Decision Document DD2011-83 Determination of the Safety of Syngenta Seeds Canada Inc.'s Cotton (Gossypium hirsutum) Event COT67B
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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 based on the environmental criteria in regulatory directive 94-08 (Dir 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 advice from the Plant and Biotechnology Risk Assessment (PBRA) Unit of the Science Strategies Directorate, has evaluated information submitted by Syngenta Seeds Canada Inc.'s regarding cotton event COT67B, which is resistant to certain lepidopteran insects. 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 COT67B is therefore authorized by the Animal Feed Division of the Animal Health Directorate as of June 17, 2011. Cotton event COT67B and any cotton lines derived from it may be used as livestock feed provided that:
- no inter-specific crosses are performed;
- the intended uses are similar;
- 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
- the novel genes are expressed at a level similar to that of the authorized line.
Cotton event COT67B is subject to the same phytosanitary import requirements as its unmodified counterpart.
Please note, that the livestock feed safety of novel feeds is a 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, Ontario K1A 0Y9
Table of Contents
- Brief Identification of the Modified Plant
- Background Information
- Description of the Novel Trait
- Criteria for the Environmental Assessment
- Potential of Cotton Event COT67B to Become a Weed of Agriculture or be Invasive of Natural Habitats
- Potential for Gene Flow from Cotton Event COT67B to Wild Relatives Whose Hybrid Offspring May Become More Weedy or More Invasive
- Altered Plant Pest Potential of Cotton Event COT67B
- Potential Impact of Cotton Event COT67B on Non-Target Organisms
- Potential Impact of Cotton Event COT67B on Biodiversity
- Potential for Development of Insect Resistance to Cotton Event COT67B
- 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 COT67B, OECD Unique Identifier SYN-IR67B-1|
|Applicant:||Syngenta Seeds Canada Inc.|
|Plant Species:||Cotton (Gossypium hirsutum)|
|Novel Traits:||Resistance to lepidopteran pests including tobacco budworm (Heliothus virescens), cotton bollworm (Helicoverpa zea), the pink bollworm (Pectinophora gossypiella) and cabbage looper (Trichoplusia ni).|
|Trait Introduction Method:||Agrobacterium-mediated transformation|
|Proposed 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
Syngenta Seeds Canada Inc. developed, through the use of recombinant DNA techniques, a cotton event resistant to a number of lepidopteran pests of cotton. The cotton event, designated as COT67B, was developed to provide a method to control yield losses from insect feeding damage caused by lepidopteran larvae. The target lepidopteran pests include the tobacco budworm, cotton bollworm, the pink bollworm and cabbage looper.
Cotton event COT67B was developed using recombinant DNA technology, resulting in the introduction of the full length cry1Ab (flcry1Ab) gene, which encodes the Cry1Ab protein of Bacillus thuringiensis subsp. kurstaki plus an additional 26 amino acid segment in the C-terminal portion of the protein, and an aminoglycoside phosphotransferase (aph4) gene of Escherichia coli, which encodes the enzyme hygromycin-B phosphotransferase. The 26 amino acid segment of FLCry1Ab is present in other Cry proteins and is thought to have been lost during evolution of the Cry1Ab gene. This segment was inserted into FLCry1Ab to enhance protein expression during plant development but has no apparent functionality in plants and is not contained in the region of the protein responsible for insecticidal activity. The FLCry1Ab protein imparts resistance to lepidopteran larvae to cotton event CTO67B. The hygromycin-B phosphotransferase enabled selection of transformed plant cells by growing the cells on artificial medium containing hygromycin B but the aph4 marker gene segregated away from the flcry1Ab gene during breeding and is not present in cotton event COT67B.
Syngenta Seeds Canada Inc. has provided data on the identity of cotton event COT67B, a description of the transformation method, data and information on the gene insertion site, gene copy number and levels of FLCry1Ab protein in the plant and the role of the inserted gene and regulatory sequences. The FLCry1Ab protein from COT67B was shown to be equivalent to the protein produced in an Escherichia coli expression system developed to produce the protein. The Escherichia coli produced FLCry1Ab was used to generate sufficient quantities of pure protein for safety studies. Data was provided for the evaluation of the potential toxicity of the novel protein to livestock and non-target organisms and potential allergenicity of the novel protein to humans and to livestock.
