Decision Document DD2013-97
Determination of the Safety of Dow AgroSciences Canada Inc.'s Soybean (Glycine max (L.) Merr.) Event DAS-44406-6

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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.) Merr. (Soybean) 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 Dow AgroSciences Canada Inc. This information is in regard to the herbicide tolerant soybean event DAS-44406-6. 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 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 DAS-44406-6 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 6, 2013. Any soybean lines derived from soybean event DAS-44406-6 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 (iv) the novel genes are expressed at a level similar to that of the authorized line.

Additionally, with respect to its use as livestock feed, soybean event DAS-44406-6 must meet the restrictions specific to 2,4-dichlorophenoxyacetic acid (2,4-D)-treated forage and hay set out in the authorization.

Soybean event DAS-44406-6 is subject to the same phytosanitary import requirements as unmodified soybean varieties. Soybean event DAS-44406-6 is required to meet the requirements of other jurisdictions, including but not limited to, the Food & Drugs Act and the Pest Control Products Act.

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

(publié aussi en français)

June 6, 2013

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

1-800-442-2342
59 Camelot Drive, Ottawa
Ontario K1A 0Y9

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 2,4-Dichlorophenoxyacetic Acid (2,4-D)
    3. Tolerance to Glufosinate-Ammonium
    4. Tolerance to Glyphosate
    5. Stable Integration into the Plant Genome
  4. Criteria for the Environmental Assessment
    1. Potential of Soybean Event DAS-44406-6 to Become a Weed of Agriculture or be Invasive of Natural Habitats
    2. Potential for Gene Flow from Soybean Event DAS-44406-6 to Sexually Compatible Plants Whose Hybrid Offspring May Become More Weedy or More Invasive
    3. Altered Plant Pest Potential of Soybean Event DAS-44406-6
    4. Potential Impact of Soybean Event DAS-44406-6 on Non-Target Organisms
    5. Potential Impact of Soybean Event DAS-44406-6 on Biodiversity
  5. Criteria for the Livestock Feed Assessment
    1. Potential Impact of Soybean Event DAS-44406-6 on Livestock Nutrition
    2. Potential Impact of Soybean Event DAS-44406-6 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 DAS-44406-6
OECD Unique Identifier DAS-44406-6
Applicant: Dow AgroSciences Canada Inc.
Plant Species: Soybean (Glycine max L.)
Novel Traits: Tolerance to 2,4-dichlorophenoxyacetic acid (2,4-D) herbicide; tolerance to glufosinate-ammonium herbicide; tolerance to glyphosate herbicide
Trait Introduction Method: Agrobacterium-mediated transformation
Intended Use of the Modified Plant: Soybean event DAS-44406-6 is intended to be grown for traditional soybean human food and livestock feed uses. Soybean event DAS-44406-6 is not intended to be grown outside the normal production area for soybean in Canada.

II. Background Information

Dow AgroSciences Canada Inc. has developed a soybean event that is tolerant to 2,4-dichlorophenoxyacetic acid (2,4-D), glufosinate-ammonium and glyphosate herbicides.

Soybean event DAS-44406-6 was developed by Dow AgroSciences Canada Inc. using recombinant deoxyribonucleic acid (DNA) technology, resulting in the introduction of the aryloxyalkanoate dioxygenase-12 (aad-12) gene, the phosphinothricin acetyltransferase (pat) gene and a modified 5-enolpyruvylshikimate-3-phosphate synthase (2mepsps) gene. The aad-12 gene is derived from the soil bacterium Delftia acidovorans (D. acidovorans) and encodes an aryloxyalkanoate dioxygenase-12 (AAD-12) enzyme which inactivates the herbicide 2,4-D. 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. The 2mepsps gene is derived from corn and encodes a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme which contains two amino acid mutations that allow the enzyme to function in the presence of the herbicide glyphosate.

Dow AgroSciences Canada Inc. has provided information on the identity of soybean event DAS-44406-6, a detailed description of the transformation method 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. 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 provided for the evaluation of herbicide residues in the feed commodities derived from the crop, following the intended herbicide application.

Soybean event DAS-44406-6 was field tested at ten 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.

