DD2005-51: Determination of the Safety of Dow AgroSciences Canada Inc. Insect Resistant Cotton (Gossypium hirsutum L.) Event 3006-210-23

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Issued: 2005-07

This Decision Document has been prepared to explain the regulatory decision reached under the regulatory directive Dir95-03 Guidelines for the Assessment of Novel Feeds: Plant Sources and based on the environmental criteria in regulatory directive Dir94-08 Assessment Criteria for Determining Environmental Safety of Plants with Novel Traits.

The Canadian Food Inspection Agency (CFIA), specifically the Animal Feed Division of the CFIA, with advice from the Plant Biosafety Office of the CFIA has evaluated information submitted by Dow AgroSciences Canada Inc. regarding insect resistant cotton event 3006-210-23. 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 3006-210-23 is therefore authorized as of July 28, 2005. Cotton event 3006-210-23 and any cotton lines derived from it may be imported and/or released, 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.

Cotton event 3006-210-23 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 Traits

  1. Development Method
  2. Resistance to Lepidopteran pests of Cotton
  3. Glufosinate Ammonium Tolerance – Selection Marker
  4. Stable Integration into the Plant's Genome

IV. Criteria for the Environmental Assessment

  1. Potential of Cotton Event 3006-210-23 to Become a Weed of Agriculture or be 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
  6. Potential for Development of Lepidopteran Resistance to the cotton event 3006-210-23

V. Criteria for the Livestock Feed Assessment

  1. Potential Impact on Livestock Nutrition
  2. Potential Impact on Livestock and Workers/By-standers

VI. New Information Requirements

VII. Regulatory Decision

I. Brief Identification of the Novel feed

Designation(s) of the novel feed: Cotton Event 3006-210-23
OECD identifier DAS-21023-5

Applicant: Dow AgroSciences Canada Inc.

Plant Species: Cotton (Gossypium hirsutum L.)

Novel Traits: Resistance to lepidopteran pests of cotton, including cotton bollworm, tobacco budworm and pink bollworm

Trait Introduction Method: Agrobacterium mediated gene transfer

Proposed Use of the novel feed: Production of cotton for fibre, cottonseed and cottonseed meal 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 line resistant to certain lepidopteran pests. The cotton line, designated as event 3006-210-23, was developed to provide a method to control yield losses from insect feeding damage caused by certain lepidopteran pests.

Cotton event 3006-210-23 was developed using Agrobacterium mediated gene transfer technology, resulting in the introduction of bacterial genes conferring lepidopteran resistance and tolerance to glufosinate-ammonium.

Dow AgroSciences Canada Inc. has provided data on the identity of cotton event 3006-210-23, 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. Each novel protein was identified, the mode of action described, characterized and compared to the original donor bacterial proteins. An evaluation of their potential toxicity to livestock and non-target organisms and potential allergenicity to humans and to livestock was provided. Relevant scientific publications were also supplied.

Cotton event 3006-210-23 was field tested in multiple locations (6) in the United States under confined research field trial conditions in 2001.

Agronomic characteristics of cotton event 3006-210-23 such as plant morphology, disease susceptibility, agronomic performance and reproductive fitness were compared to those of unmodified cotton counterparts.

Nutritional components of cotton event 3006-210-23 such as proximates, amino acids and fatty acids were compared with unmodified cotton counterparts.

The Animal Feed Division, CFIA, with input from the Plant Biosafety Office, 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 3006-210-23 on livestock nutrition,
  • potential impact of cotton event 3006-210-23 on livestock and workers/by-standers,
  • potential of cotton event 3006-210-23 to become a weed of agriculture or be invasive of natural habitats,
  • potential for gene flow from cotton event 3006-210-23 to wild relatives whose hybrid offspring may become more weedy or more invasive,
  • potential of cotton event 3006-210-23 to become a plant pest,
  • potential impact of cotton event 3006-210-23 or their gene products on non-target species, including humans, and
  • potential impact of cotton event 3006-210-23 on biodiversity.

Additionally, CFIA has reviewed a method submitted by Dow AgroSciences Canada Inc. for the detection and identification of cotton containing the Cry1Ac protein.

Note: For commercialization, cotton event 3006-210-23 (Cry1Ac) will be stacked with cotton event 281-24-236 (Cry1F – see Decision Document 2005-52) forming cottonseed event 281-24-236/3006-210-23, which contains the genes for expression of Cry1F, Cry1Ac, and PAT proteins. This stacked event will be commercialized under the name of WideStrike® cotton. Some data presented in the application for authorization for cotton event 3006-210-23 came from the WideStrike® cotton.

III. Description of the Novel Traits

1. Development Method

The cotton acala germplasm line GC510 was transformed with a plasmid vector carrying the synthetic cry1Ac and pat genes. The DNA sequence was introduced by Agrobacterium mediated gene transfer. Transformants were selected based on tolerance to the herbicide glufosinate-ammonium in the culture medium. Event 3006-210-23 was identified as a successful transformant and was chosen for further development.

2. Resistance to Lepidoperan pests of Corn

Bacillus thuringiensis var kurstaki and Bacillus thuringiensis var entomocidus are common gram-positive soil-borne bacterium. In their spore forming stage, they produce several insecticidal protein crystals such as Cry1A(c) (i.e. cry endotoxins), that are active against certain Lepidopteran insect pests, such as the cotton bollworm, tobacco budworm and pink bollworm. This protein has been shown to be non-toxic to humans, other vertebrates and non-lepidopteran invertebrates. Foliar insecticides based on Cry endotoxins (generally known as B.t.) have been registered for over 30 years in Canada and have a long history of safe use.

