Decision Document 2012-93:
Determination of the Safety of Dow AgroSciences Canada Inc.'s Soybean (Glycine max (L.) Merr.) Event DAS-68416-4

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October 5, 2012

This Decision Document has been prepared to explain the regulatory decision reached under Directive 94-08 (Dir94-08), entitled "Assessment Criteria for Determining Environmental Safety of Plants with Novel Traits", its companion biology document BIO1996-10, "The Biology of Glycine max (L.) Merr. (Soybean)", and Chapter 2.6 of the Regulatory Guidance: Feed Registration Procedures and Labelling Standards, entitled "Guidelines for the Assessment of Novel Feeds: Plant Sources".

The Canadian Food Inspection Agency (CFIA), specifically the Plant Biosafety Office of the Plant Health and Biosecurity Directorate, the Plant and Biotechnology Risk Assessment Unit of the Plant Health Science Directorate and the Animal Feed Division of the Animal Health Directorate, has evaluated information submitted by Dow AgroSciences Canada Inc. This information is in regard to the herbicide tolerant soybean event DAS-68416-4. 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 currently commercialized soybean varieties in Canada.

Taking into account these evaluations, unconfined release into the environment and use as livestock feed of soybean event DAS-68416-4 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 October 5, 2012. Any soybean lines derived from soybean event DAS-68416-4 may also be released into the environment and used as livestock feed, provided that (i) no inter-specific crosses are performed, (ii) the intended uses are similar, (iii) it is known based on characterization that these plants do not display any additional novel traits and are substantially equivalent to currently grown soybean 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-68416-4 must meet the restrictions specific to dichlorophenoxyacetic acid (2,4-D) treated feed set out in this authorization.

Soybean event DAS-68416-4 is subject to the same phytosanitary import requirements as its unmodified counterparts. Soybean event DAS-68416-4 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.

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
613-225-2342
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. Stable Integration into the Plant Genome
  4. Criteria for the Environmental Assessment
    1. Potential of Soybean Event DAS-68416-4 to Become a Weed of Agriculture or be Invasive of Natural Habitats
    2. Potential for Gene Flow from Soybean Event DAS-68416-4 to Sexually Compatible Plants Whose Hybrid Offspring May Become More Weedy or More Invasive
    3. Altered Plant Pest Potential of Soybean Event DAS-68416-4
    4. Potential Impact of Soybean Event DAS-68416-4 on Non-Target Organisms
    5. Potential Impact of Soybean Event DAS-68416-4 on Biodiversity
  5. Criteria for the Livestock Feed Assessment
    1. Potential Impact of Soybean Event DAS-68416-4 on Livestock Nutrition
    2. Potential Impact of Soybean Event DAS-68416-4 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-68416-4, OECD Unique Identifier DAS-68416-4
Applicant Dow AgroSciences Canada Inc.
Plant Species Soybean (Glycine max (L.) Merr.)
Novel Traits Tolerance to 2,4-dichlorophenoxyacetic acid (2,4-D) herbicide; tolerance to glufosinate-ammonium herbicide
Trait Introduction Method Agrobacterium-mediated transformation
Intended Use of the Modified Plant Human consumption and livestock feed use. Soybean event DAS-68416-4 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 the herbicides 2,4-dichlorophenoxyacetic acid (2,4-D) and glufosinate-ammonium. This soybean event, designated DAS-68416-4, was developed to provide growers with new options for weed control using 2,4-D and glufosinate-ammonium herbicides.

Soybean event DAS-68416-4 was developed by Dow AgroSciences Canada Inc. using recombinant DNA technology, resulting in the introduction of the aryloxyalkanoate dioxygenase-12 (aad-12) and the phosphinothricin acetyltransferase (pat) genes. The aad-12 gene encodes an aryloxyalkanoate dioxygenase-12 (AAD-12) enzyme derived from the soil bacterium Delftia acidovorans, which metabolises the herbicide 2,4-D, conferring resistance to the plant. The pat gene encodes a phosphinothricin acetyltransferase enzyme from the soil bacterium Streptomyces viridochromogenes. This enzyme inactivates the glufosinate-ammonium, conferring resistance to the plant.

