DD2008-72: Determination of the Safety of Bayer CropScience's GlyTol™ Cotton Event GHB614

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Issued: 2008-04

This Decision Document has been prepared to explain the regulatory decision reached under Directive 95-03, entitled "Guidelines for the Assessment of Novel Feeds: Plant Sources" and based on the environmental criteria in regulatory 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 Products Directorate, with advice from the Biotechnology Environmental Release Assessment Unit (BERA) of the Science Strategies Directorate, has evaluated information submitted by Bayer CropScience regarding GlyTol™ cotton event GHB614, a herbicide tolerant cotton. 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 GHB614 is therefore authorized as of April 4, 2008. Cotton event GHB614 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 GHB614 is subject to the same phytosanitary import requirements as its unmodified counterparts.

Table of Contents

I. Brief Identification of the Novel feed

II. Background Information

III. Description of the Novel Trait

  1. Development Method
  2. Glyphosate Tolerance
  3. Stable Integration into the Plant's Genome

IV. Criteria for the Environmental Assessment

  1. Potential of cotton event GHB614 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
  3. Altered Plant Pest Potential
  4. Potential Impact on Non-Target Organisms
  5. Potential Impact on Biodiversity

V. Criteria for the Livestock Feed Assessment

  1. Potential Impact on Livestock Nutrition
  2. Potential Impact on Livestock and Workers/Bystanders

VI. New Information Requirements

VII. Regulatory Decision

I. Brief Identification of the Novel feed

Designation of the Modified Plant: Cotton Event GHB614, OECD Identifier BCS-GHØØ2-5

Applicant: Bayer CropScience

Plant Species: Cotton (Gossypium hirsutum)

Novel Traits: Herbicide tolerance (glyphosate)

Trait Introduction Method: Agrobacterium-mediated gene transfer

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

Bayer CropScience has developed a cotton line, designated GHB614, which is tolerant to glyphosate, the active ingredient in the herbicides Roundup, Glfos or Touchdown, for example. The herbicide tolerant trait in cotton event GHB614 provides a method for the control or suppression of weeds in cotton production.

Cotton event GHB614 was developed using Agrobacterium mediated gene transfer technology, resulting in the introduction of a 5-enolpyruvyl shikimate-3-phosphate synthase (epsps) gene containing two amino acid substitutions. This modified gene was designated 2mepsps. EPSPS is the sixth enzyme in the shikimate pathway implicated in the synthesis of aromatic amino acids. The modified 2mEPSPS protein is insensitive to glyphosate inhibition, but still retains its function in the shikimate pathway and thus aromatic amino acid synthesis continues as well as the production of secondary metabolites. The 2mEPSPS protein binds to normal EPSPS substrates (shikimate-3-phosphate and phosphoenolpyruvate) in a manner similar to maize EPSPS, but is significantly less sensitive to glyphosate inactivation. The introduction of the two amino acid substitutions in the EPSPS protein confers the tolerance to glyphosate.

Bayer CropScience has provided data on the identity of cotton event GHB614, a description of the transformation method, data and information on the gene insertion site, gene copy number and levels of 2mEPSPS protein in the plant and the role of the inserted gene and regulatory sequences. The 2mEPSPS protein from GHB614 was shown to be equivalent to the protein produced in an Escherichia coli expression system developed to produce the protein. The E. coli produced 2mEPSPS was used to generate sufficient quantities of pure protein for safety studies. References to relevant scientific publications were included in the submission. 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.

Phenotypic data for cotton event GHB614 were collected from nine replicated field trials conducted in 2005 in typical cotton producing areas in the United States and were compared to the non-transgenic cotton control Coker 312.

Agronomic characteristics of cotton event GHB614 such as plant morphology, disease susceptibility, agronomic performance and reproductive fitness were compared to those of unmodified cotton counterparts.

Nutritional components of cotton event GHB614 such as proximates, amino acids and fatty acids were compared with unmodified cotton counterparts.

The Animal Feed Division, CFIA, with input from the Biotechnology Environmental Release Assessment Unit, CFIA, has reviewed the above information. The following assessment criteria as described in regulatory directives Dir95-03 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 GHB614 on livestock nutrition;
  • potential impact of cotton event GHB614 on livestock and workers/by-standers;
  • potential impact of cotton event GHB614 to become a weed or be invasive of natural habitats;
  • potential for gene flow from cotton event GHB614 to wild relatives whose hybrid offspring may become more weedy or more invasive;
  • potential of cotton event GHB614 to become a plant pest;
  • potential impact of cotton event GHB614 or their gene products on non-target species, including humans, and
  • potential impact of cotton event GHB614 on biodiversity.

III. Description of the Novel Trait

1. Development Method

Cotton GHB614 was created through the Agrobacterium mediated insertion of a fragment of DNA containing a copy of the 2mepsps gene, which imparts field level tolerance to glyphosate, the active ingredient in Roundup, Glyfos or Touchdown herbicides, for example.

