Decision document DD2014-105: Determination of the safety of Stine Seed Farm, Inc.'s corn (Zea mays L.) event HCEM485

This decision document has been prepared to explain the regulatory decisions reached under Directive 94-08 - Assessment Criteria for Determining Environmental Safety of Plants with Novel Traits, its companion document BIO1994-11 - The Biology of Zea mays (L.) (Corn) and Section 2.6 - Guidelines for the Assessment of Novel Feeds: Plant Sources, of Chapter 2 of the RG-1 Regulatory Guidance: Feed Registration Procedures and Labelling Standards.

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 Stine Seed Farm, Inc. This information concerns the herbicide-tolerant corn event HCEM485. The CFIA has determined that this plant with a novel trait (PNT) does not present altered environmental risk nor, as a novel feed, does it present livestock feed safety or nutrition concerns when compared to corn varieties currently grown and permitted to be used as livestock feed in Canada.

Corn event HCEM485 is subject to the same phytosanitary import requirements as unmodified corn. Corn event HCEM485 must also meet the requirements of other Canadian legislation, including but not limited to the requirements set out in the Food and Drugs Act and the Pest Control Products Act.

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

September 12, 2014

This bulletin was created by the Canadian Food Inspection Agency. For further information, please contact the Plant Biosafety Office or the Animal Feed Division by visiting the contact page.

Table of contents

1. Brief identification of the modified plant
2. Background information
3. Description of the novel traits
   1. Development method
   2. Tolerance to glyphosate herbicide
   3. Stable integration into the plant genome
4. Criteria for the environmental assessment
   1. Potential of corn event HCEM485 to become a weed of agriculture or be invasive of natural habitats
   2. Potential for gene flow from corn event HCEM485 to sexually compatible plants whose hybrid offspring may become more weedy or more invasive
   3. Potential for corn event HCEM485 to become a plant pest
   4. Potential impact of corn event HCEM485 and its gene products on non-target organisms, including humans
   5. Potential impact of corn event HCEM485 on biodiversity
5. Criteria for the livestock feed assessment
   1. Potential impact of corn event HCEM485 on livestock nutrition
   2. Potential impact of corn event HCEM485 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

Designation of the modified plant: Corn event HCEM485 OECD Unique Identifier SSF-HC485-9

Applicant: Stine Seed Farm, Inc.

Plant species: Corn (Zea mays L.)

Novel traits: Tolerance to glyphosate herbicide

Trait introduction method: Aerosol beam injection

Intended use of the modified plant: Corn event HCEM485 is intended for traditional corn human food and livestock feed uses. Corn event HCEM485 is not intended to be grown outside the normal production area for corn in Canada.

II. Background information

Stine Seed Farm, Inc. has developed a corn event that is tolerant to the herbicide glyphosate.

Corn event HCEM485 was developed using recombinant deoxyribonucleic acid (rDNA) technology, resulting in the introduction of a 5-enolpyruvylshikimate-3-phosphate synthase (epsps) gene from corn which was modified by site-directed mutagenesis. The resulting 2mepsps gene encodes a 2mEPSPS protein that contains two amino acid substitutions that allow the enzyme to function in the presence of the herbicide glyphosate.

Stine Seed Farm, Inc. has provided information on the identity of corn event HCEM485, a detailed description of the transformation method, information on the gene insertion site and gene copy number, and information on the role of the inserted gene and regulatory sequences. The novel protein was identified and characterized. Information was 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. Information was also provided for the evaluation of herbicide residues in the feed commodities derived from the crop, following the intended herbicide application.

A hybrid, consisting of a cross between corn event HCEM485 and an unmodified corn variety (hereafter referred to as the HCEM485 hybrid), was field tested at 15 sites in the United States (US) in 2007. The locations of these trials share similar environmental and agronomic conditions to corn production areas in Canada and were considered representative of major Canadian corn growing regions. Three unmodified control corn hybrids were also grown during these field trials as comparators. These unmodified control corn hybrids were of a similar genetic background to the HCEM485 hybrid.

