Decision Document DD2015-109
Determination of the Safety of Dow AgroSciences Canada Inc.'s Cotton (Gossypium hirsutum) Event DAS-81910-7

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This Decision Document has been prepared to explain the regulatory decisions reached under 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, and based on the environmental criteria in Directive 94-08 - Assessment Criteria for Determining Environmental Safety of Plants with Novel Traits.

The Canadian Food Inspection Agency (CFIA), specifically the Animal Feed Division of the Animal Health Directorate, with input from the Plant and Biotechnology Risk Assessment Unit of the Plant Health Science Directorate, has evaluated information submitted by Dow AgroSciences Canada Inc. This information concerns the herbicide tolerant cotton event DAS-81910-7. The CFIA has determined that feed derived from these modified plants does not present a significant risk to the environment, nor does it present livestock feed safety or nutrition concerns when compared to currently commercialized cotton varieties in Canada.

Taking into account these evaluations, use as livestock feed of cotton event DAS-81910-7 is therefore authorized by the Animal Feed Division of the Animal Health Directorate, respectively, as of March 19, 2015. Any cotton lines derived from cotton event DAS-81910-7 may also be used as livestock feed, provided that:

  1. no inter-specific crosses are performed,
  2. the intended uses are similar,
  3. it is known based on characterization that these plants do not display any additional novel traits and are substantially equivalent to cotton varieties that are currently grown and permitted to be used as livestock feed in Canada, in terms of their potential environmental impact and livestock feed safety and nutrition, and
  4. the novel genes are expressed at a level similar to that of the authorized line.

Additionally, with respect to its use as livestock feed, cotton event DAS-81910-7 must meet the restrictions specific to 2,4-dichlorophenoxyacetic acid (2,4-D)-treated forage and hay set out in the authorization.

Cotton event DAS-81910-7 is subject to the same phytosanitary import requirements as unmodified cotton varieties. Cotton event DAS-81910-7 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 safety of novel feeds is a critical step in the potential commercialization of these plant types. Other requirements, such as the assessment of novel foods by Health Canada, have been addressed separately from this review.

March 19, 2015

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

Table of Contents

I. Brief Identification of the Modified Plant

Designation of the Modified Plant: Cotton event DAS-81910-7 OECD Unique Identifier DAS-8191Ø-7

Applicant: Dow AgroSciences Canada Inc.

Plant Species: Cotton (Gossypium hirsutum)

Novel Traits: Tolerance to 2,4-dichlorophenoxyacetic acid (2,4-D) and glufosinate ammonium herbicides

Trait Introduction Method: Agrobacterium-mediated transformation

Intended Use of the Modified Plant: Production of cotton for fibre, cottonseed and cottonseed meal (cake, grain, flakes, pellets) or roughage for livestock feed, and cottonseed oil for human consumption. These materials will be grown outside Canada, in the usual production areas for cotton. Cottonseed and cottonseed meal will be imported into Canada for livestock feed use only.

II. Background Information

Dow AgroSciences Canada Inc. has developed a cotton event that is tolerant to 2,4-D and glufosinate ammonium herbicides. Cotton event DAS-81910-7 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 and thephosphinothricin acetyltransferase (pat) gene. The aad-12 gene is derived from the soil bacterium Delftia acidovorans (D. acidovorans) and encodes an aryloxyalkanoate dioxygenase-12 (AAD-12) protein 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) protein. This protein inactivates the herbicide glufosinate-ammonium.

Dow AgroSciences Canada Inc. has provided information on the identity of cotton event DAS-81910-7, a detailed description of the transformation method, data and information on insert copy number and intactness, levels of protein expression in the plant and the role of the inserted sequences. The novel proteins were identified and characterized. Information was provided for the evaluation of the potential toxicity of the novel proteins to livestock and non-target organisms and potential allergenicity of the novel proteins to humans and to livestock. Information was also provided for the evaluation of herbicide residues in the feed commodities derived from the crop, following the intended herbicide application.

