Decision Document DD2014-103
Determination of the Safety of Syngenta Canada Inc.'s and Bayer CropScience Inc.'s Soybean (Glycine max L.) Event SYHT0H2

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June 4, 2014

This Decision Document has been prepared to explain the regulatory decisions reached under Directive 94-08 (Dir94-08) - Assessment Criteria for Determining Environmental Safety of Plants with Novel Traits, its companion document BIO1996-10 - The Biology of Glycine max L.). 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 Syngenta Canada Inc. and Bayer CropScience Inc. This information concerns the herbicide tolerant soybean event SYHT0H2. 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 soybean varieties currently grown and permitted to be used as livestock feed in Canada.

Taking into account these evaluations, unconfined release into the environment and use as livestock feed of soybean event SYHT0H2 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, respectively, as of June 4, 2014. Any soybean lines derived from soybean event SYHT0H2 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 soybean varieties that are currently grown and permitted to be used as livestock feed in Canada, in terms of their potential environmental impact and livestock feed safety and nutrition, (iv) the novel genes are expressed at levels similar to that of the authorized line and (v) data used to establish the substantial equivalence of lines derived from soybean event SYHT0H2 be made available to the CFIA upon request.

Additionally, with respect to its use as livestock feed, soybean event SYHT0H2 must meet the restrictions specific to HPPD inhibiting herbicide-treated forage and hay set out in the authorization.

Soybean event SYHT0H2 is subject to the same phytosanitary import requirements as unmodified soybean varieties. Soybean event SYHT0H2 is required to meet the requirements of other Canadian legislation, including but not limited to the requirements set out in the Food & 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 food assessment by Health Canada, have been addressed separately from this review.

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 Mesotrione and Isoxaflutole Herbicides
    3. Tolerance to Glufosinate-Ammonium Herbicide
    4. Stable Integration into the Plant Genome
  4. Criteria for the Environmental Assessment
    1. Potential of Soybean Event SYHT0H2 to Become a Weed of Agriculture or be Invasive of Natural Habitats
    2. Potential for Gene Flow from Soybean Event SYHT0H2 to Sexually Compatible Plants Whose Hybrid Offspring May Become More Weedy or More Invasive
    3. Potential for Soybean Event SYHT0H2 to Become a Plant Pest
    4. Potential Impact of Soybean Event SYHT0H2 and Its Gene Products on Non-Target Organisms, Including Humans
    5. Potential Impact of Soybean Event SYHT0H2 on Biodiversity
  5. Criteria for the Livestock Feed Assessment
    1. Potential Impact of Soybean Event SYHT0H2 on Livestock Nutrition
    2. Potential Impact of Soybean Event SYHT0H2 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: Soybean event SYHT0H2 OECD Unique Identifier SYN-ØØØH2-5
Applicant: Syngenta Canada Inc. and Bayer CropScience Inc.
Plant Species: Soybean (Glycine max L.)
Novel Traits: Tolerance to mesotrione, isoxaflutole and glufosinate-ammonium herbicides
Trait Introduction Method: Agrobacterium-mediated transformation
Intended Use of the Modified Plant: Soybean event SYHT0H2 is intended to be grown for traditional soybean human food and livestock feed uses. Soybean event SYHT0H2 is not intended to be grown outside the normal production area for soybean in Canada.

II. Background Information

Syngenta Canada Inc. and Bayer CropScience Inc. have developed a soybean event that is tolerant to the p-hydroxyphenylpyruvate dioxygenase (HPPD) inhibiting herbicides mesotrione and isoxaflutole and to the herbicide glufosinate-ammonium. Soybean event SYHT0H2 was developed using recombinant deoxyribonucleic acid (DNA) technology, resulting in the introduction of the avhppd-03 and pat genes. The avhppd-03 gene is derived from the common oat, Avena sativa (A. sativa) and encodes a version of the HPPD enzyme (AvHPPD-03) which has lower binding affinities for the HPPD-inhibiting herbicides mesotrione and isoxaflutole. The pat gene is derived from the soil bacterium Streptomyces viridochromogenes (S. viridochromogenes) and encodes a phosphinothricin acetyltransferase (PAT) enzyme. This enzyme inactivates the herbicide glufosinate-ammonium.

Syngenta Canada Inc. and Bayer CropScience Inc. have provided information on the identity of soybean event SYHT0H2; a detailed description of the transformation method; and information on the gene insertion site, gene copy number, levels of gene expression in the plant and the role of the inserted genes and regulatory sequences. The novel proteins were identified and characterized. Information 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. Information was also provided for the evaluation of herbicide residues in the feed commodities derived from the crop, following the application of mesotrione, isoxaflutole and/or glufosinate-ammonium herbicides.

Soybean event SYHT0H2 was field tested at six sites in the United States (US) in 2010. The locations of these trials share similar environmental and agronomic conditions to soybean production areas in Canada and were considered representative of major Canadian soybean growing regions. An unmodified control soybean variety, which shares the same genetic background as soybean event SYHT0H2, was included in the trials to act as a comparator for soybean event SYHT0H2. Several reference soybean varieties were also included in the field trials to establish a typical soybean reference range for phenotypic parameters.