Agronomic and phenotypic evaluations were conducted on cotton event COT67B and non-transgenic control cotton lines grown at 22 locations throughout the U.S. cotton belt in 2002, 2004 and 2005. Characteristics of cotton event COT67B such as height-to-node ratios, node of first fruiting branch, days to 50% flowering, days to 50% open boll, and yield of seed cotton per acre were compared to those of unmodified cotton counterparts.
Nutritional components of cotton event COT67B such as proximates, amino acids and fatty acids were compared with unmodified cotton counterparts.
The Animal Feed Division, CFIA, with input from the PBRA of the CFIA, has reviewed the above information. The following assessment criteria as described in Chapter 2.6 of the Regulatory Guidance: Feed Registration Procedures and Labeling Standards and Dir94-08 were used to determine the safety and efficacy as livestock feed and the environmental safety of this novel feed:
- potential impact of cotton event COT67B on livestock nutrition;
- potential impact of cotton event COT67B on livestock and workers/by-standers;
- potential impact of cotton event COT67B to become a weed or be invasive of natural habitats;
- potential for gene flow from cotton event COT67B to wild relatives whose hybrid offspring may become more weedy or more invasive;
- potential of cotton event COT67B to become a plant pest;
- potential impact of cotton event COT67B or their gene products on non-target species, including humans;
- potential impact of cotton event COT67B on biodiversity; and
- potential for development of insect resistance to cotton event COT67B.
III. Description of the Novel Trait
1. Development Method
The flcry1Ab and aph4 genes were introduced in cotton event COT67B via Agrobacterium-mediated transformation of cells from cotton line Coker 312. Transformed cells containing the aph4 marker gene were selected for growing the cells on artificial medium containing hygromycin B. Cotton event COT67B was identified as a successful transformant and was chosen for further development as a cultivated line.
2. Resistance to Lepidopteran Insects
B.thuringiensis subsp. kurstaki is a common gram-positive soil-borne bacterium. In its spore forming stage, it produces several insecticidal protein crystals, including the ð-endotoxin Cry1Ab which is active against certain lepidopteran insects. This protein has been shown to be non-toxic to humans, other vertebrates and beneficial insects. B.thuringiensis subsp. kurstaki-based foliar insecticides have been registered for over 30 years in Canada and have a long history of safe use. The FLCry1Ab protein produced in cotton event COT67B was shown to be equivalent to the original microbial Cry1Ab protein plus a 26 amino acid segment which was inserted into the wild type gene prior to transformation. This segment, which promotes FLCry1Ab protein production in vitro, is present in other B.thuringiensis Cry proteins.
Unlike typical allergens, the FLCry1Ab protein is present at low levels in cotton event COT67B (approximately 0.0025% of seed), is not stable when digested by simulated gastric fluid, and is not glycosylated. Following incubation in simulated gastric fluid it was shown, through western blotting, that the FLCry1Ab protein was digested within one minute. The FLCry1Ab protein was also degraded in five minutes in simulated intestinal fluid.
The amino acid sequence of the FLCry1Ab protein was compared to several protein sequence databases and was shown to share no significant structural similarity with any known toxin or allergen. An acute mouse study reported no deleterious side effects when animals were administered FLCry1Ab protein by oral gavage at 1830 mg protein/kg body weight.
Due to the low levels of FLCry1Ab protein expressed in cotton event COT67B it was necessary to produce FLCry1Ab protein by bacterial fermentation to obtain sufficient quantities to conduct some of the safety studies (e.g., acute oral mouse toxicity study, simulated gastric and intestinal fluid digestion studies). The bacterial produced protein was compared to the plant produced protein and shown to be equivalent on the basis of its molecular weight, immunological reactivity, peptide mapping profile, glycosylation status, and functional activity.
Syngenta Seeds Canada Inc. has provided to the CFIA a method for detection and identification of cotton event COT67B.