Cotton event DAS-81910-7 was field tested at 8 sites in the United States (US) in 2012. An unmodified control cotton variety, which shares the same genetic background as cotton event DAS-81910-7, was included in the trials to act as a comparator for cotton event DAS-81910-7. Six conventional cotton varieties were also included in the field trials to establish ranges of comparative values that are representatives of currently grown cotton varieties.

Agronomic, phenotypic and ecological characteristics of cotton event DAS-81910-7, such as early population, seedling vigor, flower initiation, nodes above white flower, plant height, percent open bolls, lint yield, disease incidence and insect damage, were compared to those of the unmodified control cotton variety and to the range established by the conventional cotton varieties.

Nutritional components of cotton event DAS-81910-7 seed, such as protein, fat, moisture, ash, fibre, amino acids, fatty acids, vitamins, minerals and anti-nutrients were compared to those of the unmodified control cotton variety and to the range established by the conventional cotton varieties.

The Animal Feed Division (AFD) of the Animal Health Directorate, CFIA, with input from the Plant and Biotechnology Risk Assessment Unit of the Plant Health Science Directorate, CFIA, has reviewed the above information. The following assessment criteria, 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 and Directive 94-08 - Assessment Criteria for Determining Environmental Safety of Plants with Novel Traits were used to determine the safety and efficacy as livestock feed and the environmental safety of this novel feed:

  • the potential impact of cotton event DAS-81910-7 on livestock nutrition;
  • the potential impact of cotton event DAS-81910-7 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 potential for cotton event DAS-81910-7 to become a weed of agriculture or to be invasive of natural habitats;
  • the potential for gene flow from cotton event DAS-81910-7 to sexually compatible plants whose hybrid offspring may become more weedy or more invasive;
  • the potential for cotton event DAS-81910-7 to become a plant pest;
  • the potential impact of cotton event DAS-81910-7 and its gene products on non-target organisms, including humans; and
  • the potential impact of cotton event DAS-81910-7 on biodiversity.

The AFD has also considered whether feeds derived from cotton event DAS-81910-7 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 cotton event DAS-81910-7.

III. Description of the Novel Traits

1. Development Method

Cotton event DAS-81910-7 was developed through Agrobacterium-mediated transformation of cells from cotton variety Coker 310 and contains the aad-12 and pat genes and associated regulatory elements. Transformed cells were selected on the basis of tolerance to glufosinate-ammonium and regenerated to produce plants. Cotton event DAS-81910-7 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. Cotton event DAS-81910-7 contains the aad-12 gene from the gram-negative soil bacterium D. acidovorans, which encodes AAD-12 protein, an enzyme that degrades the herbicide 2,4-D into the herbicidally inactive form 2,4-dichlorophenol (DCP). Introduction of the aad-12 gene into cotton event DAS-81910-7 therefore confers commercial-level tolerance to the herbicide 2,4-D.

The AAD-12 protein produced in cotton event DAS-81910-7 is identical to the native protein except for the addition of an alanine at position number 2, which was the result of DNA modifications during cloning. The same AAD-12 protein produced in other 2,4-D tolerant crops has previously been authorized for unconfined release and livestock use in Canada by CFIA including DAS-68416-4 (DD2012-93) and DAS-44406-6 (DD2013-97).

AAD-12 protein expression in cotton event DAS-81910-7 is driven by a constitutive promoter. Samples of cotton tissues were collected from six field sites in US during 2012. Tissues were collected from unsprayed plants and plants sprayed with 2,4-D and glufosinate-ammonium herbicides. The average AAD-12 protein expression levels in micrograms of protein per gram of dry weight tissue (μg/g dwt) evaluated by enzyme-linked immunosorbent assay (ELISA), were as follows: 71.17 μg/g dwt in leaf, 70.71 μg/g dwt in pollen, 38.33 μg/g dwt in squares, 30.63 μg/g dwt in flower, 18.75 μg/g dwt in seed, 17.17 μg/g dwt in bolls, 16.42 μg/g dwt in whole plant and 10.74 μg/g dwt in roots. The AAD-12 protein concentrations observed in cotton tissues were comparable for both unsprayed and herbicide-treated plants.

