler Feeding Study:

To assess the nutritional value of corn event MIR162, a 44 day broiler feeding study was conducted to compare the effects of diets prepared from transgenic corn (MIR162 +ve) to near-isogenic control (MIR162 -ve) and commercial (NC 2006) grain corn. In a randomized complete block design, 540 birds were distributed into 36 pens (12 pens per diet) with 15 birds of the same sex in a pen. Birds were fed starter (day 0-17), grower (day 17-35) and finisher (day 35-44) diets for each diet type. Body weight, feed intake, mortality and carcass and yield were recorded. No statistically significant differences were observed between birds on corn event MIR162 (+ve) diets and MIR162 (-ve) or commercial control diets for growth, feed conversion efficiency and carcass yield. There were no statistically significant differences in mortality rate of birds between diets.

Anti-Nutrients:

Replicated trials of corn event MIR162 and non- transgenic controls were conducted at six locations in the US in 2005 to generate anti-nutrient data. No statistically significant differences were observed between corn event MIR162 and the control for inositol, phytic acid, raffinose and trypsin inhibitor. All means were within literature ranges for commercial corn.

The evidence provided by Syngenta Seeds Canada Inc. supports the conclusion that the nutritional composition of corn event MIR162 is substantially equivalent to commercial corn varieties.

2. Potential Impact of Corn Event MIR162 on Livestock and workers/ Bystanders

PMI proteins are present ubiquitously in nature. They have been found in a variety of plants, animals, and prokaryotes. As such it is likely that both livestock and humans are exposed to PMI proteins on an ongoing basis. The PMI protein expressed in corn event MIR162 did not have homology with any known allergens or toxins. It was also heat labile and rapidly degraded under conditions similar to those encountered in the gastrointestinal tract. This information suggests that the PMI protein in corn event MIR162 is unlikely to be a novel toxin or allergen.

The Vip3Aa20 protein expressed in corn event MIR162 is identical to the Vip3Aa1 protein of the common soil bacterium B. thuringiensis except for two amino-acid substitutions that were demonstrated to have no apparent impact on the protein's structure and insecticidal activity. The Vip3Aa1 protein is highly toxic to a range of important lepidopteran pests but the mode of action of Vip3Aa proteins is highly specific to insects and is not relevant to mammalian species. The Vip3Aa20 protein did not have homology with any known allergens or toxins. It was also heat labile and rapidly degraded under conditions similar to those encountered in the gastrointestinal tract. This information suggests that Vip3Aa20 protein in corn event MIR162 is unlikely to be a novel toxin or allergen.

No adverse effects from the PMI protein was observed in an acute oral toxicity study in mice using approximately 2610 times the highest predicted livestock dose level per kg body weight. No toxic effects were observed when the Vip3Aa20 protein was ingested by mice at a single oral dose of 1,250 mg/kg body weight. For some livestock species, however, corn is consumed at very high levels, which greatly increases the exposure levels to the Vip3Aa20 protein. At these very high exposure levels, the 1,250 mg/kg body weight Vip3Aa20 protein in this study was found to be only 74.5 times the highest predicted livestock dose level, and the study was found to be not sufficiently robust to demonstrate safety with a sufficient margin of safety for all livestock species. An additional acute oral toxicity was submitted by Syngenta Seeds Canada Inc. in which mice were exposed to 2,700 mg/kg body weight Vip3Aa1 protein, which represents approximately 160 times the highest predicted livestock dose level of Vip3Aa20 in corn event MIR162. Syngenta Seeds Canada Inc. adequately demonstrated that the Vip3Aa20 and Vip3Aa1 proteins were equivalent, and the Vip3Aa1 acute oral toxicity study was accepted as supporting evidence of the safety of the Vip3Aa20 protein in corn event MIR162 for livestock. In addition to these acute oral toxicity studies, no adverse effects were observed in a 44 day broiler feeding study in which corn event MIR162 was used to prepare the diet.

The evidence provided by Syngenta Seeds Canada Inc. indicates that the corn event MIR162 is unlikely to cause an increased risk to livestock and workers/by-standers when compared to commercialized corn lines.

VI. New Information Requirements

If at any time, Syngenta Seeds Canada Inc. becomes aware of any information regarding risk to the environment, including risk to human or animal health, which could result from release of corn event MIR162 materials in Canada or elsewhere, Syngenta Seeds Canada Inc. will immediately provide such information to the CFIA. On the basis of such new information, the CFIA will re-evaluate the potential impact of corn event MIR162 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 corn event MIR162.

VII. Regulatory Decision

Based on the review of the data and information submitted by Syngenta Seeds Canada Inc., and through comparisons of corn event MIR162 with unmodified corn counterparts, the Plant and Biotechnology Release Assessment Unit, CFIA, has concluded that corn event MIR162 is substantially equivalent to currently commercialized corn varieties. The novel genes and their corresponding traits in corn event MIR162 do not alter or confer any characteristics that would result in unintended environmental effects following unconfined release.

Based on the review of submitted data and information by Syngenta Seeds Canada Inc., including comparisons of corn event MIR162 with it's unmodified corn counterparts, the Animal Feed Division of the Animal Health Directorate has concluded that the introduced genes and their corresponding traits will not confer to corn event MIR162 any characteristic that would raise any concerns regarding the safety or nutritional composition of corn event MIR162. Grain corn, its byproducts and corn oil are currently listed in Schedule IV of the Feeds Regulations and are, therefore, approved for use in livestock feeds in Canada. Corn event MIR162 has been assessed and found to be as safe as and as nutritious as traditional corn varieties. Corn event MIR162 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 corn event MIR162 is therefore authorized by Plant Biosafety Office of the Plant Health and Biosecurity Directorate and the Animal Feed Division of the Animal Health Directorate as of February 11, 2010. Any corn lines derived from event MIR162 may also be released into the environment and used as livestock feed, provided no inter-specific crosses are performed, provided the intended uses are similar, provided it is known based on characterization that these plants do not display any additional novel traits and are substantially equivalent to currently grown corn varieties in Canada in terms of their potential environmental impact and livestock feed safety, and provided the novel genes are expressed at a level similar to that of the authorized line. Before corn event MIR162 is cultivated in Canada as a single event, an IRM plan must be submitted to and approved by the PBO.