Decision Document DD2011-84 Determination of the Safety of Syngenta Seeds Canada Inc.'s Cotton Event COT102
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This Decision Document has been prepared to explain the regulatory decision reached under Chapter 2.6 of the Regulatory Guidance: Feed Registration Procedures and Labelling Standards, entitled "Guidelines for the Assessment of Novel Feeds: Plant Sources" and Directive 94-08 (Dir94-08), entitled "Assessment Criteria for Determining Environmental Safety of Plants with Novel Traits".
The Canadian Food Inspection Agency (CFIA) has evaluated information submitted by Syngenta Seeds Canada Inc. regarding cotton event COT102, an insect resistant cotton. The CFIA has determined that feed derived from this modified plant does not present a significant risk to the environment, nor does it present livestock feed safety concerns when compared to currently commercialized cotton varieties in Canada.
Livestock feed use of cotton event COT102 is therefore authorized as of March 25, 2011. Cotton event COT102 and any cotton lines derived from it may be used as livestock feed provided (i) no inter-specific crosses are performed, (ii) the intended use(s) are similar, (iii) it is known, following thorough characterization, that these plants do not display any additional novel traits and are substantially equivalent to currently commercialized cotton, in terms of their specific use and safety for the environment and for human and animal health and (iv) the novel genes are expressed at a level similar to that of the authorized line.
Cotton event COT102 is subject to the same phytosanitary import requirements as its unmodified counterparts.
Please note, that the livestock feed and environmental safety 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 Animal Feed Division at:
Animal Feed Division
Animal Health Directorate
59 Camelot Drive
Ottawa ON K1A 0Y9
Table of Contents
- Brief Identification of the Modified Plant
- Background Information
- Description of the Novel Trait
- Criteria for the Environmental Assessment
- Potential to Become a Weed of Agriculture or be Invasive of Natural Habitats
- Potential for Gene Flow to Wild Relatives Whose Offspring May Become More Weedy or More Invasive
- Altered Plant Pest Potential
- Potential Impact on Non-Target Organisms
- Potential Impact on Biodiversity
- Potential for Development of Insect Resistance
- Criteria for the Livestock Feed Assessment
- New Information Requirements
- Regulatory Decision
I. Brief Identification of the Modified Plant
|Designation of the Modified Plant:||Cotton Event COT102, OECD Identifier SYN-IR1Ø2-7|
|Applicant:||Syngenta Seeds Canada Inc.|
|Plant Species:||Cotton (Gossypium hirsutum)|
|Novel Traits:||Insect resistance (various lepidopteran species)|
|Trait Introduction Method:||Agrobacterium-mediated transformation|
|Proposed Use of the Modified Plant:||Importation of cottonseed for processing, or the direct importation of processed cottonseed oil or linters for use in food products, or processed cottonseed meal for use in livestock feed.|
II. Background Information
Syngenta Seeds Canada Inc. has developed a cotton line, designated COT102, which is resistant to various lepidopteran insect species. The insect resistance trait in cotton event COT102 provides a method to control yield losses and decreased product quality from insect feeding damage caused by various lepidopteran insect species including cotton bollworm (Helicoverpa zea), tobacco budworm (Heliothis virescens), pink bollworm (Pectinophora gossypiella), fall armyworm (Spodoptera frugiperda), beet armyworm (Spodoptera exigua), soybean looper (Pseudoplusia includens), and cabbage looper (Trichoplusia ni), and cotton leaf perforator (Bucculatrix thurberiella).
Cotton event COT102 was developed using Agrobacterium mediated gene transfer technology, resulting in the introduction of a synthetic version of the vip3A(a) gene and the aph4 gene. The vip3A(a) gene is derived from Bacillus thuringiensis (strain AB88) and encodes the vegetative insecticidal protein (VIP)3A. The aph4 gene from Escherichia coli encodes the hygromycin B phosphotransferase (APH4) enzyme. Expression of this enzyme confers hygromycin tolerance to plant cells. This trait was not introduced for agronomical or livestock feed purposes but was used to select transformed from non-transformed plants during the development phase of cotton event COT102.
Syngenta Seeds Canada Inc. has provided data on the identity of cotton event COT102, 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 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.
Cotton event COT102 has been field tested in the United States and the data for trial years 2001 and 2002 were submitted.