To obtain sufficient quantities of AAD-12 protein for assessment of environmental and feed safety, it was necessary to express the aad-12 gene in a P. fluorescens production system. Equivalency was demonstrated between AAD-12 protein in cotton event DAS-81910-7 and P. fluorescens-produced AAD-12 by comparing their molecular weights, immunoreactivity, glycosylation, N-terminal sequence analysis, MALDI-TOF mass spectrometry, and functional activity. Based on the results, the proteins were found to be equivalent. Demonstration of equivalence between the AAD-12 protein produced in P. fluorescens and the AAD-12 protein produced in cotton event DAS-81910-7 allows the AAD-12 protein produced in P. fluorescens to be used in studies to confirm the safety of the AAD-12 protein produced in cotton event DAS-81910-7.

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 and toxic, based on the following information. The source of the aad-12 gene, D. acidovorans, is not known to produce allergens. A bioinformatics evaluation of the AAD-12 proteins amino acid sequence confirmed the lack of relevant similarities between the AAD-12 proteins and known allergens. P. fluorescens-produced AAD-12 protein safety studies indicated that, unlike many allergens, the AAD-12 protein is rapidly degraded in simulated gastric fluid, is not heat stable and is not glycosylated. It was also concluded that the AAD-12 protein is unlikely to be toxic to livestock or non-target organisms as 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, 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). The livestock or non-target organisms exposure to the AAD-12 protein is expected to be negligible as the AAD-12 proteins are expressed at very low levels in cotton event DAS-81910-7 and are rapidly degraded under conditions which simulate the mammalian digestive tract.

For a more detailed discussion of the potential allergenicity and toxicity of the AAD-12 protein, see Section V, part 2: Potential Impact of Cotton Event DAS-81910-7 on Animal Health and Human Safety as it Relates to the Potential Transfer of Residues into Foods of Animal Origin and Worker/Bystander Exposure to the Feed.

3. Tolerance to Glufosinate-Ammonium

Phosphinothricin, the active ingredient in glufosinate-ammonium herbicide inhibits the plant enzyme glutamine synthetase that is responsible for assimilation of ammonia produced by plants during photorespiration. The inactivation of glutamine synthetase causes rapid accumulation of lethal levels of ammonia in susceptible plants, resulting in death. Cotton event DAS-81910-7 contains the pat gene from the gram-positive soil bacterium S. viridochromogenes, which encodes PAT protein. The PAT protein is an enzyme that acetylates the primary amino group of glufosinate-ammonium, rendering it inactive. Introduction of the pat gene into cotton event DAS-81910-7 confers commercial-level tolerance to the herbicide glufosinate-ammonium.

The PAT protein produced in cotton event DAS-81910-7 is identical to the native protein derived from S. viridochromogenes and is the same PAT protein produced in other glufosinate-ammonium tolerant crops already authorized for unconfined release and livestock use in CFIA including DAS-68416-4 (DD2012-93) and DAS-44406-6 (DD2013-97).

PAT protein expression in cotton event DAS-81910-7 is driven by a constitutive promoter. Samples of cotton tissues were collected from six field sites in US during 2012. Tissues were collected from unsprayed plants and plants sprayed with 2,4-D and glufosinate-ammonium herbicides. The average PAT protein expression levels in micrograms of protein per gram of dry weight tissue (μg/g dwt) evaluated by enzyme-linked immunosorbent assay (ELISA), were as follows: 13.29 μg/g dwt in leaf, 7.91 μg/g dwt in squares, 5.30 μg/g dwt in flower, 3.85 μg/g dwt in seed, 3.16 μg/g dwt in bolls, 1.67 μg/g dwt in roots, 0.97 μg/g dwt in whole plant and 0.11 μg/g dwt in pollen. The PAT protein concentrations observed in cotton tissues were comparable for both unsprayed and herbicide-treated plants.