Agronomic characteristics of the HCEM485 hybrid, such as percent barren plants, dropped ears, early growth, early stand count, seedling vigour, ear height, early root lodging, grain moisture, final stand count, heat units to 50% pollen shed, heat units to 50% silking, leaf colour, plant height, root lodging, stalk lodging, test weight and grain yield, were compared to those of one of the unmodified control corn hybrids in 15 field trials.

Nutritional components of grain and forage from the HCEM485 hybrid, such as protein, fat, moisture, ash, carbohydrates, fiber, amino acids, fatty acids, vitamins, minerals, secondary metabolites and anti-nutrients, were compared with those of a composite sample of the three unmodified control corn hybrids in four field trials.

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

• the potential of corn event HCEM485 to become a weed of agriculture or to be invasive of natural habitats
• the potential for gene flow from corn event HCEM485 to sexually compatible plants whose hybrid offspring may become more weedy or more invasive
• the potential for corn event HCEM485 to become a plant pest
• the potential impact of corn event HCEM485 and its gene products on non-target organisms, including humans; and
• the potential impact of corn event HCEM485 on biodiversity

The Animal Feed Division (AFD) of the CFIA has also reviewed the above information with respect to the assessment criteria for determining the safety and nutrition of livestock feed, as described in Section 2.6 - Guidelines for the Assessment of Novel Feeds: Plant Sources, of Chapter 2 of the RG-1 Regulatory Guidance: Feed Registration Procedures and Labelling Standards.

The AFD has considered both intended and unintended effects and similarities and differences between corn event HCEM485 and unmodified corn varieties relative to the safety and nutrition of feed ingredients derived from corn event HCEM485 for their intended purpose, including:

• the potential impact of corn event HCEM485 on livestock nutrition; and
• the potential impact of corn event HCEM485 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 AFD has also considered whether feeds derived from corn event HCEM485 meet the definitions and requirements of feeds as listed in Schedule IV of the Feeds Regulations.

Stine Seed Farm, Inc. has provided the CFIA with a method for the detection and identification of corn event HCEM485.

III. Description of the novel trait

1. Development method

Corn event HCEM485 was developed through aerosol beam injection of immature corn embryos with a 6.0 kilobase (kb) corn genomic fragment. In addition to other endogenous corn DNA sequences, the 6.0 kb corn genomic fragment contains a 2mepsps gene, derived from the native corn epsps gene, and the native regulatory elements associated with the native epsps gene. To develop the 2mepsps gene, the native epsps gene was modified by site-directed mutagenesis to introduce two nucleotide substitutions, resulting in two amino acid substitutions in the sequence of the 2mEPSPS protein. These two amino acid substitutions in the 2mEPSPS protein confer tolerance to the herbicide glyphosate.

Transformed cells were selected on the basis of tolerance to glyphosate and regenerated to produce plants. Corn event HCEM485 was identified as a successful transformant based on molecular analyses, herbicide efficacy and agronomic evaluations and was thus chosen for further development.

2. Tolerance to glyphosate

EPSPS is an enzyme involved in the plant shikimic acid metabolic pathway, which is essential for the production of aromatic amino acids. The herbicide disrupts the shikimic acid pathway by binding to the EPSPS protein, leading to interference in aromatic amino acids production and growth suppression or death of the plant. The modified 2mEPSPS version of this enzyme expressed in corn event HCEM485 contains two amino acid substitutions that reduce binding of the 2mEPSPS protein to glyphosate, compared to native corn EPSPS. The 2mEPSPS protein confers commercial-level glyphosate tolerance to corn event HCEM485 since it continues to catalyze the production of aromatic amino acids in the presence of glyphosate.

The expression of the 2mEPSPS protein in corn event HCEM485 is driven by the native promoter of the corn epsps gene. Thus, the pattern of expression of the 2mEPSPS protein in tissues of corn event HCEM485 is expected to be the same as that of the native 2mEPSPS protein in unmodified corn tissues. Expression of the 2mEPSPS protein was measured in leaf and seed tissues collected from the HCEM485 hybrid grown in three field trials in the US. The average 2mEPSPS protein expression at different developmental stages, as evaluated by ELISA, was 2.0 to 3.0 micrograms per gram fresh weight (μg/g fwt) in leaves, and undetectable to 0.3 μg/g fwt in seed. In a second study, expression of the 2mEPSPS protein was measured in tissues collected from corn event HCEM485 grown in a single field trial in the US. The average 2mEPSPS protein expression at different developmental stages, as evaluated by ELISA, was 0.5 to 3.0 μg/g fwt in leaves, 0.16 to 0.79 μg/g fwt in stems, 0.29 to 1.87 μg/g fwt in roots, and 0.26 to 0.46 μg/g fwt in forage.