Agronomic characteristics of soybean event SYHT0H2, such as early stand count, seedling vigour, plant height, lodging, days to 50% flowering, flower colour, days to maturity, seed test weight, seed moisture, seed yield, final stand count and pod shattering, were compared to those of the unmodified control soybean variety and to the range established from the reference soybean varieties.

Nutritional components of soybean event SYHT0H2 seed and forage, such as proximate (protein, fat, moisture and ash), carbohydrates, acid detergent fiber (ADF), neutral detergent fiber (NDF), amino acids, fatty acids, vitamins, minerals and anti-nutrients were compared to those of the unmodified control soybean variety and to the range established by the reference soybean varieties.

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 Directive 94-08 (Dir94-08) - Assessment Criteria for Determining Environmental Safety of Plants with Novel Traits. The PBRA Unit has considered:

  • the potential of soybean event SYHT0H2 to become a weed of agriculture or to be invasive of natural habitats;
  • the potential for gene flow from soybean event SYHT0H2 to sexually compatible plants whose hybrid offspring may become more weedy or more invasive;
  • the potential for soybean event SYHT0H2 to become a plant pest;
  • the potential impact of soybean event SYHT0H2 and its gene products on non-target organisms, including humans; and
  • the potential impact of soybean event SYHT0H2 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 soybean event SYHT0H2 and unmodified soybean varieties relative to the safety and nutrition of feed ingredients derived from soybean event SYHT0H2 for their intended purpose, including:

  • the potential impact of soybean event SYHT0H2 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; and
  • the potential impact of soybean event SYHT0H2 on livestock nutrition.

The AFD has also considered whether feeds derived from soybean event SYHT0H2 meet the definitions and requirements of feeds as listed in Schedule IV of the Feeds Regulations.

Syngenta Canada Inc. and Bayer CropScience Inc. have provided the CFIA with a method for the detection and identification of soybean event SYHT0H2.

III. Description of the Novel Trait

1. Development Method

Soybean event SYHT0H2 was developed through Agrobacterium-mediated transformation of soybean cells and contains the avhppd-03 gene of A. sativa and the pat gene of S. viridochromogenes and associated regulatory elements. Transformed cells were selected on the basis of tolerance to glufosinate-ammonium and regenerated to produce plants. Soybean event SYHT0H2 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 Mesotrione and Isoxaflutole

The HPPD enzyme of soybean and other plants catalyzes the formation of homogentisic acid, the aromatic precursor of plastoquinone and vitamin E. Mesotrione and isoxaflutole inhibit the HPPD enzyme in many plants, resulting in depletion of plant plastoquinone and vitamin E levels, leading to bleaching and plant death. The HPPD enzymes of oat and other grass species are relatively insensitive to the effects of HPPD-inhibiting herbicides. Soybean event SYHT0H2 was developed to be tolerant to the HPPD-inhibiting herbicides mesotrione and isoxaflutole by incorporation of the avhppd-03 gene of common oat, which encodes the AvHPPD-03 enzyme that can function in the presence of the HPPD-inhibiting herbicides mesotrione or isoxaflutole. Introduction of the avhppd-03 gene into soybean event SYHT0H2 confers commercial-level tolerance to the herbicides mesotrione and isoxaflutole. The AvHPPD-03 enzyme of soybean event SYHT0H2 is 99.7 % identical to the native HPPD enzyme of common oat.

AvHPPD-03 protein expression in soybean event SYHT0H2 is driven by a constitutive promoter. Samples of soybean tissues were collected from plants from four field trials in Argentina. Tissues were collected from unsprayed plants and plants sprayed with mesotrione and glufosinate-ammonium. The average AvHPPD-03 protein expression in micrograms per gram dry weight (μg/g dwt) from unsprayed plants, as evaluated by ELISA, was as follows: 242.00 μg/g dwt in V4 leaf, 218.98 μg/g dwt in V8 leaf, 165.14 μg/g dwt in V10 leaf, 105.32 μg/g dwt in R6 leaf, 79.49 μg/g dwt in V8 root, 22.50 μg/g dwt in R6 root, 79.66 μg/g dwt in R6 forage and 8.18 μg/g dwt in R8 seed. Similar AvHPPD-03 protein levels were observed in the tissues of herbicide-treated plants.

To obtain sufficient quantities of AvHPPD-03 protein for assessment of environmental and feed safety, it was necessary to express the avhppd-03 gene in an Escherichia coli (E. coli) production system. Equivalency was demonstrated between the soybean event SYHT0H2-produced AvHPPD-03 protein and the E. coli-produced AvHPPD-03 protein by comparing their molecular weights, immunoreactivity, glycosylation, N-terminal sequence analysis, MALDI-TOF mass spectrometry and functional activity. Based on the results, the proteins were found to be equivalent. Demonstration of equivalence between the AvHPPD-03 protein produced in E. coli and the AvHPPD-03 protein produced in soybean event SYHT0H2 allows the AvHPPD-03 protein produced in E. coli to be used in studies to confirm the safety of the AvHPPD-03 protein produced in soybean event SYHT0H2.