To obtain sufficient quantities of PAT protein for assessment of environmental and feed safety, it was necessary to express the pat gene in a P. fluorescens production system. Equivalency was demonstrated between the cotton event DAS-81910-7-produced PAT protein and a P. fluorescens-produced PAT protein by comparing their molecular weights, immunoreactivity, glycosylation, N-terminal sequence analysis, MALDI-TOF mass spectrometry, and functional activity. Based on the results, the proteins were found to be equivalent. Demonstration of equivalence between the PAT protein produced in P. fluorescens and the PAT protein produced in cotton event DAS-81910-7 allows the PAT protein produced in P. fluorescens to be used in studies to confirm the safety of the PAT protein produced in cotton event DAS-81910-7.

The potential allergenicity and toxicity of the PAT protein to livestock and non-target organisms were evaluated. The weight of evidence indicates that the PAT protein is unlikely to be allergenic, based on the following information. The source of the pat gene, S. viridochromogenes, is not commonly associated with allergenicity, the PAT protein amino acid sequence lacks relevant similarities to known allergens. Unlike many allergens, the P. fluorescens -produced PAT protein was shown experimentally to be rapidly degraded in simulated gastric fluid, heat labile and not glycosylated. It was also concluded that the PAT protein is unlikely to be toxic to livestock and non-target organisms because it lacks a mode of action to suggest that it is intrinsically toxic to livestock or non-target organisms. A bioinformatics evaluation of the PAT protein amino acid sequence confirmed the lack of relevant similarities between the PAT protein and known toxins. In addition PAT has a long history of safe use in livestock feed. The livestock exposure to the PAT proteins are expected to be negligible as the PAT proteins are expressed at very low levels in cotton event DAS-81910-7 and are rapidly degraded under conditions which simulate the mammalian digestive tract.

For a more detailed discussion of the potential allergenicity and toxicity of the PAT protein, see Section V, part 2: Potential Impact of Cotton Event DAS-81910-7 on Animal Health and Human Safety as it Relates to the Potential Transfer of Residues into Foods of Animal Origin and Worker/Bystander Exposure to the Feed.

4. Stable Integration into the Plant Genome

Molecular characterization by Southern blot analysis demonstrated that cotton event DAS-81910-7 contains one intact copy of the gene cassette containing the aad-12 and pat gene and its regulatory elements inserted at a single site in the cotton genome. No additional elements, including intact or partial DNA fragments of the gene cassette or backbone sequences from the plasmid vector were detected in cotton event DAS-81910-7. A total of eleven putative open reading frames (ORFs) spanning the junctions between the insert and its flanking border regions in DAS-81910-7 cotton were identified. The upstream DNA sequences of the putative ORFs were evaluated for potential regulatory sequence. Five of these ORFs contained promoter-like regulatory elements but either lacked start codon sequence or a typical ribosome binding sites sequence that are critical elements for translation into protein. Additionally, bioinformatics analyses confirmed the lack of relevant sequence similarities with known toxins and allergens.

The stability of the insert within cotton event DAS-81910-7 was verified by Southern blot analysis and by detection of the AAD-12 and PAT proteins over five generations. The inheritance pattern of the insert and the AAD-12 and PAT proteins expression trait across five segregating generations of cotton event DAS-81910-7 showed that the insert segregates according to Mendelian rules of inheritance for a single genetic locus.

IV. Criteria for the Environmental Assessment

Lines derived from cotton event DAS-81910-7 will not be grown in Canada. However, Canada imports cottonseed, as well as a wide range of other cotton products, that are used as human food, livestock feed or other industrial products.