Agronomic characteristics of soybean event DAS-44406-6, such as early population, seedling vigor, days to 50% flowering, disease incidence, insect damage, days to maturity, lodging, plant height, final population, number of pods, number of seeds, shattering, yield and 100 seed weight, were compared to those of an unmodified control soybean variety, which shares the same genetic background as soybean event DAS-44406-6 but has not been modified. Several conventional control soybean varieties were also included in these trials to establish a reference range for typical soybean behaviour.

Nutritional components of soybean event DAS-44406-6 grain and forage, such as proximate, amino acids, fatty acids, vitamins, minerals and anti-nutrients were compared with those of the unmodified control soybean variety and the conventional control 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 DAS-44406-6 to become a weed of agriculture or to be invasive of natural habitats;
  • the potential for gene flow from soybean event DAS-44406-6 to sexually compatible plants whose hybrid offspring may become more weedy or more invasive;
  • the potential for soybean event DAS-44406-6 to become a plant pest;
  • the potential impact of soybean event DAS-44406-6 and its gene products on non-target organisms, including humans; and
  • the potential impact of soybean event DAS-44406-6 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 efficacy 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 DAS-44406-6 and unmodified soybean varieties relative to the safety and efficacy of feed ingredients derived from soybean event DAS-44406-6 for their intended purpose, including:

  • the potential impact of soybean event DAS-44406-6 on livestock nutrition; and
  • the potential impact of soybean event DAS-44406-6 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 soybean event DAS-44406-6 meet the definitions and requirements of feeds as listed in Schedule IV of the Feeds Regulations.

Dow AgroSciences Canada Inc. has provided the CFIA with a method for the detection and identification of soybean event DAS-44406-6.

III. Description of the Novel Traits

1. Development Method

Soybean event DAS-44406-6 was developed through Agrobacterium-mediated transformation of soybean cells and contains the aad-12, pat and 2mepsps genes and associated regulatory elements. Transformed cells were selected on the basis of tolerance to glufosinate-ammonium and regenerated to produce plants. Soybean event DAS-44406-6 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 2,4-Dichlorophenoxyacetic Acid (2,4-D)

The herbicide 2,4-D is a synthetic auxin, which disrupts new cell growth thus inhibiting new growth in susceptible plants. Soybean event DAS-44406-6 contains the aad-12 gene from the gram-negative soil bacterium D. acidovorans, which encodes AAD-12, an enzyme that degrades the herbicide 2,4-D into the inactive form 2,4-dichlorophenol (DCP). Introduction of the aad-12 gene into soybean event DAS-44406-6 therefore confers commercial-level tolerance to the herbicide 2,4-D.

The AAD-12 protein produced in soybean event DAS-44406-6 is identical to the native enzyme except for the addition of an alanine at position number 2, which was the result of DNA modifications during cloning. The AAD-12 protein has been subject to a previous CFIA safety assessment in Dow AgroSciences Canada Inc.'s soybean event DAS-68416-4 (DD2012-93).

AAD-12 protein expression in soybean event DAS-44406-6 is driven by a constitutive promoter. Samples of soybean tissues were collected from plants from 10 field trials in the US. Tissues were collected from unsprayed plants and plants sprayed with 2,4-D, glufosinate-ammonium, glyphosate or all three herbicides. The average AAD-12 protein expression levels in micrograms of protein per gram of dry weight tissue (μg/g dwt) from unsprayed plants, as evaluated by enzyme-linked immunosorbent assay (ELISA), were as follows: 112.61 μg/g dwt in leaf at the V5 stage, 118.57 μg/g dwt in leaf at the V10-12 stage, 23.52 μg/g dwt in root, 73.47 μg/g dwt in forage and 27.37 μg/g dwt in grain. Very similar AAD-12 protein levels were observed in the tissues of herbicide-treated plants.

The potential allergenicity and toxicity of the AAD-12 protein to livestock and non-target organisms were evaluated. The weight of evidence indicates that the AAD-12 protein is unlikely to be allergenic. The source of the aad-12 gene, D. acidovorans, is not known to produce allergens, the AAD-12 protein amino acid sequence lacks relevant similarities to those of known allergens and microbial AAD-12 protein was shown experimentally to be rapidly degraded in simulated gastric fluid and not to be heat stable. It was also concluded that the AAD-12 protein is unlikely to be toxic to livestock or 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 AAD-12 protein amino acid sequence lacks relevant similarities to known toxins and because no adverse effects were observed when microbial AAD-12 protein was ingested by mice at doses of approximately 2000 milligrams protein per kilograms body weight (mg/ kg bwt). For a more detailed discussion of the potential allergenicity and toxicity of the AAD-12 protein, see Section V, part 2.