3. Stable Integration into the Plant Genome
Molecular characterization by Southern blot analysis demonstrated that cotton event COT67B contains one copy of the flcry1Ab gene cassette inserted at a single site in the cotton genome. No additional elements, including intact or partial DNA fragments of the flcry1Ab cassette or backbone sequences from the plasmid vector, linked or unlinked to the intact insert, were detected in cotton event COT67B. Sequencing of the introduced DNA confirmed the organization of the genetic elements and revealed that the right and left borders of the insert were truncated. These deletions have no effect on the functionality of the DNA insert or the plant and this phenomenon has been previously observed with Agrobacterium transformation.
Studies confirmed the stability of the DNA insertion across several generations. The inheritance pattern of the flcry1Ab gene showed that this gene segregates according to Mendelian rules of inheritance for a single genetic locus.
IV. Criteria for the Environmental Assessment
Lines derived from COT67B cotton 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 of Cotton Event COT67B 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 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. COT67B cotton has not been modified to have altered cold-tolerance and information supplied by Syngenta Seeds Canada indicates that the reproductive and survival biology of COT67B cotton is unchanged compared to unmodified counterparts.
CFIA has concluded that COT67B cotton 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 (G. barbadense and G. tomentosum) are found only in tropical and sub-tropical regions.
The CFIA has therefore determined that gene flow from COT67B cotton to wild relatives in Canada is not possible.
3. Altered Plant Pest Potential of Cotton Event COT67B
The intended effect of the novel trait is unrelated to plant pest potential, and cotton is not a plant pest in Canada. In addition, agronomic characteristics of COT67B cotton are similar to those described for currently commercialized cotton varieties.
The CFIA has therefore determined that COT67B cotton does not present a plant pest concern.
4. Potential Impact of Cotton Event COT67B on Non-Target Organisms
COT67B cotton will not be grown in Canada. In the event that COT67B cotton seed was accidentally released into the environment, any resulting plants would not be expected to set seed. Therefore, exposure to the Cry1Ab protein in COT67B cotton is expected to be minimal to non-existent.
Cry1Ab has a history of safe-use in Canada with no evidence of adverse effect. The Cry1Ab protein shows no amino acid homology to known protein toxins other than other Cry proteins and is rapidly degraded with loss of insecticidal activity under conditions that simulate mammalian digestion. Cry1Ab produces no adverse effects at high dose in acute oral rodent toxicity studies and is safe to non-targets such as fish, birds, mammals and beneficial insects. Cry1Ab does not have a direct or indirect effect on the arthropod population, abundance of beneficial insect community structure and earthworm population.
The CFIA has determined that the use of COT67B cotton, when compared with currently commercialized cotton varieties, will not result in altered impacts on interacting organisms, including humans, with the exception of sensitive lepidopteran caterpillars feeding on cotton.
5. Potential Impact of Cotton Event COT67B 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. COT67B cotton event has not been modified to have altered cold-tolerance, and therefore is not expected to enter or survive in unmanaged ecosystems.
The CFIA has therefore concluded that COT67B cotton does not present any adverse impacts on biodiversity in Canada.
6. Potential for Development of Insect Resistance to Cotton Event COT67B
Since no lines derived from COT67B cotton will be grown in Canada, the potential development of field resistance need not be considered. A small possibility exists that lepidopteran caterpillars feeding on stored cotton seed may develop resistance to the insecticidal protein, however none of these insects are pests of cultivated crops, nor are they normally controlled with B.thuringiensis pesticides.
V. Criteria for the Livestock Feed Assessment
1. Potential Impact of Cotton Event COT67B on Livestock Nutrition
Comparison of nutrients was made between transgenic cotton event COT67B and the non-transgenic control, Coker 312. Cottonseed samples were collected from replicated plots in four US locations during the 2004 growing season and were analyzed for crude protein, ash, fat, crude fibre (CF), acid detergent fibre (ADF), neutral detergent fibre (NDF), total dietary fibre (TDF), fatty acids, amino acids, minerals and Vitamin E. There were no statistically significant differences between cotton event COT67B and the control cotton for crude protein, ash, fat, crude fibre, ADF, NDF, TDF and amino acids. All means were within literature ranges of commercial cotton varieties. No statistically significant differences were found between cotton event COT67B and Coker 312 cotton for myristic, palmitoleic, stearic, linoleic and linolenic acids. Statistically significant differences between transgenic and the control cottonseed were observed for palmitic, stearic and oleic acids, however the means were within the literature ranges of commercial cotton varieties. Calcium and vitamin E levels in cotton event COT67B cottonseed were higher than those of the control cottonseed, however the means were comparable to literature values.