Dow AgroSciences Canada Inc. has provided data on the identity of soybean event DAS-68416-4, a detailed description of the transformation method, data and information on the gene insertion site, gene copy number and levels of gene expression in the plant and the role of the inserted genes and regulatory sequences. The novel proteins were identified and characterized. Data were 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. Data were provided for the evaluation of herbicide residues in the feed commodities derived from the crop, following the intended herbicide application.

Soybean event DAS-68416-4 was field tested at six test sites located within the major soybean-producing regions of Canada and the United States in 2008 as well as 21 sites in the United States in 2009. The Canadian locations were representative of soybean growing regions in Canada.

Agronomic characteristics of soybean event DAS-68416-4 such as stand count, seed dormancy, emergence, seedling vigour, plant height, lodging, yield, days to flowering, days to maturity, shattering, final population, 100 seed weight, and susceptibilities to various soybean pests and pathogens were compared to those of the unmodified control.

Nutritional components of soybean event DAS-68416-4, such as protein, fat, carbohydrates, fibre, ash, moisture, amino acids, fatty acids, vitamins, and anti-nutrients were compared with those of the unmodified control.

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

The PBRA Unit has considered:

  • the potential of soybean event DAS-68416-4 to become a weed of agriculture or be invasive of natural habitats;
  • the potential for gene flow from soybean event DAS-68416-4 to sexually compatible plants whose hybrid offspring may become more weedy or more invasive;
  • the potential for soybean event DAS-68416-4 to become a plant pest;
  • the potential impact of soybean event DAS-68416-4 or its gene products on non-target species, including humans; and
  • the potential impact of soybean event DAS-68416-4 on biodiversity.

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

The AFD has considered both intended and unintended effects and similarities and differences between the modified plant and its counterpart relative to the safety and efficacy of feed ingredients derived from soybean event DAS-68416-4 for their intended purpose; including:

  • potential impact of soybean event DAS-68416-4 on livestock nutrition and
  • potential impact of soybean event DAS-68416-4 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-68416-4 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 containing the soybean event DAS-68416-4.

III. Description of the Novel Traits

1. Development Method

Soybean event DAS-68416-4 was developed through Agrobacterium-mediated transformation of soybean cotyledonary node explants with a T-DNA plasmid containing the aad-12 and pat genes, including their regulatory elements. Soybean event DAS-68416-4 was selected based on tolerance to glufosinate-ammonium. The presence of one or both of the inserted genes was confirmed by molecular analyses. Soybean event DAS-68416-4 was identified as a successful transformant and was 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-68416-4 contain the aad-12 gene, which encodes the enzyme AAD-12, an alpha ketoglutarate-dependent dioxygenase activity that can degrade the herbicide 2,4-D into the inactive form 2,4-dichlorophenol (DCP). Introduction of the aad-12 gene into soybean event DAS-68416-4 therefore confers commercial-level tolerance to the herbicide 2,4-D. The aad-12 gene was derived from Delftia acidovorans, a gram-negative soil bacterium. The AAD-12 protein produced in soybean event DAS-68416-4 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.

AAD-12 protein expression in soybean event DAS-68416-4 is driven by a constitutive promoter. Samples of soybean tissues were collected from plants from six field trials in the United States and Canada. Tissues were collected from unsprayed plants and plants sprayed with 2,4-D, glufosinate-ammonium, or both herbicides. The average AAD-12 protein expression in micro-grams of protein per gram of dry weight tissue (µg/g dwt) from unsprayed plants, as evaluated by enzyme-linked immunosorbent assay (ELISA), are as follows: 51.42 µg/g dwt in leaf at the V5 stage, 53.95 µg/g dwt in leaf at the V10 stage, 17.10 µg/g dwt in root, 41.11 µg/g dwt in forage, and 16.47 µg/g dwt in grain. Very similar AAD-12 protein levels were observed in the tissues of herbicide-treated plants.