1. Potential for Cotton Event DAS-81910-7 to Become a Weed of Agriculture or be Invasive of Natural Habitats

Cotton (Gossypium hirsutum) is a member of the family Malvaceae. It is a perennial species cultivated as an annual and grown in the United States, mostly in areas from Virginia southward and westward to California. Cotton is not grown in Canada as it is not adapted to environmental conditions found at these latitudes.

Cotton is not considered a weed pest in the regions where it is grown, nor is it invasive of unmanaged habitats in Canada. Cotton event DAS-81910-7 has not been modified to have altered cold-tolerance and information supplied by Dow AgroSciences Canada Inc. indicates that the reproductive and survival biology of cotton event DAS-81910-7 is unchanged compared to unmodified counterparts.

The CFIA has concluded that cotton event DAS-81910-7 is unlikely to become a weed of agriculture or invasive of natural habitats.

2. Potential for Gene Flow from Cotton Event DAS-81910-7 to Sexually Compatible Plants Whose Hybrid Offspring May Become More Weedy or More Invasive

Cotton is predominately self-pollinated. Although cross-pollination may occur at low levels, particularly in the presence of pollinators such as honeybees, cotton has no wild relatives native to Canada. Wild relatives of commercial cotton, such as G. barbadense and G. tomentosum, are found only in tropical and sub-tropical regions.

This information, together with the fact that the novel traits have no intended effects on cotton reproductive biology, led the CFIA to conclude that that gene flow from cotton event DAS-81910-7 to wild relatives in Canada is not possible.

3. Potential for Cotton Event DAS-81910-7 to Become a Plant Pest

Cotton is not a plant pest in Canada and the intended effect of the novel traits is unrelated to plant pest potential. In addition, agronomic characteristics of cotton event DAS-81910-7 are similar to those described for currently grown cotton varieties.

The CFIA has therefore determined that cotton event DAS-81910-7 does not present a plant pest concern.

4. Potential Impact of Cotton Event DAS-81910-7 and Its Gene Products on Non-Target Organisms, including Humans

The 2,4-D and glufosinate-ammonium herbicide tolerance traits introduced into cotton event DAS-81910-7 are unrelated to a potential impact on non-target organisms. AAD-12 and PAT proteins naturally occur in the soil bacteria Delftia acidovorans and Streptomyces viridochromogenes, respectively. In addition, the aad-12 and pat expression cassettes introduced into cotton event DAS-81910-7 are the same as those introduced into soybean event DAS-68416-4 (DD2012-93) and soybean event DAS-44406-6 (DD2013-97), both of which have been previously authorized for unconfined environmental release in Canada.

Detailed characterization of the AAD-12 and PAT proteins expressed in cotton event DAS-81910-7 led to the conclusion that none of these proteins displays any characteristic of a potential toxin or allergen (see Section III: Description of the Novel Traits). Therefore, no negative impacts resulting from exposure of organisms to the AAD-12 and PAT proteins expressed in cotton event DAS-81910-7 are expected.

Composition analyses showed that the levels of key nutrients and anti-nutrients in seed from cotton event DAS-81910-7 are comparable to those in conventional cotton varieties (see Section V, part 1: Potential Impact of Cotton Event DAS-81910-7 on Livestock Nutrition). Therefore, it is very unlikely that the introduction of the novel traits may have caused unintended changes to the composition of cotton event DAS-81910-7 that would negatively impact organisms interacting with cotton event DAS-81910-7.

Cotton event DAS-81910-7 will not be grown in Canada. In the event that seed from cotton event DAS-81910-7 is accidentally released into the environment, any resulting plants would not be expected to set seed. Therefore, exposure of non-target organisms to the AAD-12 and PAT proteins from cotton event DAS-81910-7 is expected to be minimal to non-existent.

Based on the above, the CFIA has determined that the use of cotton event DAS-81910-7 will not result in altered impacts on interacting organisms, including humans, when compared to currently commercialized cotton varieties.