The potential allergenicity and toxicity of the AvHPPD-03 protein to livestock and non-target organisms were evaluated. The weight of evidence indicates that the AvHPPD-03 protein is unlikely to be allergenic, based on the following information. The source of the avhppd-03 gene, A. sativa, is not commonly associated with allergenicity. The AvHPPD-03 protein amino acid sequence lacks relevant similarities to known allergens. Unlike many allergens, E. coli-produced AvHPPD-03 protein was shown experimentally to be rapidly degraded in simulated gastric fluid and not to be heat stable. Finally, unlike many allergens, soybean event SYHT0H2 AvHPPD-03 protein was shown experimentally to be nonglycosylated. It was also concluded that the AvHPPD-03 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 AvHPPD-03 protein amino acid sequence lacks relevant similarities to known toxins and because no adverse effects were observed when E. coli–produced AvHPPD-03 protein was ingested by mice at doses of approximately 2000 mg/kg bwt. For a more detailed discussion of the potential allergenicity and toxicity of the AvHPPD-03 protein, see Section V, part 2: Potential Impact of Soybean Event SYHT0H2 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. Tolerance to Glufosinate-Ammonium

Glufosinate-ammonium herbicide inhibits the plant enzyme glutamine synthetase, resulting in the accumulation of lethal levels of ammonia in susceptible plants. Ammonia is produced by plants as a result of normal metabolic processes. Soybean event SYHT0H2 was developed to be tolerant to the herbicide glufosinate-ammonium by incorporation of the pat gene. The pat gene encodes the enzyme PAT, which can acetylate the primary amino group of glufosinate-ammonium, rendering it inactive. Introduction of the pat gene into soybean event SYHT0H2 confers commercial-level tolerance to the herbicide glufosinate-ammonium. The pat gene was derived from S. viridochromogenes, a gram-positive soil bacterium and the PAT protein produced in soybean event SYHT0H2 is identical to the native enzyme.

The PAT protein produced in soybean event SYHT0H2 possesses the same amino acid sequence as the PAT protein previously characterized in the published study authored by Hérouet et al. in 2005Footnote 1.

PAT protein expression in soybean event SYHT0H2 is driven by a constitutive promoter. Samples of soybean tissues were collected from plants from four field trials in Argentina. Tissues were collected from unsprayed plants and plants sprayed with mesotrione and glufosinate-ammonium. The average PAT protein expression in micrograms per gram dry weight (μg/g dwt) from unsprayed plants, as evaluated by ELISA, was as follows: 52.21 μg/g dwt in V4 leaf, 23.00 μg/g dwt in V8 leaf, 38.23 μg/g dwt in V10 leaf, 29.41 μg/g dwt in R6 leaf, 21.16 μg/g dwt in V8 root, 9.12 μg/g dwt in R6 root, 19.17 μg/g dwt in R6 forage and 2.70 μg/g dwt in R8 seed. Similar PAT protein levels were observed in the tissues of herbicide-treated plants.

Soybean event SYHT0H2 did not express the PAT protein in sufficient quantities for evaluation of environmental and feed safety, however, purified E. coli-expressed PAT protein was previously characterized in a published study by Hérouet et al. (2005). Equivalency was demonstrated between the soybean event SYHT0H2-produced PAT protein and the E. coli-produced PAT protein by comparing their molecular weights, immunological reactivities, glycosylation status, peptide mapping profiles, N-terminal amino acid sequence and enzyme activities. Based on the results, the proteins were found to be equivalent. Demonstration of equivalence between the PAT protein produced in E. coli and the PAT protein produced in soybean event SYHT0H2 allows the use of the published study by Hérouet et al. (2005) to support the safety of the PAT protein produced in soybean event SYHT0H2.

The potential allergenicity and toxicity of the PAT protein to livestock and non-target organisms were evaluated. The weight of evidence indicates that the PAT protein is unlikely to be allergenic, based on the following information. The source of the pat gene, S. viridochromogenes, is not commonly associated with allergenicity. The pat protein amino acid sequence lacks relevant similarities to known allergens. Unlike many allergens, E. coli-produced PAT protein that had previously been characterized by Hérouet et al. (2005) was shown experimentally to be rapidly degraded in simulated gastric fluid and not to be heat stable. Finally, unlike many allergens, soybean event SYHT0H2 PAT protein was shown experimentally to be non-glycosylated. It was also concluded that the PAT 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 PAT protein amino acid sequence lacks relevant similarities to known toxins and because no adverse effects were observed when the E. coliPAT protein that had been previously characterized by Hérouet et al. (2005) was ingested by mice at doses of approximately 2000 mg/kg bwt. For a more detailed discussion of the potential allergenicity and toxicity of the PAT protein, see Section V, part 2: Potential Impact of Soybean Event SYHT0H2 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.