Cotton line Coker 312 was transformed with a plasmid vector (pTEM2-T-DNA) carrying the synthetic 2mepsps gene fused with a plant-derived coding sequence expressing an optimized plastid peptide. This peptide targets the mature protein to the plastid, where the wild type protein is located. The presence of 2mEPSPS enzyme results in a plant that is considerably less sensitive to inhibition by glyphosate than a plant with the endogenous EPSPS enzyme.

2. Glyphosate Tolerance

EPSPS is an enzyme involved in a cotton plant's shikimic acid metabolic pathway which is essential for the production of aromatic amino acids. The native cotton EPSPS enzyme is sensitive to glyphosate. The herbicide disrupts the shikimic acid pathway by binding to the EPSPS enzyme, leading to interference in aromatic amino acids production and growth suppression or death of the plant. The 2mEPSPS version of this enzyme is expressed in cotton event GHB614 and confers glyphosate tolerance since it continues to catalyze the production of aromatic amino acids in the presence of glyphosate due to a reduction in the binding of glyphosate to the 2mEPSPS in comparison to the native EPSPS.

Unlike typical allergens, the 2mEPSPS protein is present at low levels in cotton event GHB614 (less than 0.0100% of total protein in seed), is not stable when digested, is not glcosylated and the 2 amino acid substitutions are not potential sites for glycosylation. The protein was shown to be labile to digestion. Following incubation in simulated gastric fluid it was shown, through Western Blotting, that the 2mEPSPS protein was digested within 30 seconds. Also, the protein was degraded in under 30 seconds in simulated intestinal fluid.

The amino acid sequence of the 2mEPSPS was compared to several protein sequence databases and was shown to share no significant structural similarity with any known toxic, allergenic. An acute mouse study reported no deleterious side effects when animals were administered 2mEPSPS protein by oral gavage at doses up to 2000 mg protein/kg body weight.

Due to the low levels of 2mEPSPS protein expressed in cotton event GHB614 it was necessary to produce 2mEPSPS protein by bacterial fermentation to obtain sufficient quantities to conduct some of the safety studies (acute oral mouse toxicity study, simulated gastric and intestinal fluid digestion study). The bacterial produced protein was compared to the plant produced protein and shown to be of similar molecular weight, immunological reactivity and to have similar functional activity as the plant produced protein.

Bayer CropScience has provided to the CFIA a method for detection and identification of cotton event GHB614.

3. Stable Integration into the Plant's Genome

Southern Blot analysis of cotton event GHB614 indicated that there is one site of integration of the introduced DNA and that the 2mEPSPS gene expression cassette is intact. Southern Blot analysis also indicated that backbone plasmid sequences were absent in the genome of GHB614. Polymerase chain reaction (PCR) was used to confirm the organization of the elements within the DNA insert.

Southern Blot analysis showed stability of the introduced DNA in the first four generations removed from the original transformant. Data presented also demonstrated that the genes segregated according to Mendelian inheritance in these generations.

IV. Criteria for the Environmental Assessment

Lines derived from cotton event GHB614 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 GHB614 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. Cotton event GHB614 has not been modified to have altered cold-tolerance and information supplied by Bayer CropScience indicates that the reproductive and survival biology of cotton event GHB614 is unchanged compared to unmodified counterparts.

The CFIA has concluded that cotton event GHB614 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 GHB614 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 GHB614 are similar to those described for currently commercialized cotton varieties.

The CFIA has therefore determined that cotton event GHB614 does not present a plant pest concern.

4. Potential Impact on Non-Target Organisms

EPSPS proteins naturally occur in plant and microbial-based foods that have a history of safe consumption by humans and animals. An acute oral toxicity study using E. coli-produced 2mEPSPS protein was administered to mice. No adverse effects were observed at 2000 mg of 2mEPSPS protein/kg mouse body weight. During the study, there were no clinical signs, mortalities or treatment-related effects on body weight. Amino acid sequence homology searches were also performed. The 2mEPSPS protein did not display any characteristics of a potential toxin or allergen.

Rapid degradation of the 2mEPSPS protein in simulated gastric fluid or intestinal fluids indicates a minimal likelihood that the protein could survive and be absorbed through the gastrointestinal system. In case the protein survives the stomach, it would be rapidly degraded in the intestine. Consequently, the 2EPSPS protein 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 the range of those of the unmodified counterparts.

Cotton event GHB614 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 GHB614 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 GHB614 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 GHB614 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 cotton event GHB614 does not present any adverse impacts on biodiversity in Canada.