5. Potential Impact of Cotton Event DAS-81910-7 on Biodiversity

No varieties of cotton, nor any wild relatives that can readily interbreed with cotton, can grow in the Canadian environment since cotton is not adapted to the environmental conditions encountered in Canadian agricultural environments. In addition, cotton event DAS-81910-7 has no observed or expected modifications that would allow it to survive in the Canadian environment and, as a result, is not expected to enter or survive in managed or unmanaged ecosystems in Canada. Furthermore, there is no evidence to suggest that cotton event DAS-81910-7 would cause negative impacts on interacting organisms.

The CFIA has therefore concluded that cotton event DAS-81910-7 does not present any adverse impacts on biodiversity in Canada.

V. Criteria for the Livestock Feed Assessment

The AFD considered nutrient and anti-nutrient profiles; the safety of feed ingredients derived from cotton event DAS-81910-7, 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 cotton event DAS-81910-7 meet the definitions and requirements of feeds as listed in Schedule IV of the Feeds Regulations.

1. Potential Impact of Cotton Event DAS-81910-7 on Livestock Nutrition

Nutrient and anti-nutrient composition

The nutritional equivalence of unsprayed cotton event DAS-81910-7 plants to those of the unsprayed, unmodified control cotton varieties and six unsprayed, conventional control cotton varieties was determined from eight replicated field trials in the US during the 2012 growing season. Also in these field trials, the effect of herbicide spraying of cotton event DAS-81910-7 with 2,4-D plus glufosinate-ammonium on cottonseed nutritional composition was assessed by comparing data of sprayed to unsprayed cotton event DAS-81910-7. Cottonseed samples were analyzed for moisture, ash, protein, crude fat, acid detergent fibre (ADF), neutral detergent fibre (NDF), minerals, vitamins, amino acids, fatty acids, and anti-nutrients (dihydrosterculic acid, sterculic acid, malvalic acid, total and free gossypol) as recommended by the OECD consensus document for new varieties of cotton (OECD, 2009). Composition data was analyzed statistically using a mixed model analysis of variance, and statistical differences among cotton seed types were identified and assessed (P<0.05). The biological relevance of any significant difference among cottonseed types was assessed by comparing the observed mean values to the range of values observed for the conventional control cotton varieties grown in the trials and those in the published scientific literature (ILSI, 2010).

No statistically significant differences were observed between cotton event DAS-81910-7 (unsprayed) and the unmodified control cotton varieties for moisture, ash, protein, crude fat, ADF and NDF. All means fell within the ranges of the conventional control cotton varieties and published scientific literature (ILSI, 2010). No statistically significant differences were observed between cotton event DAS-81910-7 and the unmodified control cotton varieties for all minerals except for manganese. The observed differences in manganese was not considered to be meaningful from a feed safety or nutritional perspective because the mean value was within the ranges of the conventional control cotton varieties and published scientific literature (ILSI, 2010). For all eighteen amino acids measured, no statistically significant differences were found between cotton event DAS-81910-7 (unsprayed) and the unmodified control cotton varieties. The mean results for cotton event DAS-81910-7 analytes fell within the ranges of the conventional control cotton varieties and published scientific literature (ILSI, 2010). Except for myristic, palmitoleic, oleic, and linoleic acids, there were no statistically significant differences were observed between cotton event DAS-81910-7 (unsprayed) and the unmodified control cotton varieties for other fatty acids analyzed. The differences observed in myristic, palmitoleic, oleic, and linoleic acids were considered biologically not relevant since the mean results in DAS-81910-7 (unsprayed) fell within ranges of the conventional control cotton varieties and/or published scientific literature (ILSI, 2010). No statistically significant differences were observed between cotton event DAS-81910-7 (unsprayed) and the unmodified control cotton varieties for all vitamins analyzed. Malvalic acid in cotton event DAS-81910-7 (unsprayed) was significantly higher than the level in the unmodified control cotton varieties, while total gossypol in cotton event DAS-81910-7 (unsprayed) was significantly lower than the level in the unmodified control cotton varieties. These observed differences were however not considered biologically relevant; given that the mean values in cotton event DAS -81910-7 were within the range of the conventional control cotton varieties and/or published scientific literature (ILSI, 2010). For dihydrosterculic acid, sterculic acid and free gossypol the mean results for cotton event DAS-8190-7 (unsprayed) fell within the ranges of the conventional control cotton varieties and published scientific literature (ILSI, 2010), and no statistical significant differences were found between the levels in cotton event DAS-8190-7 (unsprayed) and the unmodified control cotton varieties. There were no statistically significant differences between sprayed and unsprayed cotton event DAS-81910-7 for all 59 analytes measured, indicating that spraying DAS-81910-7 with 2,4-D plus glufosinate-ammonium has no significant effect on the nutritional composition of cotton event DAS-81910-7.