4. Stable Integration into the Plant Genome

Molecular characterization by Southern blot analysis demonstrated that soybean event SYHT0H2 contains a single DNA insert consisting of one copy of the avhppd-03 gene, four copies of the pat gene and regulatory elements associated with these genes. No additional elements, including intact or partial DNA fragments of the gene cassettes or backbone sequences from the plasmid vector, linked or unlinked to the intact insert, were detected in soybean event SYHT0H2.

The stability of the insert within soybean event SYHT0H2 was verified by Southern blot analysis over three generations. The inheritance of the avhppd-03 and pat genes was measured by polymerase chain reaction over three segregating generations. The results indicated that the avhppd-03 and pat genes segregate according to Mendelian rules of inheritance for a single genetic locus.

IV. Criteria for the Environmental Assessment

1. Potential of Soybean Event SYHT0H2 to Become a Weed of Agriculture or Be Invasive of Natural Habitats

The biology of soybean, as described in the CFIA biology document BIO1996-10 - The Biology of Glycine max (L.)) Merr (Soybean), is such that unmodified plants of this species are not invasive of unmanaged habitats in Canada. Soybean does not possess an intrinsic potential to become weedy in Canada due to its lack of seed dormancy and the poor competitive ability of its seedlings. According to the information provided by Syngenta Canada Inc. and Bayer CropScience Inc., soybean event SYHT0H2 was determined not to be significantly different from unmodified soybean varieties in this respect.

The CFIA evaluated data submitted by Syngenta Canada Inc. and Bayer CropScience Inc. on the reproductive biology and life history traits of soybean event SYHT0H2. As previously mentioned, soybean event SYHT0H2 was field tested in the US at six locations in 2010. It was determined that the US locations share similar environmental and agronomic conditions to southwestern Ontario and were considered to be representative of major Canadian soybean growing regions. During the field trials, soybean event SYHT0H2 was compared to the unmodified control soybean variety. Reference soybean varieties were also included in these trials to establish ranges of comparative values that are representative of currently grown soybean varieties. Soybean event SYHT0H2 plants were either unsprayed or sprayed with mesotrione and glufosinate-ammonium herbicides. Phenotypic and agronomic traits were evaluated, covering a broad range of characteristics that encompass the entire life cycle of the soybean plant. The traits included early stand count, seedling vigour, plant height, lodging, days to 50% flowering, flower colour, days to maturity, seed test weight, seed moisture, seed yield, final stand count and pod shattering. Although instances of statistically significant differences were observed between soybean event SYHT0H2 and the unmodified control soybean variety for some traits in the individual-site analyses, there was no consistent trend in the data across locations that would indicate the differences were due to the genetic modification. In addition, the values for soybean event SYHT0H2 were within the reference range established from the reference soybean varieties included in the same field trials. Therefore, the statistical analysis of these observations showed no biologically meaningful differences between the agronomic characteristics of soybean event SYHT0H2 and the unmodified control soybean variety and supports a conclusion of phenotypic and agronomic equivalence to currently grown soybean varieties.

Syngenta Canada Inc. and Bayer CropScience Inc. evaluated the germination of soybean event SYHT0H2 seed under warm and cool temperature regimes. No difference was detected between soybean SYHT0H2 and the unmodified control soybean variety at either temperature regime. Therefore the introduction of the novel traits did not impact the germination of the soybean seed and did not confer dormancy to the soybean seed.

Soybean event SYHT0H2 was exposed to soil compaction, drought stress, excess water and wind in the field during the agronomic characteristic studies. No trend in increased or decreased susceptibility to these abiotic stressors was observed in soybean event SYHT0H2 compared to the unmodified control soybean variety.

The susceptibility of soybean event SYHT0H2 to various soybean pests and pathogens was evaluated in the field at the same locations as the agronomic characteristic studies (further detail provided below in Section IV, part 3: Potential for Soybean Event SYHT0H2 to Become a Plant Pest). No trend in increased or decreased susceptibility to pests or pathogens was observed in soybean event SYHT0H2 compared to the unmodified control soybean variety.

No competitive advantage was conferred to plants of soybean event SYHT0H2, other than that conferred by tolerance to mesotrione, isoxaflutole and glufosinate-ammonium herbicides, as the reproductive characteristics, growth characteristics and tolerance to abiotic and biotic stresses of soybean event SYHT0H2 were comparable to those of the unmodified control soybean variety. Tolerance to these herbicides provides a competitive advantage only when they are used and will not, in and of itself, make a herbicide tolerant plant weedier or more invasive of natural habitats. Soybean event SYHT0H2 plants growing as volunteers will not be controlled if mesotrione, isoxaflutole and/or glufosinate-ammonium herbicides are used as the only weed control tools. However, control of soybean event SYHT0H2 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 traits have no intended or observed effects on weediness or invasiveness. The CFIA has therefore concluded that soybean event SYHT0H2 has no altered weediness or invasiveness potential in Canada compared to currently grown soybean 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, 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.

