ARCHIVED - Novel Food Information - Insect Resistant Cotton (COT102)

Health Canada has notified Sygenta Seeds Canada Inc. that it has no objection to the sale of food derived fromInsect Resistant Cotton (COT102). The Department conducted a comprehensive assessment of this cotton event according to its Guidelines for the Safety Assessment of Novel Foods. These Guidelines are based upon internationally accepted principles for establishing the safety of foods with novel traits.

Background:

The following provides a summary of the notification from Sygenta Seeds Canada Inc. and the evaluation by Heath Canada and contains no confidential business information.

1. Introduction

Cotton line COT 102 was genetically modified using recombinant DNA techniques to introduce the coding sequence (vip3a) for the Vegetative Insecticidal Protein 3A (VIP3A) derived from the common soil bacterium Bacillus thuringiensis sp. strain AB88. VIP3A is an insecticidal enzyme effective against lepidopteran pests. This cotton line was also modified through in the introduction of the coding sequence for the antibiotic resistance gene hygromycin B phosphotransferase (aph4) derived from E.coli strain K12. This gene was introduced to act as a selectable marker.

The assessment conducted by Food Directorate evaluators assessed how Insect Resistant Cotton COT102 was developed; how its composition and nutritional quality compares to traditional cotton varieties; and the potential for the presence of any toxicants, anti-nutrients, or allergens. Syngenta has provided data which demonstrates that Insect Resistant Cotton COT102 is as safe and nutritious as conventional cotton varieties sold in Canada.

The Food Program has a legislated responsibility for pre-market assessment of novel foods and novel food ingredients as detailed in the Food and Drug Regulations (Division 28). Food use of Insect Resistant Cotton COT102 is considered a novel food under the following part of the definition of novel foods: "c) a food that is derived from a plant, animal or microorganism that has been genetically modified such that

(i) the plant, animal or microorganism exhibits characteristics that were not previously observed in that plant, animal or microorganism"

2. Development of the Modified Plant

The petitioner has provided information describing the methods used to develop COT102 cotton and molecular biology data that characterizes the genetic change which confers insect resistance. Commercial cotton variety coker312 was genetically modified using Agrobacterium tumefaciens transformation. The transforming plasmid pCOt1 carried a transfer DNA (T-DNA) sequence comprised of the vip3a gene cassette and the aph4 gene cassette.

The vip3a gene cassette contained the actin-2 (ACT2) promoter from Arabidopsis thaliana, a synthetic copy of the coding region of vip3a from Bacillus thuringiensis strain AB88 (vip3a) and the termination sequence of the nopaline synthase gene from Agrobacterium tumefaciens (Nos 3')

The aph4 gene cassette contained the promoter plus the first intron of the ubiquitin-3 gene of Arabidopsis thaliana, the coding region of aph4 from E.coli strain K12 and the termination sequence of the nopaline synthase gene from Agrobacterium tumefaciens (Nos 3')

3. Characterization of the Modified Plant

Southern blot analysis of COT102 cotton demonstrated the insertion of single copies of both the vip3a and aph4 gene cassettes in the cotton genome at a single locus. Southern blot analysis also demonstrated the integrity of the vip3a and the aph4 genes and their regulatory elements. Southern blot analysis also demonstrated, as expected, the absence of any plasmid derived sequences outside the T-DNA region, such as the spectinomycin resistance gene found in the plasmid backbone. The elements contained in both cassettes have been shown to be stable with no rearrangements through Southern blot and sequence analysis. These analyses of the insert show that the cassette is entirely integrated into the genome and that all the elements are intact.

The petitioner has provided data for five generations of COT102 cotton that demonstrated that the trait is stable and is inherited in the expected Mendelian manner.

4. Product Information

Cotton event COT102 differs from conventional cotton by the insertion of two novel genes; vip3A and aph4 and their associated regulatory elements. The insertion of these genes results in the expression of two novel proteins in MON87460; VIP3A and APH4. The expression of VIP3A confers resistance to lepidopteran pests. The expression of APH4 in COT102, confers resistance to the antibiotic hygromycin B phosphotransferase and was used a selectable marker for transformed plants.

The petitioner has provided data to demonstrate the level of expression of the VIP3A in COT102. This study used plant samples from three field sites planted in the major cotton growing locations in the United States. Each site was planted in three replicated plots using an complete block design. The quantities of VIP3A protein were determined by an enzyme-linked immunosorbent assay (ELISA) in nine tissue types at six developmental stages. Protein quantities for the tissues were calculated on a microgram (µg) per gram (g) fresh weight (fwt) basis. The range of VIP3A expressed in the whole plant from US-grown COT 102 was 1 - 13 µg/g fwt, with a mean value of 1.76 µg/g fwt.