Conclusion

It was concluded based on the evidence of data provided by Dow AgroSciences Inc. that the nutritional composition of cotton event DAS-81910-7 (unsprayed or sprayed with 2,4-D plus glufosinate-ammonium) cottonseed is not different from conventional control cottonseed varieties grown in the trial and to those reported in published scientific literature. Feed ingredients derived from cotton event DAS-81910-7 are considered to meet present ingredient definitions found in Schedule IV of the Feeds Regulations.

2. Potential Impact of Cotton Event DAS-81910-7 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

Cotton event DAS-81910-7 is tolerant to 2,4-D and glufosinate ammonium herbicides due to production of the AAD-12 and PAT proteins. The assessment of cotton event DAS-81910-7 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

To obtain sufficient quantities of AAD-12 protein and PAT protein for assessment of environmental and feed safety, it was necessary to express the aad-12 and pat gene in a P. fluorescens production system. Equivalency was demonstrated between cotton event DAS-81910-7-produced AAD-12 and PAT protein and a P. fluorescens-produced AAD-12 and PAT protein by comparing their molecular weights, immunoreactivity, glycosylation, N-terminal sequence analysis, MALDI-TOF mass spectrometry, and functional activity. Based on the results, the proteins were found to be equivalent. Demonstration of equivalence between the AAD-12 and PAT protein produced in P. fluorescens and the AAD-12 and PAT protein produced in cotton event DAS-81910-7 allows the AAD-12 and PAT protein produced in P. fluorescens to be used in studies to confirm the safety of the AAD-12 and PAT protein produced in cotton event DAS-81910-7.

The potential allergenicity and toxicity of the AAD-12 and PAT protein to livestock were evaluated. With respect to its potential allergenicity, no single experimental method yields decisive evidence, thus a weight-of-evidence approach was taken, taking into account information obtained with various test methods. The source of the pat gene, S. viridochromogenes, is not known to produce allergens. A bioinformatics evaluation of the AAD-12 and PAT proteins amino acid sequence confirmed the lack of relevant similarities between the AAD-12 and PAT proteins and known allergens. P. fluorescens-produced AAD-12 and PAT protein safety studies indicated that, unlike many allergens, these proteins are rapidly degraded in simulated gastric fluid and are not heat stable. The weight of evidence thus indicates that the AAD-12 and PAT proteins are unlikely to be allergenic.

For AAD-12 protein, its potential toxicity to livestock, 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. AAD-12 protein is rapidly degraded in simulated gastric fluid and is readily denatured by heat. In addition, P. fluorescens-produced AAD-12 protein safety studies 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.

In terms of its potential toxicity to livestock, the PAT protein lacks a mode of action to suggest that it is intrinsically toxic to livestock and a bioinformatics evaluation of the PAT protein amino acid sequence confirmed the lack of relevant similarities between the PAT protein and known toxins. In addition PAT has a long history of safe use in livestock feed. This information indicates that the PAT protein is unlikely to be toxic to livestock.
The livestock exposure to the AAD-12 and PAT proteins are expected to be negligible as the AAD-12 and PAT proteins are expressed at very low levels in cotton event DAS-81910-7 and are rapidly degraded under conditions which simulate the mammalian digestive tract.