Syngenta Canada Inc. and Bayer CropScience Inc. have submitted independent herbicide tolerance management plans to the CFIA, which were both determined to be satisfactory when evaluated by the PBRA Unit. Syngenta Canada Inc. and Bayer CropScience Inc. indicated that they do not intend to promote or support the use of HPPD-inhibiting herbicides for use on soybean event SYHT0H2 other than those specified in the herbicide tolerance management plans. Should other HPPD-inhibiting herbicides be used on soybean event SYHT0H2 in the future, additional information may be required by the CFIA, such as a revised herbicide tolerance management plan.

Syngenta Canada Inc. and Bayer CropScience Inc. will make these herbicide tolerance management plans readily available to growers and agriculture extension personnel, in both private and public sectors, to promote careful management practices for soybean event SYHT0H2. Syngenta Canada Inc. and Bayer CropScience Inc. will provide an efficient mechanism for growers to report agronomic problems to the company, which will facilitate the ongoing monitoring of soybean event SYHT0H2. Syngenta Canada Inc. and Bayer CropScience Inc. will monitor grower implementation to determine the effectiveness of the herbicide tolerance management plans and make any changes to the plans as appropriate.

2. Potential for Gene Flow from Soybean Event SYHT0H2 to Sexually Compatible Plants Whose Hybrid Offspring May Become More Weedy or More Invasive

Natural hybridization between cultivated soybean and the wild annual species G. soja can occur. However, G. soja is not naturalized in North America and although this species is occasionally grown in research plots, there are no reports of its escape to unmanaged habitats, nor of it becoming a weed in Canadian agro-ecosystems. The biology of soybean, as described in the CFIA biology document BIO1996-10 - The Biology of Glycine max (L.) Merr. (Soybean), shows that soybeans exhibit a high degree of self-fertilization. Cross pollination is usually less than one percent, suggesting that any pollen flow from cultivated soybeans to related species is minimal.

This information, together with the fact that the novel traits have no intended effects on soybean reproductive biology, led the CFIA to conclude that there is minimal potential for gene flow from soybean event SYHT0H2 to related species in Canada.

3. Potential for Soybean Event SYHT0H2 to Become a Plant Pest

Soybean is not considered a plant pest in Canada and the mesotrione, isoxaflutole and glufosinate-ammonium herbicide tolerance traits introduced into soybean event SYHT0H2 are unrelated to plant pest potential (e.g. the potential for the plant to harbour new or increased populations of pathogens or pests).

The susceptibility of soybean event SYHT0H2 to various soybean pests and pathogens was evaluated in the field at the same locations as the agronomic characteristic studies. The stressors observed included Japanese beetles, bean leaf beetles, green clover worms, salt marsh caterpillars, grasshoppers, stink bugs, soybean loopers, whiteflies, leafhoppers, cercospora leaf spot, frogeye leaf spot, powdery mildew and septoria leaf spot. The evaluations of soybean event SYHT0H2 did not show any increased or decreased susceptibility to these pests and pathogens compared to the unmodified control soybean variety.

The CFIA has therefore concluded that soybean event SYHT0H2 does not display any altered plant pest potential compared to currently grown soybean varieties.

4. Potential Impact of Soybean Event SYHT0H2 and Its Gene Products on Non-Target Organisms, Including Humans

The mesotrione, isoxaflutole and glufosinate-ammonium herbicide tolerance traits introduced into soybean event SYHT0H2 appear to be unrelated to a potential impact on non-target organisms. Detailed characterization of the AvHPPD-03 and PAT proteins expressed in soybean event SYHT0H2 led to the conclusion that none of these proteins display any characteristic of a potential toxin or allergen (see Section V, part 2: Potential Impact of Soybean Event SYHT0H2 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 AvHPPD-03 or PAT proteins expressed in soybean event SYHT0H2 are expected.

Compositional analysis showed that the levels of key nutrients and anti-nutrients in seed and forage from soybean event SYHT0H2 are comparable to those in conventional soybean varieties (see Section V, part 1: Potential Impact of Soybean Event SYHT0H2 on Livestock Nutrition). Therefore it is very unlikely that the introduction of the novel traits may have caused unintended changes to the composition of soybean event SYHT0H2 tissues that would negatively impact organisms consuming or otherwise interacting with the plant.

Field evaluations of soybean event SYHT0H2 did not indicate any increased or decreased resistance to insect pests or pathogens compared to the unmodified control soybean variety (see Section V, part 3: Potential for Soybean Event SYHT0H2 to Become a Plant Pest).

Collectively, these information elements indicate that the interactions between soybean event SYHT0H2 and the populations of animals and microorganisms interacting with soybean crops are expected to be similar to currently grown soybean varieties.

The CFIA has therefore determined that the unconfined release of soybean event SYHT0H2 in Canada is not expected to result in altered impacts on non-target organisms, including humans, compared to currently grown soybean varieties.