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 DAS-44406-6 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 DAS-44406-6 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 DAS-44406-6 is identical to the native enzyme.

The PAT protein produced in soybean event DAS-44406-6 is the same as the PAT protein produced in other glufosinate-ammonium-tolerant crops that have already been approved for unconfined release and animal feed use in Canada, including cotton events 3006-201-23 (DD2005-51) and 281-24-236 (DD2005-52), corn events 1507 (DD2002-41) and 59122 (DD2005-55) and soybean event DAS-68416-4 (DD2012-93).

PAT protein expression in soybean event DAS-44406-6 is driven by a constitutive promoter. Samples of soybean tissues were collected from plants from 10 field trials in the US. Tissues were collected from unsprayed plants and plants sprayed with 2,4-D, glufosinate-ammonium, glyphosate or all three herbicides. The average PAT protein expression in μg/g dwt from unsprayed plants, as evaluated by ELISA, was as follows: 8.98 μg/g dwt in leaf at the V5 stage, 10.59 μg/g dwt in leaf at the V10-12 stage, 1.56 μg/g dwt in root, 6.19 μg/g dwt in forage and 2.12 μg/g dwt in grain. Very similar PAT protein levels were observed in the tissues of herbicide-treated plants.

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. The source of the pat gene, S. viridochromogenes, is not known to produce allergens, the PAT protein amino acid sequence lacks relevant similarities to known allergens and microbial PAT protein was shown experimentally to be rapidly degraded in simulated gastric fluid. It was also concluded that the PAT protein was unlikely to be toxic to livestock or non-target organisms because it lacks a mode of action to suggest that it is intrinsically toxic to livestock and non-target organisms, because the PAT protein amino acid sequence lacks relevant similarities to known toxins and because no adverse effects were observed when microbial PAT protein was ingested by mice at doses of approximately 5000 mg/kg bwt. For a more detailed discussion of the potential allergenicity and toxicity of the PAT protein, see Section V, part 2.

4. 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 enzyme, leading to interference in aromatic amino acids production and growth suppression or death of the plant. The native soybean EPSPS enzyme is sensitive to glyphosate. The modified 5-enolpyruvylshikimate-3-phosphate synthase (2mEPSPS) version of this enzyme expressed in soybean event DAS-44406-6 contains two amino acid mutations that reduce binding of the 2mEPSPS enzyme to glyphosate, compared to native soybean EPSPS. The 2mEPSPS confers commercial-level glyphosate tolerance to soybean event DAS-44406-6 since it continues to catalyze the production of aromatic amino acids in the presence of glyphosate.

The expression of the 2mEPSPS in soybean event DAS-44406-6 is driven by a constitutive promoter. Samples of soybean event DAS-44406-6 tissues were collected from 10 field trial sites in the US. Tissues were collected from unsprayed plants and plants sprayed with 2,4-D, glufosinate-ammonium, glyphosate or all three herbicides. Average 2mEPSPS expression levels from unsprayed plants, as evaluated by ELISA, were as follows: 2368.16 μg/g dwt in leaf at the V5 stage, 2583.46 μg/g dwt in leaf at the V10-12 stage, 89.71 μg/g dwt in root, 357.09 μg/g dwt in forage and 21.97 μg/g dwt in grain. Very similar 2mEPSPS protein levels were observed in the tissues of herbicide-treated plants.

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, microbial 2mEPSPS protein was shown experimentally to be rapidly degraded in simulated gastric fluid and not to be heat stable and soybean event DAS-44406-6 2mEPSPS protein was shown experimentally to be unglycosylated. 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 microbial 2mEPSPS protein was ingested by mice at doses of approximately 5000 mg/kg bwt. For a more detailed discussion of the potential allergenicity and toxicity of the 2mEPSPS protein, see Section V, part 2.

5. Stable Integration into the Plant Genome

Molecular characterization by Southern blot analysis demonstrated that soybean event DAS-44406-6 contains one intact copy of the gene cassette containing the aad-12, pat and 2mepsps genes and their regulatory elements inserted at a single site in the soybean 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 soybean event DAS-44406-6.