Corn event HCEM485 did not express the 2mEPSPS protein in sufficient quantities for assessment of environmental and livestock feed safety. The 2mEPSPS protein was previously expressed in an E. coli production system and characterized in a published safety study by Hérouet-Guicheney et al. (2009)^Footnote1. The corn event HCEM485-produced 2mEPSPS and the E. coli-produced 2mEPSPS previously characterized by Hérouet-Guicheney et al. (2009) differ by a single amino acid that is not expected to impact the functional activity or safety of the 2mEPSPS protein. Equivalency was demonstrated between the corn event HCEM485-produced 2mEPSPS protein and the E. coli-produced 2mEPSPS protein previously characterized by Hérouet-Guicheney et al. (2009) by comparing their molecular weights and binding by antibodies specific for 2mEPSPS. Based on the results, the proteins were found to be equivalent. Demonstration of equivalence between the 2mEPSPS protein produced in E. coli and the 2mEPSPS protein produced in corn event HCEM485 allows the use of the published safety study by Hérouet-Guicheney et al. (2009) to support the safety of the 2mEPSPS protein produced in corn event HCEM485.

The potential allergenicity and toxicity of the 2mEPSPS protein to livestock and non-target organisms were evaluated. The weight of evidence indicates that the 2mEPSPS protein is unlikely to be allergenic. The source of the 2mepsps gene, corn, is not commonly associated with allergenicity, the 2mEPSPS protein amino acid sequence lacks relevant similarities to known allergens and the E. coli 2mEPSPS protein that had been previously characterized by Hérouet-Guicheney et al. (2009) was shown experimentally to be rapidly degraded in simulated gastric fluid and simulated intestinal fluid. It was also concluded that the 2mEPSPS protein is unlikely to be toxic to livestock and non-target organisms because it lacks a mode of action to suggest that it is intrinsically toxic to livestock or non-target organisms, because the 2mEPSPS protein amino acid sequence lacks relevant similarities to known toxins and because no adverse effects were observed when the E. coli 2mEPSPS protein that had been previously characterized by Hérouet-Guicheney et al. (2009) was ingested by mice at doses of approximately 2000 milligrams per kilogram body weight (mg/kg bwt). For a more detailed discussion of the potential allergenicity and toxicity of the 2mEPSPS protein, see Section V, part 2: Potential Impact of Corn Event HCEM485 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.

3. Stable integration into the plant genome

Results from molecular characterization of corn event HCEM485 by Southern blot analysis are consistent with the introduction of four intact copies of the gene cassette containing the 2mepsps gene and its regulatory elements inserted at a single site in the corn genome. No additional elements, including intact or partial DNA fragments of the gene cassette or backbone sequences from the plasmid vector, linked or unlinked to the intact insert, were detected in corn event HCEM485.

The inheritance pattern of the herbicide tolerance trait across two segregating generations of corn event HCEM485 showed that the trait segregates according to the Mendelian rules of inheritance for a single genetic locus.

IV. Criteria for the environmental assessment

1. Potential of corn event HCEM485 to become a weed of agriculture or be invasive of natural habitats

As described in the CFIA biology document BIO1994-11 - The Biology of Zea mays L. (Corn), 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 information provided by Stine Seed Farm, Inc., corn event HCEM485 was determined not to be significantly different from unmodified corn varieties in this respect.