Chemical pesticide residue profile

The safety of glufosinate ammonium herbicide residues and metabolites in cotton event DAS-81910-7, following application of glufosinate ammonium herbicide, was also evaluated as part of the feed safety assessment. It was determined that that potential glufosinate-ammonium residues and metabolites in livestock feeds derived from cotton event DAS-81910-7 would not present levels of concern to livestock, nor humans via the potential transfer into foods of animal origin.

As cotton event DAS-81910-7 is also intended to be sprayed with herbicide 2,4-D, the safety of herbicide 2,4-D's residues and metabolites was also evaluated as part of the feed safety assessment. Herbicide tolerant cotton 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 cotton event DAS-81910-7 has not been fully assessed due to no appropriate regulatory points of reference at the time of assessment. The AFD has therefore placed a provisional restriction on feed commodities derived from cotton event DAS-81910-7 following the application of 2,4-D until an authorization for 2,4-D application on cotton event DAS-81910-7 has been granted by Health Canada's Pest Management Regulatory Agency (PMRA).

Conclusion

It was concluded, based on the evidence provided by Dow AgroSciences Canada Inc. that the novel AAD-12 and PAT proteins-based herbicide tolerance traits will not confer to cotton event DAS-81910-7 any characteristic that would raise concerns regarding the safety of cotton event DAS-81910-7. Feed ingredients derived from cotton event DAS-81910-7, without the application of 2,4-D are considered to meet present ingredient definitions for cotton 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 the unconfined environmental release or livestock feed use of cotton event DAS-81910-7 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 cotton event DAS-81910-7 on the environment, livestock and human health and may re-evaluate its decision with respect to the livestock feed use and unconfined environmental release authorizations of cotton event DAS-81910-7.

VII. Regulatory Decision

Cotton event DAS-81910-7 will not be grown in Canada nor can the seed overwinter; therefore, the release of the feed into the environment would result in neither intended nor unintended environmental effects.

Based on the review of the data and information submitted by Dow AgroSciences Canada Inc. and input from other relevant scientific sources, the AFD of the Animal Health Directorate, CFIA, has concluded that the novel AAD-12 and PAT protein-based herbicide tolerance traits will not confer to cotton event DAS-81910-7 any characteristic that would raise concerns regarding the safety or nutrition of cotton event DAS-81910-7.

Livestock feeds derived from cotton are currently listed in IV of the Feeds Regulations. Cotton event DAS-81910-7 has been found to be as safe as and as nutritious as currently and historically grown cotton varieties. Cotton event DAS-81910-7 and its products are considered to meet present ingredient definitions.

Taking into account these evaluations, use as livestock feed of cotton event DAS-81910-7 is therefore authorized by the Animal Feed Division of the Animal Health Directorate, respectively, as of March 19, 2015. Any cotton lines derived from cotton event DAS-81910-7 may also be used as livestock feed, provided that:

  1. no inter-specific crosses are performed,
  2. the intended uses are similar,
  3. it is known based on characterization that these plants do not display any additional novel traits and are substantially equivalent to cotton varieties that are currently grown and permitted to be used as livestock feed in Canada, in terms of their potential environmental impact and livestock feed safety and nutrition, and
  4. the novel genes are expressed at a level similar to that of the authorized line.

Additionally, with respect to its use as livestock feed, cotton event DAS-81910-7 must meet the restrictions specific to 2,4-dichlorophenoxyacetic acid (2,4-D)-treated forage and hay set out in the authorization.

Cotton event DAS-81910-7 is subject to the same phytosanitary import requirements as unmodified cotton varieties. Cotton event DAS-81910-7 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 cotton event DAS-81910-7.

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