The petitioner has provided data to demonstrate the level of expression of the APH4 in COT102. This study used plant samples from three field sites planted in the major cotton growing locations in the United States. Each site was planted in three replicated plots using an complete block design. The quantities of APH4 protein were determined by an enzyme-linked immunosorbent assay (ELISA) in nine tissue types at six developmental stages. Protein quantities for the tissues were calculated on a microgram (µg) per gram (g) fresh weight (fwt) basis. APH4 quantification results were consistent across sampling locations, with quantifiable levels of protein only be found in pollen (mean 2.25 µg/g fwt). In all other tissue types, APH4 was detected, but was below the LOQ of 150 ng/g fwt.

The petitioner also provided an analysis of the presence of VIP3A and APH4 in processed cotton products (cottonseed oil and linters) used for human food. The petitioner provided data demonstrating that neither VIP3A or APH4 were detectable in cotton fibre (ie. linters). The petitioner has also provided ELISA analysis of the cottonseed linters for the presence of VIP3A, which indicated that the protein was not detectable. Additionally, the petitioner has indicated that further testing revealed that no protein of any kind was detectable in cottonseed oil.

5. Dietary Exposure

Cottonseed from Insect Resistant Cotton COT102 will be used in applications similar to those derived from other cotton varieties. The use of cotton for human consumption is limited to refined cottonseed oil and linters, short fibres which are processed as a source of food grade cellulose and found in products such as bologna and sausage casings. The use of these linters in human food is routine, safe and well documented.

6. Nutrition

The nutrient data for this submission was obtained from a study conducted at two locations in the United States in 2002. COT 102 and control Coker 312 cotton lines were grown in four replicates at each site. The samples were analyzed for proximates, 18 amino acids, nine fatty acids, nine minerals, total and free gossypol, and cyclopropenoid fatty acids. Combined literature ranges were also provided for comparison.

The chemical and nutritional composition of COT 102 was similar to the control Coker 312, with very few statistical differences noted. In the few cases where significant differences were noted (for two amino acids (lysine and serine), copper, iron, and zinc), each difference was observed at only one site, and the concentrations of these nutrients were within the typical, published range for cotton.

7. Toxicology

The novel genes present in COT102 cotton were isolated from microbial organisms with no known pathogenicity to humans. Both the VIP3A and APH4 proteins were not acutely toxic to mice when administered by oral gavage at doses of 2700 mg/kg bw and 779 mg/kg bw, respectively. No mortality or clinical signs were observed with either protein. Neither protein showed any amino acid sequence homology with any known toxins.

The primary human food uses of cotton are limited to refined cottonseed oil and cottonseed linters. Both refined cottonseed oil and cotton fibre are essentially devoid of protein. Therefore, anticipated human dietary exposure to either of the novel proteins, VIP3A or APH4, in COT102 cotton through the direct consumption of cotton products would be negligible. Indirect human exposure to VIP3A and APH4 through the consumption of food animals would be non-existent.

Cotton contains two categories of naturally occurring toxicants (antinutrients), cyclopropenoid fatty acids (CPFAs) and several terpenoid phytoalexins (mainly gossypol), that limit its use as a food source. There are no significant differences in the levels of total and free gossypol in cottonseed from event COT102 and its non-trangenic parent. The levels and distribution of all antinutrients were comparable in the parent and transgenic plants. Therefore, in this regard, the novel plant does not raise any additional health concerns when compared to the non-transgenic parent from which it was derived.

Neither novel protein is considered likely to be an allergen since they do not share the characteristics of proteins that are food allergens. Unlike many food allergens, the novel proteins constitute a negligible amount of the total protein in food. No significant homology was found between the amino acid sequences of VIP3A and APH4 and those of any known allergen. Both novel proteins are rapidly degraded in simulated gastric fluid, as detected by western blot analysis, suggesting that both the VIP3A and APH4 proteins would be digested in the mammalian digestive tract.

Conclusion:

Health Canada's review of the information presented in support of the food use of Cotton event COT102 concluded that derived food products do not raise concerns related to safety. Health Canada is of the opinion that COT102 is similar to regular commodity cotton in terms of being an acceptable food source.

Health Canada's opinion deals only with the human food use of Cotton event COT102. Issues related to the environmental safety of COT102 in Canada and its use as livestock feed have been addressed separately through existing regulatory processes in the Canadian Food Inspection Agency.


This Novel Food Information document has been prepared to summarize the opinion regarding the subject product provided by the Food Directorate, Health Products and Food Branch, Health Canada. This opinion is based upon the comprehensive review of information submitted by the petitioner according to the Guidelines for the Safety Assessment of Novel Foods.

(Également disponible en français)

For further information, please contact:

Novel Foods Section
Food Directorate
Health Products and Food Branch
Health Canada, PL2204A1
251 Frederick Banting Driveway
Ottawa, Ontario K1A 0K9

Telephone: (613) 941-5535
Facsimile: (613) 952-6400

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