The CFIA evaluated data submitted by Stine Seed Farm, Inc. on the reproductive biology and life history traits of corn event HCEM485. As previously mentioned, the HCEM485 hybrid was field tested at 15 locations in the US in 2007. It was determined that the US locations share similar environmental and agronomic conditions to corn production areas in southern Ontario and Quebec, and were considered representative of major Canadian corn growing regions. During the field trials, the HCEM485 hybrid was compared to one of the three unmodified control corn hybrids previously mentioned. Phenotypic and agronomic traits were evaluated, covering a broad range of characteristics that encompass the entire life cycle of the corn plant. The traits included percent barren plants, dropped ears, early growth, early stand count, seedling vigour, ear height, early root lodging, grain moisture, final stand count, heat units to 50% pollen shed, heat units to 50% silking, leaf colour, plant height, root lodging, stalk lodging, test weight and grain yield. Although instances of statistically significant differences were observed between the HCEM485 hybrid and the unmodified control corn hybrid for some traits in the individual-site analyses, there were no consistent trends in the data across locations that would indicate the differences were due to the genetic modification. Therefore, the statistical analysis of these observations showed no biologically meaningful differences between the HCEM485 hybrid and the unmodified control corn hybrid and supports a conclusion of phenotypic and agronomic equivalence to currently grown corn varieties.

Stine Seed Farm, Inc. provided information on the germination of the HCEM485 hybrid seed under warm and cool temperature regimes. No difference was detected between the HCEM485 hybrid and the unmodified control corn hybrid at either temperature regime. Therefore, the introduction of the novel trait did not impact the germination of the corn seed and did not confer dormancy to the corn seed.

The susceptibility of the HCEM485 hybrid to corn pathogens was evaluated in the field at the same locations as the agronomic characteristics studies (further detail provided below in Section IV, part 3: Potential for Corn Event HCEM485 to Become a Plant Pest). No trend in increase or decrease of susceptibility to pathogens was observed in the HCEM485 hybrid compared to the unmodified control corn hybrid.

No competitive advantage was conferred to plants of corn event HCEM485, other than that conferred by tolerance to the glyphosate herbicide, as the reproductive characteristics, growth characteristics and tolerance to pathogens of the HCEM485 hybrid were comparable to those of the unmodified control corn hybrid. 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. Corn event HCEM485 plants growing as volunteers will not be controlled if glyphosate is used as the only weed control tool. However, control of corn event HCEM485 as a volunteer weed in subsequent crops or in fallow ground can 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 corn event HCEM485 has no altered weediness or invasiveness potential in Canada compared to currently grown corn varieties.

The CFIA considers the changes in usual agronomic practices that may arise from volunteer plants with novel herbicide tolerances. Similarly, the CFIA considers the potential that continued application of the same herbicide in subsequent rotations may lead to increased selection pressure for herbicide-tolerant weed populations. In order to address these issues, a herbicide tolerance management plan, which includes integrated weed management strategies, must be implemented. This plan includes recommendations to rotate or combine weed control products with alternate modes of action, and to employ other weed control practices.

2. Potential for gene flow from corn event HCEM485 to sexually compatible plants whose hybrid offspring may become more weedy or more invasive

As described in the CFIA biology document BIO1994-11 - The Biology of Zea mays (L.) (Corn), there are no sexually compatible species in Canada that can hybridize with corn. The glyphosate tolerance trait introduced into corn event HCEM485 has no intended effects on corn reproductive biology.

The CFIA has therefore concluded that gene flow from corn event HCEM485 to wild relatives is not possible in Canada.

3. Potential for corn event HCEM485 to become a plant pest

Corn is not considered a plant pest in Canada and the glyphosate tolerance trait introduced into corn event HCEM485 is unrelated to plant pest potential (i.e., the potential for the plant to harbor new or increased populations of pathogens or pests).

The susceptibility of corn event HCEM485 to corn pathogens was evaluated in the field at the same locations as the agronomic characteristic studies. The stressors observed included southern rust disease, grey leaf spot, northern corn blight, common rust and smut. The evaluations of the HCEM485 hybrid did not show any increased or decreased susceptibility to these corn pathogens compared to the unmodified control corn hybrid.

Based on the above information, the CFIA has concluded that corn event HCEM485 does not display any altered plant pest potential compared to currently grown corn varieties.

4. Potential impact of corn event HCEM485 and its gene products on non-target organisms, including humans

The glyphosate tolerance trait introduced into corn event HCEM485 is unrelated to a potential impact on non-target organisms.