5. Potential Impact of Soybean Event SYHT0H2 on Biodiversity

Soybean event SYHT0H2 expresses no novel phenotypic characteristics that would extend its range beyond the current geographic range of soybean production in Canada. Soybean's only sexually compatible wild relative in Canada (G. soja) does not occur in unmanaged habitats and the possibility of soybean outcrossing to G. soja is very low. Soybean event SYHT0H2 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 soybean event SYHT0H2 will have any direct effects on biodiversity, in comparison to the effects that would be expected from the cultivation of the soybean varieties that are currently grown in Canada.

Soybean event SYHT0H2 has tolerance to mesotrione, isoxaflutole and glufosinate-ammonium herbicides.The use of these herbicides 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, soybean event SYHT0H2 or the cultivation of soybean. It is therefore unlikely that soybean event SYHT0H2 will have any indirect effects on biodiversity, in comparison to the effects that would be expected from cultivation of currently grown soybean varieties in Canada.

The CFIA has concluded that the introduced genes and their corresponding novel traits do not confer to soybean event SYHT0H2 any characteristic that would result in unintended environmental effects following unconfined release. The CFIA has therefore concluded that the potential impact on biodiversity of soybean event SYHT0H2 is unlikely to be different from that of the soybean 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 soybean event SYHT0H2, 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 soybean event SYHT0H2 meet the definitions and requirements of feeds as listed in Schedule IV of the Feeds Regulations.

1. Potential Impact of Soybean Event SYHT0H2 on Livestock Nutrition

Nutrient and anti-nutrient composition:

The nutritional equivalence of soybean event SYHT0H2 plants (unsprayed plants and plants sprayed with mesotrione and glufosinate-ammonium), to those of the unsprayed, unmodified control soybean variety and six unsprayed, reference soybean varieties was determined from eight replicated field trials in the US during the 2010 growing season. Forage and seed samples were analysed for proximate (moisture, ash, protein and fat), carbohydrate, acid detergent fibre (ADF) and neutral detergent fibre (NDF). Seed samples were further analysed for amino acids, fatty acids, vitamins, minerals and anti-nutrients (phytic acid, trypsin inhibitor, lectin, raffinose, stachyose, daidzein, glycitein and genistein) as recommended by the OECD consensus document for new varieties of soybean (OECD, 2001). Composition data was analysed statistically using analysis of variance (ANOVA), using generalized linear model and statistical differences among treatments were identified and assessed (P<0.05). The biological relevance of any statistically significant difference among soybean varieties was assessed by comparing the observed values to the range of the values observed in the reference soybean varieties grown in the trials and in the published scientific literature (ILSI, 2010).

No statistically significant differences were observed between forage and seed samples of the unmodified control soybean variety and soybean event SYHT0H2, either untreated or herbicide-treated, for protein, fat, ash, carbohydrates, ADF and NDF, except for levels of fat in forage samples from herbicide-treated plants and ADF from seed samples from herbicide-treated plants. These differences were not biologically meaningful as all means for these samples were within the range of the values observed in the reference soybean varieties grown in the trials and in the published scientific literature (ILSI, 2010).

Statistically significant differences were observed between seed samples from both untreated and herbicide-treated plants of soybean event SYHT0H2 and the unmodified control soybean variety for aspartic acid, glutamic acid, proline, methionine, phenylalanine, lysine, histidine and arginine. Statistically significant differences were also observed between seed samples from unsprayed plants for threonine, serine, alanine, leucine and tyrosine. These differences were not biologically meaningful as all means for these samples were within the range of the values observed in the reference soybean varieties grown in the trials and in the published scientific literature (ILSI, 2010). Statistically significant differences were observed between seed samples from both untreated and herbicide-treated plants of soybean event SYHT0H2 and the unmodified control soybean variety for palmitic, stearic, oleic, linoleic, arachidic and behenic acids. Statistically significant differences were also observed between unsprayed plants only of soybean event SYHT0H2 and the unmodified control soybean variety for linolenic acid. These differences were not biologically meaningful as all means for these samples were within the range of the values observed in the reference soybean varieties grown in the trials and in the published scientific literature (ILSI, 2010). No statistically significant differences were observed between seed samples of untreated and herbicide-treated plants of soybean event SYHT0H2 and the unmodified control soybean variety for vitamins or minerals except for vitamin K1 levels in herbicide-treated plants and gamma tocopherol, delta tocopherol, iron and potassium levels in both untreated and herbicide-treated plants. These differences were not biologically meaningful as all means for these samples were within the range of the values observed in the reference soybean varieties grown in the trials and in the published scientific literature (ILSI, 2010). No statistically significant differences were observed between seed samples of untreated and herbicide-treated plants of soybean event SYHT0H2 and the unmodified control soybean variety for anti-nutrients.