Anti-nutrients and Toxicants
Cyclopropenoid fatty acids (sterculic, malvalic and dihydrosterculic acids) and gossypol (total and free) were analyzed in cotton event COT67B and compared to non-transgenic control cotton Coker 312 for cottonseed, meal and refined oil. There were no statistically significant differences between cotton event COT67B and Coker 312 cotton for sterculic and malvalic acids across locations, however statistically significant differences were observed between cotton event COT67B and control cotton for dihydrosterculic acid, but the levels were comparable to literature values. No statistically significant differences were observed between cotton event COT67B and Coker 312 cottonseed for both total and free gossypol. For cottonseed oil, the means for malvalic, sterculic and dihydrostrerculic acids for both the transgenic and control were within literature ranges of commercial cotton varieties.
The evidence provided by Syngenta supports the conclusion that the nutritional composition of cotton event COT67B is comparable to conventional cotton varieties.
2. Potential Impact of Cotton Event COT67B on Livestock, Workers and Bystanders
The history of use of microbial insecticidal mixtures containing Cry proteins and literature about Cry proteins suggest that the bacterial B.thuringiensis Cry proteins are not toxic to humans and other vertebrates.
Cry1Ab-like proteins are present in previously authorized cotton lines in Canada. The FLCry1Ab protein produced in cotton event COT67B is the same as the original B.thuringiensis Cry1Ab protein except that it contains an additional 26 amino acid segment at its C-terminus. This segment, which promotes FLCry1Ab protein production in vitro, has no apparent functionality in plants and is not contained in the region of the protein responsible for insecticidal activity. This segment is present in other Cry proteins and there is no reason to believe that its presence in FLCry1Ab would affect the potential impact of cotton event COT67B on livestock and workers/ bystanders.
The FLCry1Ab protein shares no biologically relevant significant homology with known mammalian allergens and toxins, is heat-labile, is not glycosylated, and is rapidly degraded under the conditions present in the gastrointestinal tract. In addition, no toxic effects were observed when the FLCry1Ab protein was ingested by mice as a single oral dose of 1830 mg/kg body weight. Cotton is not known for the production of endogenous allergens and the transformation event which produced cotton event COT67B would not be expected to induce their synthesis.
Based on the detailed characterization provided (nutritional composition and agronomic data of the modified plant compared to unmodified comparators) it is unlikely that the introduction of the FLCry1Ab protein has had any unintended effects on the modified plant.
The evidence provided by Syngenta Seeds Canada Inc. supports the conclusion that the potential impact on livestock and workers/by-standers of cotton event 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 COT67B 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 COT67B 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 cotton event COT67B.
VII. Regulatory Decision
Based on the review of data and information submitted by Syngenta Seeds Canada Inc., including comparisons of cotton event COT67B with the unmodified parental counterparts, the Animal Feed Division of the Animal Health 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 COT67B. 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 COT67B has been assessed and found to be as safe and as nutritious as traditional cotton varieties. Cotton event COT67B and its products are considered to meet the present ingredient definitions and are approved for use as livestock feed ingredients in Canada. Cotton event COT67B will not be grown in Canada nor can the seed over winter, therefore the release of the feed into the environment would result in neither intended nor unintended environmental effects.
Livestock feed use of cotton event COT67B is therefore authorized as of June 17, 2011. Cotton event COT67B and any other cotton lines derived from it may be used as livestock feed provided no inter-specific crosses are performed, provided that no inter-specific crosses are performed, the intended use(s) are similar, 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 the novel genes are expressed at a level similar to that of the authorized line.
Cotton event COT67B 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 COT67B.
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