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 Pseudomonas fluorescens production system. The equivalency of the AAD-12 protein produced in soybean event DAS-68416-4 to the P. fluorescens-produced AAD-12 protein was evaluated by comparing their molecular weights, immunoreactivity, glycosylation status, N-terminal and C-terminal sequences, and tryptic peptide mass map. Based on the results of these tests, the proteins were found to be equivalent. Demonstration of protein equivalence between P. fluorescens and soybean event DAS-68416-4-produced AAD-12 proteins allows utilization of the AAD-12 protein produced in P. fluorescens to be used in studies to confirm the safety of the AAD-12 protein produced in soybean event DAS-68416-4.

The potential mammalian toxicity and allergenicity of the AAD-12 protein was evaluated. This protein lacks sequence similarity to known allergens and protein toxins which have adverse effects to mammals. No adverse effects were observed when the AAD-12 protein was ingested by mice at a dose of approximately 2000 mg/kg body weight in acute oral toxicity studies. In vitro digestive fate studies have shown that AAD-12 is rapidly degraded in simulated gastric fluid, unlike protein allergens which are normally resistant to digestion. The AAD-12 protein expressed in soybean event DAS-68416-4 is not glycosylated, unlike many known allergens, providing additional evidence that these proteins do not have the properties of known allergens. Therefore, based on the weight of evidence, the AAD-12 protein expressed in soybean event DAS-68416-4 is unlikely to be toxic or allergenic to mammals.

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 within hours of application. Ammonia is produced by plants as a result of normal metabolic processes. Soybean event DAS-68416-4 was developed to be tolerant to the herbicide glufosinate-ammonium by incorporation of the pat gene. The pat gene codes 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-68416-4 confers field-level tolerance to the herbicide glufosinate-ammonium. The pat gene was derived from Streptomyces viridochromogenes, a gram-positive soil bacterium, and the PAT protein produced in soybean event DAS-68416-4 is identical to the native enzyme.

The PAT protein produced in soybean event DAS-68416-4 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 and 281-24-236, canola events HCN92, HCN28 (T45) and HCR-1, corn events T14, T25, 1507 and 59122, soybean events A2704-12 and A5547-127, and sugar beet event T120-7.

PAT protein expression in soybean event DAS-68416-4 is driven by a constitutive promoter. Samples of soybean tissues were collected from plants from six field trials in the United States and Canada. Tissues were collected from unsprayed plants and plants sprayed with 2,4-D, glufosinate-ammonium, or both herbicides. The average PAT protein expression in micro-grams of protein per gram of dry weight tissue (µg/g dwt) from unsprayed plants, as evaluated by enzyme-linked immunosorbent assay (ELISA), are as follows: 9.17 µg/g dwt in leaf at the V5 stage, 10.94 µg/g dwt in leaf at the V10 stage, 1.73 µg/g dwt in root, 3.63 µg/g dwt in forage, and 2.73 µg/g dwt in grain. Very similar AAD-12 protein levels were observed in the tissues of herbicide-treated plants.

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-68416-4-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 so these studies were accepted to support the safety of soybean event DAS-68416-4. This demonstration of equivalency was based on similar molecular weights and immunoreactivities, as well as the DNA sequence of the pat gene in soybean event DAS-68416-4 and the tryptic mass map of the microbial PAT protein.

Dow AgroSciences Canada Inc. provided a bioinformatics evaluation of the PAT protein, which confirmed the lack of relevant similarities between the PAT protein sequence and sequences of known allergens and toxins. Many allergens have been reported to be expressed at high levels in plants and resistant to digestive enzymes. The PAT protein was expressed at low levels in soybean event DAS-68416-4, and the 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 previous studies also indicated that the PAT protein did not cause any adverse effects in mice at a level of 5000 mg/kg body weight. Therefore, based on the weight of evidence, the PAT protein expressed in soybean event DAS-68416-4 is unlikely to be toxic or allergenic to mammals.