The 2mEPSPS protein differs from the native corn EPSPS by only two amino acid substitutions. Detailed characterization of the 2mEPSPS protein expressed in corn event HCEM485 led to the conclusion that this protein does not display any characteristic of a potential toxin or allergen (see Section V, part 2: Potential Impact of Corn Event HCEM485 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). Therefore, no negative impacts resulting from exposure of organisms to the 2mEPSPS protein expressed in corn event HCEM485 are expected.

Composition analyses showed that the levels of key nutrients and anti-nutrients in grain and forage from the HCEM485 hybrid are comparable to those in the composite sample of the three unmodified control corn hybrids (see Section V, part 1: Potential Impact of Corn Event HCEM485 on Livestock Nutrition). Therefore, it is very unlikely that the introduction of the novel trait may have caused unintended changes to the composition of corn event HCEM485 tissues that would negatively impact organisms interacting with corn event HCEM485.

Field evaluations of the HCEM485 hybrid did not show any increased resistance to pathogens compared to the unmodified control corn hybrids (see Section IV, part 3: Potential for Corn Event HCEM485 to Become a Plant Pest).

Collectively, these information elements indicate that the interactions between corn event HCEM485 and the populations of animals and microorganisms interacting with corn crops will be similar to currently grown corn varieties.

Based on the above information, the CFIA has concluded that the unconfined release of corn event HCEM485 in Canada will not result in altered impacts on non-target organisms, including humans, compared to currently grown corn varieties.

5. Potential impact of corn event HCEM485 on biodiversity

Corn event HCEM485 expresses 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 trait to other species in unmanaged environments. Corn event HCEM485 is unlikely to cause adverse effects on non-target organisms and does not display increased weediness, invasiveness or plant pest potential. It is therefore unlikely that corn event HCEM485 will have any direct effects on biodiversity, in comparison to the effects that would be expected from the cultivation of the corn varieties that are currently grown in Canada.

Corn event HCEM485 has tolerance to the herbicide glyphosate. The use of this herbicide in cropping systems has the intended effect of reducing local weed populations within agro-ecosystems. This may result in a reduction in local weed species biodiversity, and may have effects on other trophic levels which utilize these weed species. It must be noted, however, that the goal of reduction in weed biodiversity in agricultural fields is not unique to the use of PNTs, corn event HCEM485, or the cultivation of corn. It is therefore unlikely that corn event HCEM485 will have any indirect effects on biodiversity, in comparison to the effects that would be expected from cultivation of currently grown corn varieties.

The CFIA has concluded that the introduced gene and its corresponding novel trait do not confer to corn event HCEM485 any characteristic that would result in unintended environmental effects following unconfined release. The CFIA has therefore concluded that the potential impact on biodiversity of corn event HCEM485 is unlikely to be different from that of the corn varieties that are currently grown in Canada.

V. Criteria for the livestock feed assessment

The AFD considered nutrient and anti-nutrient profiles; the safety of feed ingredients derived from corn event HCEM485, 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 corn event HCEM485 meet the definitions and requirements of feeds as listed in Schedule IV of the Feeds Regulations.

1. Potential impact of corn event HCEM485 on livestock nutrition

Nutrient and anti-nutrient composition

The nutritional equivalence of the HCEM485 hybrid (treated with glyphosate) to composite samples of three unsprayed, unmodified control corn hybrids was determined from four replicated field trial sites in US during the 2007 growing season. Grain and forage samples (from two sites) were analysed for protein, fat, moisture, ash, carbohydrates (calculated), acid detergent fibre (ADF), neutral detergent fibre (NDF), calcium and phosphorus. Grain samples were further analysed for amino acids, fatty acids, vitamins, minerals, secondary plant metabolites (ferulic acid and p-coumaric acid), phytosterols and anti-nutrients (phytic acid, trypsin inhibitor, raffinose and inositol) as recommended by the OECD consensus document for new varieties of corn (OECD, 2002 – PDF (209kb)). Composition data was analyzed statistically by analysis of variance across locations using generalized linear model, and differences among treatments were identified and assessed (P<0.05). The biological relevance of any significant difference among corn varieties was assessed by comparing the observed values to the range of the values observed in the unmodified control corn hybrids and in the published scientific literature (Agriculture and Food Systems Institute (AFSI), 2006).