The stability of the insert within soybean event DAS-44406-6 was verified by Southern blot analysis and by detection of the PAT protein over five generations. The inheritance pattern of the insert and the PAT protein expression trait across five segregating generations of soybean event DAS-44406-6 showed that the insert segregates according to Mendelian rules of inheritance for a single genetic locus.

IV. Criteria for the Environmental Assessment

1. Potential of Soybean Event DAS-44406-6 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 traits such as the lack of seed dormancy and the poor competitive ability of seedlings. According to the information provided by Dow AgroSciences Canada Inc., soybean event DAS-44406-6 was determined not to be significantly different from unmodified soybean varieties in this respect.

The CFIA evaluated data submitted by Dow AgroSciences Canada Inc. on the reproductive biology and life history traits of soybean event DAS-44406-6. This event was field tested in the US at 10 locations in 2010. 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 DAS-44406-6 was compared to the unmodified control soybean variety. Conventional control soybean varieties were also included in these trials to establish ranges of comparative values that are representative of currently grown soybean varieties. Soybean event DAS-44406-6 plants were either unsprayed or sprayed with 2,4-D, glufosinate-ammonium, glyphosate or all three 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 population, seedling vigor, days to 50% flowering, days to maturity, lodging, plant height, final population, number of pods, number of seeds, shattering, yield and 100 seed weight. Although instances of statistically significant differences were observed between soybean event DAS-44406-6 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 and the values for soybean event DAS-44406-6 were within the reference range established for the conventional control soybean varieties included in the same field trials. Therefore, the statistical analysis of these observations showed no biologically meaningful differences between soybean event DAS-44406-6 and the unmodified control soybean variety and supports a conclusion of phenotypic and agronomic equivalence to currently grown soybean varieties.

Dow AgroSciences Canada Inc. evaluated the germination of soybean event DAS-44406-6 seed under warm and cool temperature regimes. No significant difference was detected between soybean event DAS-44406-6 and the unmodified control soybean variety at either 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 DAS-44406-6 was exposed to heat, drought and excessive moisture in the field during the agronomic characteristic studies. No trend in increased or decreased susceptibility to these abiotic stressors was observed in soybean event DAS-44406-6 compared to the unmodified control soybean variety.

The susceptibility of soybean event DAS-44406-6 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 3: Altered Plant Pest Potential of Soybean event DAS-44406-6). No trend in increased or decreased susceptibility to pests or pathogens was observed in soybean event DAS-44406-6 compared to the unmodified control soybean variety.

No competitive advantage was conferred to plants of soybean event DAS-44406-6, other than that conferred by tolerance to the 2,4-D , glufosinate-ammonium and glyphosate herbicides, as the reproductive characteristics, growth characteristics and tolerance to abiotic and biotic stresses of soybean event DAS-44406-6 were comparable to those of the unmodified control soybean variety. Tolerance to 2,4-D, glufosinate-ammonium and glyphosate herbicides provides a competitive advantage only when these herbicides are used and will not, in and of itself, make a herbicide tolerant plant weedier or more invasive of natural habitats. Soybean event DAS-44406-6 plants growing as volunteers will not be controlled if 2,4-D, glufosinate-ammonium and/or glyphosate herbicides are used as the only weed control tools. However, control of soybean event DAS-44406-6 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 DAS-44406-6 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, an 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.

Dow AgroSciences Canada Inc. has submitted an herbicide tolerance management plan to the CFIA, which was determined to be satisfactory when evaluated by the PBRA Unit.

Dow AgroSciences Canada Inc. will make this herbicide tolerance management plan readily available to growers and agriculture extension personnel, in both private and public sectors, to promote careful management practices for soybean event DAS-44406-6. Dow AgroSciences Canada Inc. will provide an efficient mechanism for growers to report agronomic problems to the company, which will facilitate the ongoing monitoring of soybean event DAS-44406-6. Dow AgroSciences Canada Inc. will monitor grower implementation to determine the effectiveness of the herbicide tolerance management plan and make any changes to the plan as appropriate.

2. Potential for Gene Flow from Soybean Event DAS-44406-6 to Sexually Compatible Plants Whose Hybrid Offspring May Become More Weedy or More Invasive

Natural hybridization between cultivated soybean and the wild annual species Glycine soja (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 DAS-44406-6 to related species in Canada.