4. Stable Integration into the Plant Genome

Molecular characterization by Southern blot analysis demonstrated that soybean event DAS-68416-4 contains one intact copy of the aad-12 and pat genes at a single site in the soybean genome. No additional elements, including intact or partial DNA fragments of the aad-12 and pat genes or backbone sequences from the vector, linked or unlinked to the intact insert were detected in soybean event DAS-68416-4. DNA sequence analysis confirmed that the insertion event resulted in small insertions and deletions of genomic DNA, however no new open reading frames were created. These insertional effects are not uncommon in transformation events, and no effects on the functionality of the DNA insert or the composition or performance of the plant itself were observed as a result of these changes.

The stability of the inserted DNA in soybean event DAS-68416-4 was demonstrated by Southern blot analysis across four generations. Analysis of the inheritance pattern of the aad-12 and pat genes and the AAD-12 protein within one segregating generation of soybean event DAS-68416-4 confirmed the stability of the inserted DNA and the stability of AAD-12 protein expression. The results of the analysis are consistent with the finding of a single site of insertion that segregates according to the Mendelian laws of genetics.

IV. Criteria for the Environmental Assessment

1. Potential of Soybean Event DAS-68416-4 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-68416-4 was determined not to be significantly different from its conventional counterparts in this respect.

The PBRA evaluated data submitted by Dow AgroSciences Canada Inc. on the reproductive biology and life history traits of soybean event DAS-68416-4. This event was field tested in Canada and the United States (US) in six locations in the 2008 growing season and in the US in 21 locations in the 2009 growing season. The unmodified control was also grown in the field trials. 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, several agronomic characteristics were evaluated including stand count, emergence, seedling vigour, plant height, lodging, days to flowering, days to maturity, shattering, final population, yield, and 100 seed weight. Although instances of statistically significant differences were observed between soybean event DAS-68416-4 and the unmodified control for some traits, there were no consistent trends in the data across locations or years that would indicate that any of these differences were due to genetic modification.

Dow AgroSciences Canada Inc. provided information on the dormancy and germination of soybean event DAS-68416-4 seed under a warm and a cool temperature regime. No significant difference was detected for germination at either a warm or a cool temperature between DAS-68416-4 and the unmodified control. Therefore, the novel traits did not impact seed dormancy.

The susceptibility of soybean event DAS-68416-4 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-68416-4). No trend in increase or decrease of susceptibility was observed in DAS-68416-4 compared to the unmodified control.

The introduction of the aad-12 gene did not make soybean event DAS-68416-4 weedy or invasive of natural habitats since none of the soybean's reproductive or growth characteristics were modified, and soybean event DAS-68416-4's tolerance to abiotic and biotic stresses was unchanged as well. No competitive advantage was conferred to soybean event DAS-68416-4, other than that conferred by tolerance to 2,4-D and/or glufosinate-ammonium herbicides.

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

Dow AgroSciences Canada Inc. has submitted a Herbicide Tolerance Management (HTM) plan to the CFIA which was evaluated by the PBRA Unit. The HTM plan contains recommendations to address these concerns, as well as appropriate strategies that will allow for the environmentally safe and sustainable deployment of this product. In addition, the HTM plan contains strategies for communication to growers and an efficient mechanism allowing growers to report problems to Dow AgroSciences Canada Inc. Dow AgroSciences Canada Inc. will make this HTM plan readily available to growers to promote careful management practices for soybean event DAS-68416-4.

The above considerations, together with the fact that the novel traits have no intended effects on soybean weediness or invasiveness, led the CFIA to conclude that soybean event DAS-68416-4 has no altered weed or invasiveness potential compared to conventional soybean varieties.

2. Potential for Gene Flow from Soybean Event DAS-68416-4 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 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 agroecosystems. The biology of soybean, as described in the CFIA biology document BIO1996-10, 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 minmal potential for gene flow from soybean event DAS-68416-4 to sexually compatible plant species in Canada.

3. Altered Plant Pest Potential of Soybean Event DAS-68416-4

Soybean is not considered a plant pest in Canada. The novel traits are unrelated to plant pest potential.