Agronomic characteristics of cotton event COT102 such as seedling emergence, plant height, time to reproduction, lodging, susceptibilities to pathogens, and yield parameters were compared to those of unmodified cotton counterparts.
Nutritional components of cotton event COT102, such as proximates, amino acids and fatty acids were compared with those of unmodified cotton counterparts.
The Animal Feed Division of the Animal Health Directorate, CFIA, with input from the Plant and Biotechnology Risk Assessment (PBRA) Unit of the Plant Health Science Directorate, CFIA, has reviewed the above information with respect to the assessment criteria 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" and Directive 94-08 (Dir94-08), entitled "Assessment Criteria for Determining Environmental Safety of Plants With Novel Traits". The Animal Feed Division has considered:
- potential impact of cotton event COT102 on livestock nutrition; and
- potential impact of cotton event COT102 on livestock and workers/bystanders;
- potential of cotton event COT102 to become a weed of agriculture or be invasive of natural habitats;
- potential for gene flow from cotton event COT102 to wild relatives whose hybrid offspring may become more weedy or more invasive;
- potential for cotton event COT102 to become a plant pest;
- potential impact of cotton event COT102 or its gene products on non-target species, including humans; and
- potential impact of cotton event COT102 on biodiversity;
- potential for development of insect resistance to cotton event COT102.
Syngenta Seeds Canada Inc. has provided the CFIA with a method for the detection and identification of cotton products containing the cotton event COT102.
III. Description of the Novel Traits
1. Resistance to Lepidopteran Insects
Bacillus thuringiensis is a common gram-positive soil-borne bacterium. The VIP3A protein is produced during the vegetative bacterial growth stage of B. thuringiensis protein and secreted as a soluble protein into the extracellular environment. VIP3A is proteolytically activated to a toxin core in the lepidopteran larval midgut and forms pores in the gut membranes of sensitive species, a mechanism that appears to correlate with its toxicity. All testing to date indicates that VIP3A is a lepidopteran-specific toxin. With the exception of one amino-acid, the amino acid sequence of the VIP3A protein produced in cotton event COT102 is identical to that of the wild-type B. thuringiensis protein. The vip3A(a) coding sequence was optimized to accommodate the preferred plant codon usage.
The vip3A(a) gene is expressed in cotton event COT102 using a promoter that confers constitutive expression of proteins. Tissue samples were collected at two growth stages from COT102 grown at three representative US field trial sites. Levels of VIP3A protein were evaluated by enzyme-linked immunosorbent assays. Across all developmental stages and locations, mean uncorrected VIP3A concentrations measured in whole-plant samples ranged from ca. 1 – 13 µg/g fresh weight. Leaves had the highest mean VIP3A levels, which ranged from ca. 3 – 22 µg/g fresh wt. across sampling stages. Mean VIP3A concentrations measured in squares, roots and bolls did not exceed ca. 4 µg/g fresh wt., 2 µg/g fresh wt. and 1 µg/g fresh wt., respectively. For all test locations, mean VIP3A concentrations measured in seeds were ca. 3 µg/g on a fresh weight basis. The VIP3A concentration measured in pollen was ca. 1 µg/g air-dried pollen. VIP3A protein was present in the defatted (non-toasted) cottonseed meal from COT102 at a level of ca. 2.7 µg/g.
VIP3A protein isolated from COT102 was analyzed by mass spectral analysis. This analysis confirmed that the amino acid sequence of the VIP3A protein produced by COT102 corresponded with the predicted amino acid sequence without any evidence of post-translational modifications.
The level of VIP3A protein in cotton event COT102 tissues was too low to extract sufficient amounts for evaluation of environmental and feed safety. To obtain sufficient quantities of the VIP3A protein for safety studies, it was necessary to express the vip3A(a) gene in an E. coli production system and use VIP3A protein produced in a modified corn line. The equivalency of the COT102-produced VIP3A protein to the E. coli- and corn-produced VIP3A protein was evaluated by comparing their molecular weight, immunological reactivity, and insecticidal activity. Based on the results, the VIP3A protein produced in cotton event COT102 was found to be equivalent to the E. coli - produced counterpart.