No statistically significant differences were observed between forage from the HCEM485 hybrid and the unmodified control corn hybrids for moisture, ash, carbohydrates, ADF, NDF and phosphorus. Statistically significant effects were found for protein, fat and calcium but these differences were not biologically significant as all means were within the range of the values observed for the unmodified control corn hybrids grown in the trial and in the published scientific literature (AFSI, 2006).

No statistically significant differences were observed between grain samples from the HCEM485 hybrid and the unmodified control corn hybrids for protein, fat, moisture, ash, carbohydrates, ADF and neutral detergent fibre. Except for methionine, no statistically significant differences were observed between grain samples from the HCEM485 hybrid and the unmodified control corn hybrids for all amino acids. The difference observed for methionine was not biologically significant as the mean levels for the HCEM485 hybrid was within the range of the values observed for the unmodified control corn hybrids and in the published scientific literature (AFSI, 2006). No statistically significant differences were observed between the HCEM485 hybrid and the unmodified control corn hybrids for linoleic and arachidic fatty acids. Statistically significant differences were observed between the HCEM485 hybrid and the unmodified control corn hybrids for palmitic, oleic, stearic, eicosenic and linolenic fatty acids but these differences were not biologically significant as all means were within the range of the values observed for the unmodified control corn hybrids and in the published scientific literature (AFSI, 2006). No statistically significant differences were observed between grain from the HCEM485 hybrid and the unmodified control corn hybrids for vitamins B3 and B5. The levels of vitamins B1, B6 and B9 and alpha, beta, gamma, delta and total tocopherols were significantly different between the HCEM485 hybrid and the unmodified control corn hybrids, but these differences were not biologically significant as all means were within the range of the values observed in the unmodified control corn hybrids and in the published scientific literature (AFSI, 2006). No statistically significant differences were observed between grain from the HCEM485 hybrid and the unmodified control corn hybrids for iron, magnesium, manganese, phosphorus, zinc and sodium. Statistically significant differences were observed for calcium, copper and potassium but these differences were not biologically significant as all means were within the range of the values observed in the unmodified control corn hybrids and in the published scientific literature (AFSI, 2006). No statistically significant differences were observed between grain from the HCEM485 hybrid and the unmodified control corn hybrids for the secondary plant metabolites: ferulic acid and p-coumaric acid or the antinutrients phytic acid and raffinose. Statistically significant differences were observed for inositol and trypsin inhibitor but these differences were not biologically significant as all means were within the range of the values observed in the published scientific literature (AFSI, 2006). No statistically significant differences were observed between grain from the HCEM485 hybrid and the unmodified control corn hybrids for the phytosterols (cholesterol, campesterol, stigmasterol, β-sitosterol and stigmastanol).

Conclusion

It was concluded, based on the evidence provided by Stine Seed Farm, Inc., that the nutritional composition of the HCEM485 hybrid is similar to that of unmodified control corn hybrids grown in the same trials and to that reported for other corn in the published scientific literature. Feed ingredients derived from corn event HCEM485 are considered to meet present ingredient definitions for corn.

2. Potential impact of corn event HCEM485 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

Corn event HCEM485 is tolerant to glyphosate herbicide due to production of the 2mEPSPS protein. The assessment of corn event HCEM485 evaluated the impact of the following potential hazards relative to the safety of feed ingredients derived from this event:

• The presence of novel protein 2mEPSPS and
• The chemical pesticide residue profile

Novel 2mEPSPS protein

Corn event HCEM485 did not express the 2mEPSPS protein in sufficient quantities for assessment of environmental and feed safety; however, purified E. coli-expressed 2mEPSPS protein was previously characterized in a published study by Hérouet-Guicheney et al. (2009). Equivalency was demonstrated between the corn event HCEM485-produced 2mEPSPS protein and the E. coli-produced 2mEPSPS protein by comparing their molecular weights and binding to antibodies specific for 2mEPSPS. Demonstration of equivalence between the 2mEPSPS protein produced in E. coli and the 2mEPSPS protein produced in corn event HCEM485 allows the 2mEPSPS protein produced in E. coli to be used in studies to confirm the safety of the 2mEPSPS protein produced in corn event HCEM485.