The susceptibility of soybean event DAS-68416-4 to various soybean pests and pathogens was evaluated in the field at the same locations as the phenotypic characteristic studies in the 2008 growing season and at 16 of the 21 locations in the 2009 growing season. The stressors observed included: brown spot, frogeye leaf spot, bacterial blight, leaf blight, downy mildew, aphids, bean leaf beetle, Mexican bean beetle, grasshoppers, Japanese beetle, lady bugs, and leaf hoppers. Ecological evaluations of soybean event DAS-68416-4 did not show any increase or decrease in susceptibility to any insect or disease stressor compared to the unmodified control grown at the same locations.

The CFIA therefore concludes that soybean event DAS-68416-4 does not display any altered pest potential compared to conventional soybean varieties.

4. Potential Impact of Soybean Event DAS-68416-4 on Non-Target Organisms

The 2,4-D and glufosinate-ammonium herbicide tolerance traits introduced into DAS-68416-4 soybean are unrelated to potential impact on non-target organisms.

Detailed characterization of the novel AAD-12 protein expressed in DAS-68416-4 soybean led to the conclusion that the AAD-12 protein does not display any characteristics of a potential toxin or allergen (as summarized in Part III: Description of the Novel Traits). The PAT protein has a history of environmental safety. Therefore, no negative impacts resulting from exposure of organisms to the AAD-12 and PAT proteins expressed in DAS-68416-4 soybean are expected.

Composition analyses showed that the levels of key nutrients and anti-nutrients in DAS-68416-4 soybean grain and forage are comparable to those in conventional soybean varieties. Two IgE antibody binding analyses using the sera from clinically documented, soybean-allergic patients showed that DAS-68416-4 soybean seed does not contain increased levels of endogenous allergens compared to the unmodified control. Therefore, it is very unlikely that the genetic transformation may have caused unintended changes to the composition of DAS-68416-4 soybean tissues that would negatively impact organisms interacting with DAS-68416-4 soybean.

Field evaluations of DAS-68416-4 soybean did not show any increased or decreased resistance to damage caused by pest insects and pathogens compared to the unmodified control (see Section 3: Altered Plant Pest Potential of Soybean Event DAS-68416-4).

Collectively, these information elements indicate that no negative impacts on organisms resulting from exposure to soybean event DAS-68416-4 is expected. The CFIA has therefore determined that the unconfined release of soybean event DAS-68416-4 will not result in altered impacts on non-target organisms, including humans, compared to conventional soybean varieties currently grown in Canada.

5. Potential Impact of Soybean Event DAS-68416-4 on Biodiversity

Soybean event DAS-68416-4 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. The novel traits are unlikely to have an impact on plant pest potential or non-target organisms. It is therefore unlikely that soybean event DAS-68416-4 will have any direct effects on biodiversity, in comparison to the effects that would be expected from the cultivation of currently grown soybean varieties.

Soybean event DAS-68416-4 has tolerance to 2,4-D and glufosinate-ammonium herbicides. The use of these herbicides in cropping systems has the intended effect of reducing local weed populations within agroecosystems. 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-68416-4, or the cultivation of soybean. It is therefore unlikely that soybean event DAS-68416-4 will have significant indirect effects on biodiversity, in comparison to the effects that would be expected from cultivation of currently cultivated soybean varieties.

The CFIA has concluded that the novel gene and its corresponding traits do not confer to soybean event DAS-68416-4 any characteristics 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-68416-4 is unlikely to be different from that of soybean varieties 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-68416-4, 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-68416-4 meet the definitions and requirements of feeds as listed in Schedule IV of the Feeds Regulations.

1. Potential Impact of Soybean Event DAS-68416-4 on Livestock Nutrition

Nutrient and Anti-nutrient Composition:

The compositional equivalence of soybean event DAS-68416-4 (unsprayed or sprayed with glufosinate-ammonium, 2,4-D or glufosinate-ammonium+ 2,4-D) to an unmodified control (Maverick) was examined at six replicated field sites in US (four) and Canada (two) during the 2008 growing season. Forage and seed samples were analysed for proximate, acid detergent fibre (ADF), neutral detergent fibre (NDF), calcium and phosphorus. Seed samples were further analysed for amino acids, fatty acids, vitamins, mineral, isoflavones (diadzin, glycitin, genistin, diadzein, glycitein, genistein) and anti-nutrients (phytic acid, trypsin inhibitor, lectin, stachyose and raffinose). No statistically significant differences were observed between soybean event DAS-68416-4 forage (sprayed or unsprayed) and the unmodified control , for protein, fat, ash, carbohydrate, ADF, NDF, calcium and phosphorus.