The potential mammalian toxicity and allergenicity of the VIP3A protein was evaluated. The protein lacks sequence similarity to known allergens and protein toxins which have adverse effects to mammals. No adverse effects were observed when the VIP3A protein was ingested by mice after a single oral dosage of 2700 mg/kg body weight; the VIP3A protein is unlikely to be inherently toxic to livestock species. Furthermore, in vitro digestive fate studies have shown that the VIP3A protein is rapidly degraded when exposed to conditions present in the gastrointestinal tract indicating that oral ingestion of VIP3A is unlikely to result in systemic exposure. The lack of digestive stability further suggests that VIP3A is unlikely to be a protein allergen. There is no evidence that the VIP3A protein expressed in cotton event COT102 undergoes any post-translational modifications, unlike many known allergens, providing additional evidence that the VIP3A protein does not have the properties of known allergens.
2. Hygromycin Tolerance
The APH4 enzyme catalyzes the phosphorylation of the 4-hydroxyl group on the hyosamine moiety of hygromycin B, inactivating it. The amino acid sequence of the APH4 protein produced in cotton event COT102 is identical to that of the wild-type E. coli protein.
The aph4 gene is expressed in cotton event COT102 using a promoter that confers constitutive expression of proteins. Tissue samples were collected at the pre-harvest growth stage from COT102 grown at three representative US field trial sites. Levels of APH4 protein were evaluated by enzyme-linked immunosorbent assays. Leaves and whole-plant samples had detectable but not quantifiable amounts at all sampling times through the peak bloom stage, and APH4 was typically not detected at the later sampling stages, such as pre-harvest. APH4 was generally not detectable in cottonseeds, except for some seed samples in which the APH4 levels were below the limit of quantification (ca. 137 ng/g fresh wt tissue). APH4 concentration measured in pollen at the pre-harvest stage was ca. 2.3 µg/g air-dried pollen.
The level of APH4 protein in cotton event COT102 tissues was too low to extract sufficient amounts for further study. To obtain sufficient quantities of the APH4 protein for safety studies, it was necessary to express the aph4 gene in an E. coli production system.
Since it was not possible to extract sufficient amounts of APH4 from COT102 tissues, equivalence of the COT102 and E. coli-produced APH4 proteins could not be confirmed. However, the use of the E. coli-produced APH4 protein as a test substance was deemed acceptable given the information provided concerning the sequence identity of the E. coli-produced APH4 protein and data demonstrating that the protein was enzymatically active.
The potential mammalian toxicity and allergenicity of the APH4 protein was evaluated. The protein lacks sequence homology to known allergens and protein toxins, it lacks a mode of action that suggests that it is intrinsically toxic, and it is rapidly degraded when exposed to conditions present in the gastrointestinal tract. These factors suggest that the APH4 protein is unlikely to be inherently toxic and that systemic exposure to the protein is unlikely following oral ingestion. Furthermore, no adverse effects were observed when the E. coli-produced APH4 protein was ingested by mice after a single oral dosage of 779 mg/kg body weight supporting the apparent lack of inherent toxicity of the APH4 protein. The lack of digestive stability suggests that the APH4 protein is unlikely to be a protein allergen.
3. Development Method
The vip3A(a) and aph4 genes were introduced into cotton event COT102 via Agrobacterium-mediated transformation of variety Coker 312 hypocotyl tissue. Transformants were selected on the basis of growth on culture medium containing hygromycin B. Cotton event COT102 was identified as a successful transformant and was chosen for further development.
4. Stable Integration into the Plant Genome
Southern Blot analysis of cotton event COT102 indicated that there is one copy of both the vip3A(a) and aph4 genes at a single site of insertion. Southern Blot analysis also indicated that backbone plasmid sequences were absent in the genome of COT102. The nucleotide sequencing of the inserted DNA demonstrated that the vip3A(a) and aph4 expressions cassettes were intact in cotton event COT102.
Segregation analyses were conducted across five generations of COT102. These five generations were analysed using ELISA for expression of the vip3A(a) protein. Statistical significance for the segregation data was determined using Chi square analysis. All generations segregated in a manner consistent with the finding of a single dominant allele that segregates according to the Mendelian laws of genetics.