Broiler performance:

A feeding study was performed to examine the health and performance of birds from feeding meals from soybean event SYHT0H2 meal, the unmodified control soybean variety or a reference soybean variety. Diets were prepared with 29.0% to 33.5% meal from each soybean and fed to groups of 60 male and 60 female birds for 42 consecutive days. The parameters evaluated were survival, body weight, feed conversion and carcass yield. Broiler chickens fed diets prepared with soybean event SYHT0H2 meal did not exhibit any adverse or unexpected effects in comparison with chickens fed diets prepared with the other soybeans and performance over the 42-day test period did not differ significantly (ANOVA, P > 0.05) between chickens fed diets containing soybean event SYHT0H2 meal and chickens fed diets formulated with meal from the other soybeans. A statistically significant interaction between diet and gender was observed for thigh weight, however, in pairwise comparisons between males, thigh weight differed significantly only between male chickens fed soybean event SYHT0H2 meal and those fed meal from the reference soybean variety. The soybean event SYHT0H2 and the unmodified control soybean variety groups did not differ significantly and no effect of diet on thigh weight was detected in females so these results were not considered biologically significant. No significant diet-related differences between the SYHT0H2 and control groups were observed in the other carcass measurements.

Conclusion:

The CFIA concluded, based on the evidence provided by Syngenta Canada Inc. and Bayer CropScience Inc., that the nutritional composition of soybean event SYHT0H2 is similar to that of the reference soybean varieties grown in the trials and to that reported for other soybeans in the published scientific literature. Feed ingredients derived from soybean event SYHT0H2 are considered to meet present ingredient definitions for soybean.

2. Potential Impact of Soybean Event SYHT0H2 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

Soybean event SYHT0H2 is tolerant to the HPPD-inhibiting herbicides mesotrione and, isoxaflutole and to glufosinate-ammonium herbicide due to production of the AvHPPD-03 and PAT proteins, respectively. The assessment of soybean event SYHT0H2 evaluated the impact of the following potential hazards relative to the safety of feed ingredients derived from this event:

  • The presence of novel proteins AvHPPD-03 and PAT
  • The chemical pesticide residue profile.

Novel AvHPPD-03 protein:

To obtain sufficient quantities of AvHPPD-03 protein for assessment of environmental and feed safety, it was necessary to express the avhppd-03 gene in an E. coli production system. Equivalency was demonstrated between the soybean event SYHT0H2-produced AvHPPD-03 protein and the E. coli-produced AvHPPD-03 protein by comparing their molecular weights, immunoreactivity, glycosylation, N-terminal sequence analysis, MALDI-TOF mass spectrometry and functional activity. Based on the results, the proteins were found to be equivalent. Demonstration of equivalence between the AvHPPD-03 protein produced in E. coli and the AvHPPD-03 protein produced in soybean event SYHT0H2 allows the AvHPPD-03 protein produced in E. coli to be used in studies to confirm the safety of the AvHPPD-03 protein produced in soybean event SYHT0H2.

The potential allergenicity and toxicity of the novel AvHPPD-03 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 avhppd-03 gene, A. sativa, is not known to produce allergens and a bioinformatics evaluation of the AvHPPD-03 protein amino acid sequence confirmed the lack of relevant similarities between the AvHPPD-03 protein and known allergens. E. coli-produced AvHPPD-03 protein safety studies indicated that, unlike many allergens, this protein is rapidly degraded in simulated gastric fluid and is not heat stable and, unlike many allergens, the soybean event SYHT0H2 AvHPPD-03 protein was shown to be nonglycosylated. The weight of evidence thus indicates that the AvHPPD-03 protein is unlikely to be allergenic.

In terms of its potential toxicity to livestock, the AvHPPD-03 protein lacks a mode of action to suggest that it is intrinsically toxic to livestock and a bioinformatics evaluation of the AvHPPD-03 protein amino acid sequence confirmed the lack of relevant similarities between the AvHPPD-03 protein and known toxins. In addition, E. coli-produced AvHPPD-03 protein safety studies indicated that no adverse effects were observed when the AvHPPD-03 protein was ingested by mice at doses of approximately 2000 mg/kg bwt. This information indicates that the AvHPPD-03 protein is unlikely to be toxic to livestock.

The livestock exposure to the AvHPPD-03 protein is expected to be negligible as the AvHPPD-03 protein is expressed at very low levels in soybean event SYHT0H2 and is rapidly degraded under conditions which simulate the mammalian digestive tract.

Novel PAT protein:

Soybean event SYHT0H2 did not express the PAT protein in sufficient quantities for evaluation of environmental and feed safety, however, purified E. coli-expressed PAT protein was previously characterized in a published study by Hérouet et al. (2005). Equivalency was demonstrated between the soybean event SYHT0H2-produced PAT protein and the E. coli-produced PAT protein by comparing their molecular weights, immunological reactivities, glycosylation status, peptide mapping profiles, N-terminal amino acid sequence and enzyme activities. Based on the results, the proteins were found to be equivalent. Demonstration of equivalence between the PAT protein produced in E. coli and the PAT protein produced in soybean event SYHT0H2 allows the use of the published study by Hérouet et al. (2005) to support the safety of the PAT protein produced in soybean event SYHT0H2.