3. Altered Plant Pest Potential of Soybean Event DAS-44406-6

Soybean is not considered a plant pest in Canada and the 2,4-D, glufosinate-ammonium and glyphosate herbicide tolerance traits introduced into soybean event DAS-44406-6 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 DAS-44406-6 to various soybean pests and pathogens was evaluated in the field at the same locations as the agronomic characteristic studies. The stressors observed included grasshoppers, brown leaf beetles, stink bugs, bean leaf beetles, salt marsh caterpillars, fall armyworm, frogeye leaf spot, Septoria, Septoria brown spot, bacterial blight, mold and bean pod mottle virus. The evaluations of soybean event DAS-44406-6 did not show any increase or decrease in susceptibility to these pests and pathogens compared to the unmodified control soybean variety.

The CFIA has therefore concluded that soybean event DAS-44406-6 does not display any altered plant pest potential compared to currently grown soybean varieties.

4. Potential Impact of Soybean Event DAS-44406-6 on Non-Target Organisms

The 2,4-D, glufosinate-ammonium and glyphosate herbicide tolerance traits introduced into soybean event DAS-44406-6 are unrelated to a potential impact on non-target organisms.

Detailed characterization of the AAD-12, PAT and 2mEPSPS proteins expressed in soybean event DAS-44406-6 led to the conclusion that none of these proteins displays any characteristic of a potential toxin or allergen (see Section III). Therefore, no negative impacts resulting from exposure of organisms to the AAD-12, PAT and 2mEPSPS proteins expressed in soybean event DAS-44406-6 are expected.

Composition analyses showed that the levels of key nutrients and anti-nutrients in grain and forage from soybean event DAS-44406-6 are comparable to those in conventional soybean varieties (See Section V, part 1. Potential Impact of Soybean Event DAS-44406-6 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 DAS-44406-6 tissues that would negatively impact organisms interacting with soybean event DAS-44406-6.

Field evaluations of soybean event DAS-44406-6 did not show any increased resistance to insect pests or pathogens compared to the unmodified control soybean variety (see Section 3. Altered Plant Pest Potential of Soybean Event DAS-44406-6).

Collectively, these information elements indicate that the interactions between soybean event DAS-44406-6 and the populations of animals and microorganisms interacting with soybean crops will be similar compared to currently grown soybean varieties.

The CFIA has therefore determined that the unconfined release of soybean event DAS-44406-6 in Canada will not result in altered impacts on non-target organisms, including humans, compared to currently grown soybean varieties.

5. Potential Impact of Soybean Event DAS-44406-6 on Biodiversity

Soybean event DAS-44406-6 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 DAS-44406-6 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 DAS-44406-6 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 DAS-44406-6 has tolerance to 2,4-D, glufosinate-ammonium and glyphosate 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 DAS-44406-6 or the cultivation of soybean. It is therefore unlikely that soybean event DAS-44406-6 will have any indirect effects on biodiversity, in comparison to the effects that would be expected from cultivation of currently grown soybean varieties.

The CFIA has concluded that the introduced genes and their corresponding novel traits do not confer to soybean event DAS-44406-6 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 DAS-44406-6 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 DAS-44406-6, 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 DAS-44406-6 meet the definitions and requirements of feeds as listed in Schedule IV of the Feeds Regulations.

1. Potential Impact of Soybean Event DAS-44406-6 on Livestock Nutrition

Nutrient and anti-nutrient composition:

The nutritional equivalence of soybean event DAS-44406-6 plants (unsprayed plants and plants sprayed with 2,4-D, glufosinate-ammonium, glyphosate or all three herbicides), to those of the unsprayed, unmodified control soybean variety and six unsprayed, conventional control soybean varieties was determined from 10 replicated field trials in the US during the 2010 growing season. Forage and seed samples were analysed for proximate (crude fat, ash, protein and moisture), acid detergent fibre (ADF), neutral detergent fibre (NDF), calcium and phosphorus. Seed samples were further analysed for amino acids, fatty acids, vitamins, minerals, isoflavones (diadzein, genistein, glycitein) and anti-nutrients (phytic acid, trypsin inhibitor, lectin, raffinose and stachyose) as recommended by the OECD consensus document for new varieties of soybean (OECD, 2001 – PDF (190 kb)). Composition data was analysed statistically using a mixed model analysis of variance and statistical differences among treatments were identified and assessed (P<0.05). The biological relevance of any significant difference among soybean varieties was assessed by comparing the observed values to the range of the values observed in the conventional control soybean varieties grown in the trials and in the published scientific literature (ILSI, 2010).