IV. Criteria for the Environmental Assessment
Lines derived from cotton event COT102 will not be grown in Canada. However, Canada imports cottonseed, as well as a wide range of other cotton products, that is used as human food, livestock feed or other industrial products.
1. Potential of Cotton Event COT102 to Become a Weed of Agriculture or 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 US, 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 COT102 has not been modified to have altered cold-tolerance and information supplied by Syngenta Seeds Canada Inc. indicates that the reproductive and survival biology of cotton event COT102 is unchanged compared to unmodified counterparts.
The CFIA has concluded that cotton event COT102 is unlikely to become a weed of agriculture or invasive of natural habitats.
2. Potential for Gene Flow to Wild Relatives Whose 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.
The CFIA has therefore determined that gene flow from cotton event COT102 to wild relatives in Canada is not possible.
3. Altered Plant Pest Potential
Cotton is not a plant pest in Canada and the intended effect of the novel trait is unrelated to plant pest potential. In addition, agronomic characteristics of cotton event COT102 are similar to those described for currently commercialized cotton varieties.
The CFIA has therefore determined that cotton event COT102 does not present a plant pest concern.
4. Potential Impact on Non-Target Organisms
Separate acute oral toxicity studies were performed where VIP3A and APH4 proteins were administered to mice. No adverse effects were observed at 2700 mg/kg body weight and 779 mg/kg body weight for VIP3A and APH4 proteins, respectively. Amino acid sequence homology searches were also performed. The VIP3A and APH4 proteins did not display any characteristics of a potential toxin or allergen.
Rapid degradation of the VIP3A and APH4 proteins under the conditions present in the gastrointestinal tract indicates a minimal likelihood that the protein could survive and be absorbed through the gastrointestinal system. Consequently, the VIP3A and APH4 proteins would likely pose little risks to human and animal health.
Seed content analysis determined that proximates (moisture, fat, protein, fibre, ash), amino acids, fatty acids, minerals, gossypol and cyclopropenoid fatty acids all fall within accepted ranges.
Cotton event COT102 will not be grown in Canada and exposure to the novel gene and resulting enzyme is expected to be minimal to non-existent. In the event that cotton event COT102 seed was accidentally released into the environment, any resulting plants would not be expected to set seed.
Based on the above, CFIA has determined that the use of cotton event COT102 will not result in altered impacts on interacting organisms, including humans, when compared to currently commercialized cotton varieties.
5. Potential Impact on Biodiversity
No varieties of cotton, or wild relatives that can readily interbreed with cotton, grow in the Canadian environment. Cotton is not grown in Canada and is not adapted to the environmental conditions encountered in Canadian agricultural environments. Cotton event COT102 has not been modified to have altered cold-tolerance, and therefore is not expected to enter or survive in managed or unmanaged ecosystems.
The CFIA has therefore concluded that cotton event COT102 does not present any adverse impacts on biodiversity in Canada.
6. Potential for Development of Insect Resistance
Since cotton is not grown in Canada, as it is not adapted to environmental conditions found at these latitudes, an insect resistance management stewardship plan specific to this product is not required.
V. Criteria for the Livestock Feed Assessment
1. Potential Impact on Livestock Nutrition
Comparison of nutrients was made between transgenic cotton event COT102 and its parental non-transgenic control Coker 312. Cottonseed samples were collected from replicated plots 2001(3 locations) and 2002 (2 locations) growing seasons in the US, and analyzed for proximate, crude fibre (CF), acid detergent fibre (ADF), neutral detergent fibre (NDF) and total dietary fibre (TDF), minerals, fatty acids and amino acids. There were no statistically significant differences between COT102 and Coker 312 across locations for protein, crude fat, ash, crude fibre, ADF, NDF and TDF in both growing seasons. All means in both test and control seeds were within literature ranges. No statistically significant differences were observed between COT102 and Coker 312 for minerals analyzed in 2001. Significant differences were observed between COT102 and Coker 310 for Cu, Fe and Zn in 2002, but these levels were within literature ranges. No statistically significant differences were found between COT102 and Coker 310 for myristic, palmitic, palmitoleic, steric, oleic, linoleic, arachidic and behenic acids in both years. All fatty acids were within literature values. Lysine and serine levels in COT102 were significantly lower than the levels in Coker 310, however these differences were not consistent within locations. All amino acids measured were within literature ranges.