The potential allergenicity and toxicity of the PAT 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 pat gene, S. viridochromogenes, is not known to produce allergens and a bioinformatics evaluation of the PAT protein amino acid sequence confirmed the lack of relevant similarities between the PAT protein and known allergens. Studies with E. coli-produced PAT protein that had previously been characterized by Hérouet et al. (2005) indicated that, unlike many allergens, this protein is rapidly degraded in simulated gastric fluid and is not heat stable. Also, unlike many allergens, soybean event SYHT0H2 PAT protein was shown experimentally to be non-glycosylated. The weight of evidence thus indicates that the PAT protein is unlikely to be allergenic.

In terms of its potential toxicity to livestock, the PAT protein lacks a mode of action to suggest that it is intrinsically toxic to livestock and a bioinformatics evaluation of the PAT protein amino acid sequence confirmed the lack of relevant similarities between the PAT protein and known toxins. In addition, studies with E. coli-produced PAT protein that had previously been characterized by Hérouet-Guicheney et al. (2005) indicated that no adverse effects were observed when the PAT protein was ingested by mice at doses of approximately 2000 mg/kg bwt. This information indicates that the PAT protein is unlikely to be toxic to livestock.

The livestock exposure to the PAT protein is expected to be negligible as the PAT protein is expressed at very low levels in Soybean event SYHT0H2 and is rapidly degraded under conditions which simulate the mammalian digestive tract.

Chemical pesticide residue profile:

The safety of herbicide residues and metabolites in soybean event SYHT0H2, following application of herbicides, was also evaluated as part of the feed safety assessment. It was determined that that potential glufosinate-ammonium residues and metabolites in livestock feeds derived from soybean event SYHT0H2 would not present levels of concern to livestock, nor humans via the potential transfer into foods of animal origin.

It was also determined that potential mesotrione or isoxaflutole residues in livestock feeds derived from soybean event SYHT0H2, except forage and hay, would not present levels of concern to livestock, nor humans via the potential transfer into foods of animal origin. No authorization for forage or hay produced from soybean event SYHT0H2 treated with HPPD-inhibiting herbicides has been granted at this time, as there were not sufficient data available to support the inclusion of HPPD-inhibiting herbicide-treated forage and hay as feed. Syngenta Canada Inc. and Bayer CropScience Inc. have indicated that they will restrict the feeding of soybean event SYHT0H2 forage and hay on the Canadian mesotrione and isoxaflutole labels. The exemption for the provision of residue data is supported by current agricultural practices indicating the negligible usage of soybean by-products for forage and hay as feed.

Conclusion:

It was concluded, based on the evidence provided by Syngenta Canada Inc. and Bayer CropScience Inc., that the novel PAT and AvHPPD-03 protein-based herbicide tolerance traits will not confer to soybean event SYHT0H2 any characteristic that would raise concerns regarding the safety of soybean event SYHT0H2. Feed ingredients, with the exception of forage and hay, derived from soybean event SYHT0H2 are considered to meet present ingredient definitions for soybean.

VI. New Information Requirements

If at any time, Syngenta Canada Inc. or Bayer CropScience 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 soybean event SYHT0H2 or lines derived from it, the company which becomes aware of the new information 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 soybean event SYHT0H2 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 soybean event SYHT0H2.

VII. Regulatory Decision

Based on the review of the data and information submitted by Syngenta Canada Inc. and Bayer CropScience 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 soybean event SYHT0H2 does not present altered environmental risk when compared to soybean varieties that are currently grown in Canada.

Based on the review of the data and information submitted by Syngenta Canada Inc. and Bayer CropScience Inc. and input from other relevant scientific sources, the Animal Feed Division of the Animal Health Directorate, CFIA, has concluded that the novel PAT and AvHPPD-03 protein-based herbicide tolerance traits will not confer to soybean event SYHT0H2 any characteristic that would raise concerns regarding the safety or nutrition of soybean event SYHT0H2. Livestock feeds derived from soybeans are currently listed in Schedule IV of The Feeds Regulations. Soybean event SYHT0H2 has been found to be as safe as and as nutritious as currently and historically grown soybean varieties. Soybean event SYHT0H2 and its products are considered to meet present ingredient definitions.

Unconfined release into the environment and use as livestock feed of soybean event SYHT0H2 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 June 4, 2014. Any soybean lines derived from soybean event SYHT0H2 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 soybean varieties that are currently grown and permitted to be used as livestock feed in Canada, in terms of their potential environmental impact and livestock feed safety and nutrition, (iv) the novel genes are expressed at levels similar to those in the authorized line and (v) data used to establish the substantial equivalence of lines derived from soybean event SYHT0H2 be made available to the CFIA upon request.

Additionally, with respect to its use as livestock feed, soybean event SYHT0H2 must meet the restrictions specific to HPPD-inhibiting herbicide-treated forage and hay set out in the authorization.

Soybean event SYHT0H2 is subject to the same phytosanitary import requirements as unmodified soybean varieties. Soybean event SYHT0H2 is required to meet the requirements of other Canadian legislation, including but not limited to the requirements set out in the Food & 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 soybean event SYHT0H2.

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