Statistically significant differences were observed between soybean event DAS-68416-4 seed (sprayed with 2,4-D) and the unmodified control for protein and carbohydrates, but the means were within the range of published literature values for conventional soybean varietiesFootnote 1Footnote 2 and therefore the differences were not considered biologically relevant. Except for potassium, no statistically significant differences were observed between soybean event DAS-68416-4 seed (sprayed or unsprayed) and the unmodified control for the other minerals analysed. Potassium levels of soybean event DAS-68416-4 soybeans were within the range of published literature values for conventional soybean varieties.Footnote 1Footnote 2 There were statistically significant differences between soybean event DAS-68416-4 (sprayed or unsprayed) and the unmodified control for alanine, arginine, aspartic acid, glutamic acid, glycine, histidine, leucine, lysine, phenylalanine, and threonine, however, the means were within the range of published literature values for conventional soybean varietiesFootnote 1Footnote 2 and therefore the differences were not considered biologically relevant. Statistically significant differences were observed between soybean event DAS-68416-4 seed and the unmodified control for palmitic palmitoleic, oleic acids (untreated), linolenic acid (untreated and sprayed with glufosinate-ammonium+2,4-D), arachidic acid (untreated and sprayed with glufosinate-ammonium) and behenic acid (sprayed with glufosinate-ammonium). All mean levels of fatty acids were within the natural variation of published literature values for conventional soybean varieties.Footnote 1Footnote 2

Gamma tocopherol and folic acid were statistically significantly different between soybean event DAS 68416-4 (sprayed with 2,4-D; and glufosinate-ammonium +2,4-D) and unmodified control, however they were within the published literature ranges of conventional soybean varieties.Footnote 1Footnote 2 There were no statistically significant differences between soybean event DAS-68416-4 (untreated) and unmodified control for the isoflavones, however the soybean event DAS-68416-4 soybeans sprayed with glufosinate-ammonium +2,4-D has significantly higher levels of daidzein, genistein and glycitein, but the means were within published literature values for conventional soybean varieties.Footnote 1Footnote 2 No statistically significant differences were observed between soybean event DAS 68416-4 (sprayed and unsprayed) soybeans for lectin, phytic acid, trypsin inhibitor, raffinose and stachyose. All means were all within the published literature values for conventional soybean varieties.Footnote 1Footnote 2

Conclusion

It was concluded based on the evidence provided by Dow Agro Sciences Canada Inc. that the nutritional composition of soybean event DAS 68416-4 is similar to the conventional soybean varieties.

2. Potential Impact of Soybean Event DAS-68416-4 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-68416-4 is tolerant to 2,4-D and glufosinate-ammonium herbicides due to production of the AAD-12 and PAT proteins, respectively. The assessment of soybean event DAS-68416-4 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 and PAT
  • The chemical pesticide residue profile

Novel AAD-12 and PAT proteins

The AAD-12 protein shares no significant biologically relevant sequence homology with any known toxins or allergens and lacks a mode of action that suggests that it is intrinsically toxic. This protein is also heat labile and rapidly degraded under conditions similar to those encountered in the gastrointestinal tract. No signs of toxicity were demonstrated in single-dose oral toxicity studies in mice using purified microbial AAD-12 protein at 2000 mg/kg-bw. These factors support the lack of intrinsic toxicity or allergenicity of the AAD-12 protein.