Cyclopropenoid fatty acids (sterulic, malvalic and dihydrosterculic acids) and gossypol (total and free) were analyzed in COT102 and compared to non-transgenic parental control Coker 310. There were no statistically significant differences between COT102 and Coker 310 for sterculic, malvalic and dihydrosterculic acids in cottonseeds analyzed within and across locations in 2002. All means for these anti-nutrients were within literature values. No statistically significant differences were observed in total and free gossypol between COT102 and Coker 310 cottonseed. All means were comparable to literature values.
The evidence provided by Syngenta Seeds Canada Inc. supports the conclusion that the nutritional composition of cotton event COT102 is equivalent to conventional cotton varieties.
2. Potential Impact on Livestock and Workers/Bystanders
The VIP3A protein shares no significant biologically relevant homology with known allergens, it is heat labile, and it is rapidly degraded under the conditions present in the gastrointestinal tract. The mechanism of action of the VIP3A protein is specific to insect pests; no signs of toxicity were demonstrated in single-dose oral toxicity studies in mice using VIP3A purified protein from an E. coli over expression system or using a lyophilized preparation of proteins extracted from leaves of VIP3A-Enriched Maize. These factors support the lack of intrinsic toxicity of the VIP3A protein to humans or livestock species.
The APH4 protein shares no significant biologically relevant homology with known toxins or allergens and it lacks a mode of action that suggests that it is intrinsically toxic. E. coli-produced APH4 protein is heat labile and it is rapidly degraded under the conditions present in the gastrointestinal tract. No signs of toxicity were demonstrated in a single-dose oral toxicity study in mice using purified APH4 protein from an E. coli over expression system. These factors support the lack of intrinsic toxicity of the APH4 protein.
These findings indicate that cotton event COT102, which produces VIP3A and APH4 proteins, are unlikely to result in adverse toxicological effects in livestock fed materials derived from COT102 or in humans exposed to the proteins. Based on the lack of inherent toxicity or allergenic properties demonstrated by the proteins, no significant risk to livestock and workers/bystanders is expected from exposure to the VIP3A and APH4 proteins produced by cotton event COT102.
Based on the detailed characterization provided (nutritional composition and agronomic data of the modified plant compared to the unmodified comparator) it is unlikely that the modification has had any unintended effects on the cotton event COT102.
The evidence provided by Syngenta Seeds Canada Inc. supports the conclusion that the potential impact on livestock and workers/by-standers of cotton event COT102 is equivalent to that of currently commercialized cotton lines.
VI. New Information Requirements
If at any time, Syngenta Seeds 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 cotton event COT102 materials in Canada or elsewhere, Syngenta Seeds 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 cotton event COT102 on the environment, livestock and human health, and may re-evaluate its decision with respect to the livestock feed use authorization of cotton event COT102.
VII. Regulatory Decision
Based on the review of data and information submitted by Syngenta Seeds Canada Inc., including comparisons of cotton event COT102 with the unmodified parental counterparts, the Animal Feed Division of the Animal Products Directorate, CFIA, has concluded that the novel gene and its corresponding trait does not confer to the plants any characteristic that would raise any concerns regarding the safety or nutritional composition of cotton line COT102. Cottonseed and cottonseed meal and hulls are currently listed in Schedule IV of the Feeds Regulations and are, therefore approved for use in livestock feeds in Canada. Cotton event COT102 has been assessed and found to be as safe and as nutritious as traditional cotton varieties. Cotton event COT102 and its products are considered to meet the present ingredient definitions and are approved for use as livestock feed ingredients in Canada. Cotton event COT102 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.
Livestock feed use of cotton event COT102 is therefore authorized as of March 25, 2011. Cotton event COT102 and any other cotton lines derived from it may be used as livestock feed provided no inter-specific crosses are performed, provided the intended uses are similar, provided it is known, based on characterization, that these plants do not display any additional novel traits and are substantially equivalent to currently grown cotton, in terms of their specific use and safety for the environment and for human and animal health and provided the novel genes are expressed at a level similar to that of the authorized line.
Cotton event COT102 is subject to the same phytosanitary import requirements as its unmodified counterparts.
Please refer to Health Canada's Decisions on Novel Foods for a description of the food safety assessment of cotton event COT102.
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