No statistically significant differences were observed between the unmodified control soybean variety and soybean event DAS-44406-6 forage for protein, fat, ash, carbohydrates, ADF, NDF, calcium and phosphorus. Statistically significant effects were found between soybean event DAS-44406-6 (sprayed with all three herbicides) and the unmodified control soybean variety for protein and NDF, however all means were within the range of the values observed in the conventional control soybean varieties grown in the trials and in the published scientific literature (ILSI, 2010). No statistically significant differences were observed between the unmodified control soybean variety and soybean event DAS-44406-6 for copper, iron, magnesium, manganese, phosphorus and selenium. Statistically significant differences were observed for potassium and calcium but the mean differences were negligible and not biologically meaningful, as means were within the range of the values observed in the conventional control soybean varieties grown in the trials and in the published scientific literature (ILSI, 2010). Except for cystine, no statistically significant differences were found between the novel crop and the unmodified control soybean variety for the amino acids. Means levels of cystine were within the range of the values observed in the conventional control soybean varieties grown in the trials and in the published scientific literature (ILSI, 2010). Statistically significant differences were observed between soybean event DAS-44406-6 (sprayed and unsprayed) and the unmodified control soybean variety for palmitic, oleic, linoleic and linolenic acids, however the mean differences were negligible and not biologically meaningful as the means were within the range of the values observed in the conventional control soybean varieties grown in the trials and in the published scientific literature (ILSI, 2010). No statistically significant differences were observed between the unmodified control soybean variety and soybean event DAS-44406-6 for vitamins B1, B2, B3, B5, B6, vitamin C, E and δ-tocopherol. Vitamins B9, γ-tocopherol and total tocopherol were significantly different for some of DAS-44406-6 treatments compared with the unmodified control soybean variety, but the means were with the range for the currently grown soybean varieties. No statistically significant differences were observed between soybean event DAS-44406-6 and the unmodified control soybean variety for isoflavones (diadzein, genistein and glycitein). There were statistically significant differences between soybean event DAS-44406-6 (unsprayed and sprayed with all three herbicides) and the unmodified control soybean variety for lectin and between soybean event DAS-44406-6 (sprayed with 2,4-D and glufosinate) and the unmodified control soybean variety for trypsin inhibitor, however all means within the range of the values observed in the conventional control soybean varieties grown in the trials and in the published scientific literature (ILSI, 2010), therefore the differences were not considered biologically meaningful.

Conclusion:

It was concluded, based on the evidence provided by Dow AgroSciences Canada Inc., that the nutritional composition of soybean event DAS-44406-6 is similar to that of the conventional control soybean varieties grown in the trials and to that reported for other soybeans in the published scientific literature.

2. Potential Impact of soybean event DAS-44406-6 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 DAS-44406-6 is tolerant to 2,4-D, glufosinate-ammonia and glyphosate herbicides due to production of the AAD-12, PAT and 2mEPSPS proteins, respectively. The assessment of soybean event DAS-44406-6 evaluated the impact of the following potential hazards relative to the safety of feed ingredients derived from this event:

  • The presence of novel proteins AAD-12, PAT and 2mEPSPS
  • The chemical pesticide residue profile

Novel AAD-12 protein

To obtain sufficient quantities of AAD-12 protein for evaluation of environmental and feed safety, it was necessary to express the aad-12 gene in a microbial production system. Equivalency was demonstrated between soybean event DAS-44406-6-produced AAD-12 protein and a microbial-produced AAD-12 protein that had been used in studies previously submitted for Dow AgroSciences Canada Inc.'s soybean event DAS-68416-4 (DD2012-93), based on similar molecular weights, immunoreactivities, glycosylation status, N-terminal and C-terminal sequences and tryptic peptide mass mapping results. Demonstration of equivalence between the AAD-12 protein produced in soybean event DAS-44406-6 and the microbially-produced AAD-12 protein used in studies submitted for soybean event DAS-68416-4 allows utilization of information from these studies to confirm the safety of the AAD-12 protein produced in soybean event DAS-44406-6.