V. Criteria for the Livestock Feed Assessment

1. Potential Impact on Livestock Nutrition

Nutritional Composition

The compositional equivalence of cotton event GHB614 (unsprayed or sprayed with glyphosate herbicide) to its isogenic non-transgenic control Coker 312 was assessed from nine field trials in the US during the 2005 growing season. Cottonseed samples were collected from replicated plots at each site and analyzed for proximate, acid detergent fibre (ADF), neutral detergent fibre (NDF), amino acids, fatty acids, minerals and vitamin E. There were no statistically significant differences between transgenic cotton event GHB614 cotton (sprayed or unsprayed) and Coker 312 cotton for crude protein, ash, crude fat, carbohydrates, ADF and NDF. All means were within literature values. No statistically significant differences were observed between transgenic cotton event GHB614 (sprayed or unsprayed) and non-transgenic Coker 312 for threonine, valine, glycine, alanine, aspartic acid, glutamic acid, proline, serine, tyrosine, isoleucine, leucine, tryptophan, phenylalanine, histidine, cystine and methionine. All means were within the range of literature values. Statistically significant differences were observed between transgenic GHB614 (sprayed and unsprayed) treatments and non-transgenic Coker 312 for stearic, oleic and linolenic, while no statistically significant differences were observed for myristic, palmitic, palmitoleic, linoleic, arachidic and behenic acid. The means for stearic, oleic and linolenic acids in both cotton event GHB614 and Coker 312 were within the range of literature values. There were no statistically significant differences between cotton event GHB614 and Coker 312 for all minerals and vitamin E analyzed. All means were within the range of literature values.

Anti-nutrients

Phytic acid, total gossypol, free gossypol, and cyclopropenoid fatty acids (malvic, sterculic and dihydrosterculic acids) were analyzed in GHB614 cottonseed and compared to its isogenic non-transgenic control, Coker 312. No statistically significant differences were observed between GHB614 (sprayed and unsprayed) and Coker 312 for phytic acid, total gossypol and free gossypol. All means were within the range of literature values. There were no statistically significant differences between the transgenic and non-transgenic lines for malvic acid, while statistically significant differences were observed between GHB614 and Coker 312 cotton for sterculic and dyhdrosterculic acids. The means for sterculic and dyhdrosterculic acids for both GHB614 and Coker 312 were within the range of literature values.

The evidence provided by Bayer CropScience supports the conclusion that the nutritional composition of cotton event GHB614 is substantially equivalent to conventional cotton varieties.

2. Potential Impact on Livestock and Workers/Bystanders

The EPSPS protein is an enzyme present in many foods with a long history of safe use in Canada, and therefore would not be expected to be toxic or allergenic. The EPSPS is the sixth enzyme in the shikimate pathway, and is found in plants and microorganisms, but not animals. It is ubiquitous in nature, and as such, it is likely that both livestock and humans are exposed to EPSPS proteins on an ongoing basis. The 2mEPSPS protein shares no biologically relevant significant homology with known toxins or allergens, it is present in small amounts in the feed, it is heat labile and it is rapidly degraded under the conditions present in the gastrointestinal tract. Additionally, a single-dose oral toxicity study showed that the 2mEPSPS protein is not toxic to mice at a dose of 2000 mg 2mEPSPS/kg body weight. A further study showed that the 2mEPSPS protein was non-toxic to female mice following intravenous exposure of up to 10 mg 2mEPSPS/kg body weight. Additionally, the 2mEPSPS protein is rapidly degraded under conditions present in the gastrointestinal tract and does not have relevant sequence homology to any known toxins or allergens.

Cotton is not known for the production of endogenous allergens and the transformation event which produced cotton Event GHB614 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 the unmodified comparator) it is unlikely that the introduction of the 2mEPSPSS protein has had any unintended effects on the modified plant.

The evidence provided by Bayer CropScience supports the conclusion that the potential impact on livestock and workers/by-standers of cotton Event GHB614 is equivalent to that of currently commercialized cotton lines.

VI. New Information Requirements

If at any time, Bayer CropScience 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 GHB614 materials in Canada or elsewhere, Bayer CropScience 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 GHB614 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 GHB614.

VII. Regulatory Decision

Based on the review of data and information submitted by Bayer CropScience, including comparisons of cotton event GHB614 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 GHB614. 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 GHB614 has been assessed and found to be as safe and as nutritious as traditional cotton varieties. Cotton event GHB614 and its products are considered to meet the present ingredient definitions and are approved for use as livestock feed ingredients in Canada. Cotton event GHB614 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 GHB614 is therefore authorized as of April 4, 2008. Cotton event GHB614 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 GHB614 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 GHB614. The food safety decisions are available at the Health Canada web site.

This bulletin is published by the Canadian Food Inspection Agency. For further information, please contact the Animal Feed Division at:

Animal Feed Division
Animal Products Directorate
59 Camelot Drive
Ottawa, Ontario K1A 0Y9
Telephone: 613-225-2342
Fascimile: 613-773-7565

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