The PAT protein shares no significant biologically relevant sequence homology with any known toxins or allergens and lacks a mode of action that suggests that it is intrinsically toxic. The PAT protein was previously assessed for Dow AgroSciences Canada Inc.'s Cotton Event 3006-210-23; and it has been determined, based on the established equivalence, that the Cotton Event 3006-210-23 safety data may be used to support the safety of the PAT protein from soybean event DAS-68416-4. These studies indicated that the PAT protein is rapidly degraded under conditions similar to those encountered in the gastrointestinal tract. No signs of toxicity were demonstrated in the original single-dose oral toxicity studies in mice using purified microbial PAT protein at levels up to 5000 mg/kg bw. Furthermore, the PAT protein has history of safe use, having been approved in prior events. These factors support the lack of intrinsic toxicity or allergenicity of the PAT protein.

Chemical pesticide residue profile

The safety of herbicide residues and metabolites in soybean event DAS-68416-4, 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 commodities: soybean seeds, soybean meal, soybean oil, soybean milk, soybean hulls, aspirated grain fractions, and soybean forage and hay from soybean event DAS-68416-4, 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.

Herbicide tolerant soybean for use with 2,4-D has not at this time been the subject of any prior AFD Decisions. The safety of 2,4-D residues in feed ingredients after the application of 2,4-D to soybean event DAS-68416-4 has not been fully assessed, as there were no regulatory points of reference available at the time of the evaluation. The AFD has therefore placed a provisional restriction on feed commodities derived from soybean event DAS-68416-4 following the application of 2,4-D, until an authorization for 2,4-D application on soybean event DAS-68416-4 has been granted by Health Canada's Pest Management Regulatory Agency (PMRA).

Conclusions

Feed ingredients derived from soybean event DAS-68416-4, without the application of 2,4-D, 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 information regarding risk to the environment, including risk to human or animal health, which could result from release of soybean event DAS-68416-4 in Canada or elsewhere, Dow AgroSciences Canada Inc. will immediately provide such information to the CFIA. On the basis of such new information, the CFIA will re-evaluate the potential impact of soybean event DAS-68416-4 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-68416-4.

VII. Regulatory Decision

Based on the review of the data and information submitted by Dow AgroSciences Canada Inc. and other relevant information, the CFIA has determined that soybean event DAS-68416-4 does not present altered environmental risk when compared to currently commercialized soybean varieties in Canada.

Based on the review of submitted data and information by Dow AgroSciences Canada Inc., including comparisons of soybean event DAS-68416-4 with the unmodified control, the Animal Feed Division of the Animal Health Directorate, CFIA, has concluded that the novel genes and their corresponding traits will not confer to soybean event DAS-68416-4 any characteristic that would raise any concerns regarding the safety or nutritional composition of soybean event DAS-68416-4. 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-68416-4 has been found to be as safe as and as nutritious as traditional soybean varieties. Soybean event DAS-68416-4 and its products are considered to meet present ingredient definitions and are approved for use as livestock feed ingredients in Canada.

Please note that the safety of 2,4-D residues in feed ingredients after the application of 2,4-D to soybean event DAS-68416-4 was not fully assessed. Any feed ingredient(s) derived from the combination of soybean event DAS-68416-4 and 2,4-D may not be manufactured or sold in Canada, or imported into Canada, until an authorization for 2,4-D application on soybean event DAS-68416-4 has been granted by Health Canada's Pest Management Regulatory Agency (PMRA).

Unconfined release into the environment and use as livestock feed of soybean event DAS-68416-4 is therefore authorized by Plant Biosafety Office of the Plant Health and Biosecurity Directorate and the Animal Feed Division of the Animal Health Directorate as of October 5, 2012. Any soybean lines derived from soybean event DAS-68416-4 may also be released into the environment and used as livestock feed, provided that (i) no inter-specific crosses are performed, (ii) the intended uses are similar, (iii) it is known based on characterization that these plants do not display any additional novel traits and are substantially equivalent to currently grown soybean varieties in Canada, in terms of their potential environmental impact and livestock feed safety, 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-68416-4 must meet the restrictions specific to 2,4-D treated feed set out in this authorization

Soybean event DAS-68416-4 is subject to the same phytosanitary import requirements as its unmodified counterparts. Soybean event DAS-68416-4 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-68416-4.

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