The potential allergenicity and toxicity of the AAD-12 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 aad-12 gene, D. acidovorans, is not known to produce allergens and a bioinformatics evaluation of the AAD-12 protein amino acid sequence confirmed the lack of relevant similarities between the AAD-12 protein and known allergens. Unlike many allergens, studies with the microbial AAD-12 protein indicated that the protein is rapidly degraded in simulated gastric fluid and is not heat stable. The weight of evidence thus indicates that the AAD-12 protein is unlikely to be allergenic.

In terms of its potential toxicity to livestock, the AAD-12 protein lacks a mode of action to suggest that it is intrinsically toxic to livestock and a bioinformatics evaluation of the AAD-12 protein amino acid sequence confirmed the lack of relevant similarities between the AAD-12 protein and known toxins. In addition, microbial AAD-12 protein safety studies previously provided for soybean event DAS-68416-4 indicated that no adverse effects were observed when the AAD-12 protein was ingested by mice at doses of approximately 2000 mg/kg bwt. This information indicates that the AAD-12 protein is unlikely to be toxic to livestock.

The livestock exposure to the AAD-12 protein is expected to be negligible as the protein is expressed at very low levels in soybean event DAS-44406-6, is rapidly degraded under conditions which simulate the mammalian digestive tract and is unstable under heating conditions expected to be encountered during processing of some soybean products.

Novel PAT protein

To obtain sufficient quantities of PAT protein for evaluation of environmental and feed safety, it was necessary to express the pat gene in a microbial production system. Equivalency was demonstrated between soybean event DAS-44406-6-produced PAT protein and a microbial-produced PAT protein that had been used in studies previously submitted for Dow AgroSciences Canada Inc.'s cotton event 3006-210-23 (DD2005-51), based on similar molecular weights and immunoreactivities, as well as the DNA sequence of the pat gene in soybean event DAS-44406-6. Demonstration of equivalence between the PAT protein produced in soybean event DAS-44406-6 and the microbially-produced PAT protein used in studies submitted for cotton event 3006-210-23 allows utilization of information from these studies to confirm the safety of the PAT protein produced in soybean event DAS-44406-6.

The potential mammalian allergenicity and toxicity of the PAT protein were evaluated. With respect to its potential allergenicity, it is recognized that no single experimental method yields decisive evidence for allergenicity, thus a weight-of-evidence approach was taken, taking into account information obtained with various test methods. The source of the pat gene protein, S. viridochromogenes, is not known to produce allergens and a bioinformatics evaluation of the PAT amino acid sequence confirmed the lack of relevant similarities between the PAT protein and known allergens. Unlike many allergens, microbial PAT safety studies previously provided for cotton event 3006-210-23 indicated that the PAT protein is readily degraded in simulated mammalian gastric fluid. The weight of evidence thus indicates that the PAT protein is unlikely to be allergenic.

In terms of its potential toxicity, 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's amino acid sequence confirmed the lack of relevant similarities between the PAT protein and known toxins. In addition, microbial PAT protein safety studies previously provided for cotton event 3006-210-23 indicated that no adverse effects were observed when the PAT protein was ingested by mice at approximately 5000 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 DAS-44406-6 and is rapidly degraded under conditions which simulate the mammalian digestive tract.

Novel 2mEPSPS protein

To obtain sufficient quantities of 2mEPSPS protein for evaluation of environmental and feed safety, it was necessary to express the 2mepsps gene in a microbial production system. The equivalency of the soybean event DAS-44406-6-produced 2mEPSPS protein to the microbially-produced 2mEPSPS protein was evaluated by comparing their molecular weights, immunoreactivities, glycosylation, tryptic peptide mass mapping results and enzyme activities. Based on the results, the proteins were found to be equivalent. Demonstration of equivalence between the microbially-produced 2mEPSPS protein and the 2mEPSPS protein produced in soybean event DAS-44406-6 allows utilization of the microbially-produced 2mEPSPS protein in studies to confirm the safety of the 2mEPSPS protein produced in soybean event DAS-44406-6.