The potential allergenicity and toxicity of the 2mEPSPS protein to livestock were evaluated. With respect to its potential allergenicity, no single experimental method yields decisive evidence, thus a weight-of-evidence approach was taken, taking into account information obtained with various test methods. The source of the 2mepsps gene, corn, is not known to produce allergens and a bioinformatics evaluation of the 2mEPSPS protein amino acid sequence confirmed the lack of relevant similarities between the 2mEPSPS protein and known allergens. E. coli-produced 2mEPSPS protein safety studies, previously published by Hérouet-Guicheney et al. (2009), indicated that, unlike many allergens, this protein is rapidly degraded in simulated gastric fluid and simulated intestinal fluid. The weight of evidence thus indicates that the 2mEPSPS protein is unlikely to be allergenic.

In terms of its potential toxicity to livestock, the 2mEPSPS protein lacks a mode of action to suggest that it is intrinsically toxic to livestock and a bioinformatics evaluation of the 2mEPSPS protein amino acid sequence confirmed the lack of relevant similarities between the 2mEPSPS protein and known toxins. In addition, E. coli-produced 2mEPSPS protein safety studies, previously published by Hérouet-Guicheney et al. (2009), indicated that no adverse effects were observed when the 2mEPSPS protein was ingested by mice at doses of approximately 2000 mg/kg bwt. These data indicate that the 2mEPSPS protein is unlikely to be toxic to livestock.

The livestock exposure to the 2mEPSPS protein is expected to be negligible as the 2mEPSPS protein is expressed at very low levels in corn event HCEM485 and is rapidly degraded under conditions which simulate the mammalian digestive tract.

Chemical pesticide residue profile

The safety of pesticide residues and metabolites in corn event HCEM485, following application of herbicides, was also evaluated as part of the feed safety assessment. It was determined that potential glyphosate residues and metabolites in livestock feeds derived from corn event HCEM485 would not present levels of concern to livestock, nor humans via the potential transfer into foods of animal origin.

Conclusion

It was concluded, based on the evidence provided by Stine Seed Farm, Inc., that the novel 2mEPSPS protein-based herbicide tolerance trait will not confer to corn event HCEM485 any characteristic that would raise concerns regarding the safety of corn event HCEM485. Feed ingredients derived from corn event HCEM485 are considered to meet present ingredient definitions for corn.

VI. New information requirements

If at any time, Stine Seed Farm, Inc. becomes aware of any new information regarding risk to the environment, livestock or human health, which could result from the unconfined environmental release or livestock feed use of corn event HCEM485 or lines derived from it, Stine Seed Farm, Inc. is required to 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 HCEM485 on the environment, livestock and human health and may re-evaluate its decision with respect to the livestock feed use and unconfined environmental release authorizations of corn event HCEM485.

VII. Regulatory decision

Based on the review of the data and information submitted by Stine Seed Farm, Inc. and input from other relevant scientific sources, the Plant and Biotechnology Risk Assessment Unit of the Plant Health Science Directorate, CFIA, has concluded that the unconfined environmental release of corn event HCEM485 does not present altered environmental risk when compared to corn varieties that are currently grown in Canada.

Based on the review of the data and information submitted by Stine Seed Farm, Inc. and input from other relevant scientific sources, the Animal Feed Division of the Animal Health Directorate, CFIA, has concluded that the novel 2mEPSPS protein-based herbicide tolerance trait will not confer to corn event HCEM485 any characteristic that would raise any concerns regarding the safety or nutrition of corn event HCEM485. Grain corn, its by-products and corn oil are currently listed in IV of the Feeds Regulations and are, therefore authorized for use in livestock feeds in Canada. Corn event HCEM485 has been found to be as safe as and as nutritious as currently and historically grown corn varieties. Corn event HCEM485 and its products are considered to meet present ingredient definitions and are authorized for use as livestock feed ingredients in Canada.

Corn event HCEM485 is subject to the same phytosanitary import requirements as unmodified corn varieties. Corn event HCEM485 is required to meet the requirements of other Canadian legislation, including but not limited to the requirements set out in the Food and Drugs Act and the Pest Control Products Act.

Please refer to Health Canada's Decisions on Novel Foods for a description of the food safety assessment of corn event HCEM485.