The potential mammalian allergenicity and toxicity of the 2mEPSPS protein were evaluated. With respect to its potential allergenicity, it is recognized that no single experimental method yields decisive evidence for allergenicity, 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 commonly associated with allergenicity and a bioinformatics evaluation of the 2mEPSPS protein amino acid sequence confirmed the lack of relevant similarities between the 2mEPSPS protein and known allergens. Unlike many allergens, studies with the microbial 2mEPSPS protein indicated that the enzyme is rapidly degraded in simulated gastric fluid and is not heat stable and studies with soybean event DAS-44406-6 2mEPSPS indicated that it is not glycosylated. The weight of evidence thus indicates that the 2mEPSPS protein is unlikely to be allergenic.

In terms of its potential toxicity, 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, no adverse effects were observed when the microbial 2mEPSPS protein was ingested by mice at doses of approximately 5000 mg/kg bwt. This information indicates 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 soybean event DAS-44406-6, it is rapidly degraded under conditions which simulate the mammalian digestive tract and is unstable under heating conditions expected to be encountered during processing of some soybean products.

Chemical pesticide residue profile

The safety of herbicide residues and metabolites in soybean event DAS-44406-6, following application of herbicides, was also evaluated as part of the feed safety assessment.

It was determined that that potential glufosinate-ammonium and glyphosate residues and metabolites in livestock commodities: soybean seeds, soybean meal, soybean oil, soybean milk, soybean hulls, aspirated grain fractions and soybean forage and hay from soybean event DAS-44406-6, would not present levels of concern to livestock, nor humans, via the potential transfer into foods of animal origin.

It was determined that 2,4-D in livestock feed commodities (soybean seeds, soybean meal, soybean oil, soybean milk, soybean hulls and aspirated grain fractions) produced from soybean event DAS-44406-6, did not present levels of concern to livestock, nor humans via the potential transfer into foods of animal origin, when comparing the estimated exposure to established legal residue limits in Canada and the US.

No authorization for forage or hay produced from soybean event DAS-44406-6 treated with 2,4-D has been granted at this time, as there were not sufficient data available to support the inclusion of 2,4-D treated forage and hay as feed. Dow AgroSciences Canada Inc. has indicated that they will restrict the feeding of DAS-44406-6 soybean forage and hay on the Canadian 2,4-D label. 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:

Feed ingredients derived from soybean event DAS-44406-6, with the exception of forage or hay produced from the 2,4-D and soybean event DAS-44406-6 combination, as outlined above, are considered to meet present ingredient definitions for soybean and as such are approved for use as livestock feed in Canada.

VI. New Information Requirements

If at any time, Dow AgroSciences Canada Inc. becomes aware of any new information regarding risk to the environment livestock or human health, which could result from release or livestock feed use of soybean event DAS-44406-6 or lines derived from it, Dow AgroSciences Canada 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 soybean event DAS-44406-6 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 soybean event DAS-44406-6.

VII. Regulatory Decision

Based on the review of the data and information submitted by Dow AgroSciences Canada 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 DAS-44406-6 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 Dow AgroSciences Canada Inc. and input from other relevant scientific sources, the Animal Feed Division of the Animal Health Directorate, CFIA, has concluded that the novel AAD-12, PAT and 2mEPSPS protein-based herbicide tolerance traits will not confer to soybean event DAS-44406-6 any characteristic that would raise any concerns regarding the safety or nutritional composition of soybean event DAS-44406-6. Grain soybean, its byproducts and soybean oil are currently listed in Schedule IV of the Feeds Regulations and are, therefore approved for use in livestock feeds in Canada. Soybean event DAS-44406-6 has been found to be as safe as and as nutritious as currently and historically grown soybean varieties. Soybean event DAS-44406-6 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 soybean event DAS-44406-6 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 6, 2013. Any soybean lines derived from soybean event DAS-44406-6 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, respectively and (iv) the novel genes are expressed at levels similar to those in the authorized line.

Additionally, with respect to its use as livestock feed, soybean event DAS-44406-6 must meet the restrictions specific to 2,4-dichlorophenoxyacetic acid (2,4-D)-treated forage and hay set out in this authorization.

Soybean event DAS-44406-6 is subject to the same phytosanitary import requirements as unmodified soybean varieties. Soybean event DAS-44406-6 is required to meet the requirements of other jurisdictions, including but not limited to, 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 DAS-44406-6. The food safety decisions are available at the Health Canada web site.

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