A synthetic cry1A(c) gene was developed to maximize it's expression in cotton. The protoxin is a chimeric full length d-endotoxin comprised of the core toxin of Cry1Ac1 from Bacillus thuringiensis var kurstaki and the nontoxic portions of the Cry1Ca3 from Bacillus thuringiensis var entomocidus and the Cry1Ab1 protein from Bacillus thuringiensis var kurstaki. The Cry1Ca3 and the Cry1Ab1 comprise the chimeric C-terminal domain and are approximately those that are removed by alkaline proteases during the formation of the active Cry1Ac1 core toxin. The Cry1A(c) protoxin is 131 kDa where the truncated form is 65 kDa

This synthetic Cry1A(c) protein is insecticidal to Lepidoptera larvae after cleavage to a bio-active, trypsin resistant core. Insecticidal activity is believed to depend on the binding of the active fragment to specific receptors on the insect, midgut epithelial cells, forming a pore which disrupts osmotic balance and eventually results in cell lysis and insect death. Current knowledge indicates that only sensitive insects possess such receptors. Specific Lepidopteran pests of cotton, sensitive to the protein are: cotton bollworm, tobacco budworm and pink bollworm.

The synthetic cry1A(c) gene expressed in cotton line 3006-210-23 is linked to a constitutive promoter, (i.e. results in expression in all cotton tissues). Cry1A(c) protein expression was determined from plants grown in various locations across the USA. Mean Cry1A(c) expression in cotton line 3006-210-23 was 1.92 ng/mg dry weight and 0.57 ng /mg dry weight in young leaf and seed tissue respectively. In the whole plant (seedling stage) the Cry1A(c) protein averaged 1.59 ng/mg dry weight.

Protein allergens are normally resistant to digestion and heat processing unlike the Cry1A(c) protein which was shown to degrade readily in simulated gastric fluid, and be deactivated after exposure to 75 ºC to 90 ºC for 30 minutes.

The full nucleotide sequence of the synthetic cry1A(c) gene and corresponding amino acid sequence of Cry1A(c) protein were provided. A search for amino acid sequence similarity between the Cry1A(c) protein and known allergens which included food allergens, as well as non-food and wheat gluten allergens, using a database assembled from the public domain databases was conducted. The Cry1A(c) protein was compared to two protein allergen databases (one generated from literature sequences and SWISSPROT, and FAARP databases, the second containing all entries in the first database as well as additional entries from public protein databanks including protein database at NCBI). This comparison revealed that the Cry1A(c) protein does not share a linear epitope of 8 or more contiguous amino acids with known allergens. A search for homology with known toxins indicated no amino acid sequence homologies between known toxins and the Cry1A(c) protein, with the exception of homologies to other B.t. insecticidal delta-endotoxins. Additionally, the insecticidal protein was demonstrated not to be glycosylated, as many known allergens are, providing additional evidence that Cry1A(c) does not have the properties of known allergens.

Due to the low levels of Cry1A(c) protein expressed in the cotton plant it was necessary to produce Cry1A(c) protein by bacterial fermentation to obtain sufficient quantities to conduct some of the safety studies (e.g. acute oral toxicity study in mice, simulated gastric fluid digestion study and simulated 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. Neither the plant or bacterially expressed Cry1A(c) is glycosylated.

3. Glufosinate-Ammonium Tolerance – Selection Marker

Phosphinothricin, the active ingredient of glufosinate-ammonium herbicide, inhibits the plant enzyme glutamine synthetase, resulting in the accumulation of lethal levels of ammonia in susceptible plants within hours of application. Ammonia is produced by plants as a result of normal metabolic processes.

The glufosinate-ammonium tolerance gene engineered into cotton line 3006-210-23 codes for the enzyme phosphinothricin acetyltransferase (PAT). This enzyme detoxifies phosphinothricin by acetylation into an inactive compound. PAT has extremely high substrate specificity and data included in the submission indicates that it does not acetylate other enzymes or proteins.

The pat gene was originally isolated from Streptomyces viridochromogenes, an aerobic soil actinomycete bacterium. The PAT enzyme is therefore naturally occurring in the soil. More generally, acetyltransferase enzymes are ubiquitous in nature. The PAT protein expressed in event 3006-210-23 was used for selection of initial transformants only, and will not be used as a herbicide-tolerance trait in the field.

The pat gene is linked to a constitutive promoter. The expression of the pat gene in line 3006-210-23 was evaluated in leaf, flower, whole plant, root, pollen, nectar and seed. For all tissue samples taken in various locations across the USA, the levels of PAT protein were below the limit of detection except for the whole plant and seed with values of 0.11 ng/mg dry weight and 0.06 ng/mg dry weight respectively. The limit of quantification of the method used for the analysis was 0.002 to 0.4 ng/mg PAT protein/dry sample weight.

Unlike typical allergens, PAT protein is present at low levels in the plant. Protein allergens are normally resistant to digestion unlike the PAT protein which was shown to degrade readily in simulated gastric fluid (digested within 1 minute).

The enzyme amino acid sequences for the PAT protein was provided in the submission. A search for amino acid sequence similarity between the PAT protein and known allergens using a database assembled from the public domain databases was performed. The PAT protein was compared to two protein allergen databases (one generated from literature sequences and SWISSPROT, and FAARP databases, the second containing all entries in the first database as well as additional entries from public protein databanks including protein database at NCBI). This comparison revealed that the PAT protein does not share a linear epitope of 8 or more contiguous amino acids with known allergens. Additionally, a search for homology with known toxins indicated no amino acid sequence homologies between known toxins and the PAT protein. PAT only had high similarity with other Acetyl transferase proteins.

Due to the low levels of PAT protein expressed in the cotton plant it was necessary to produce PAT protein by bacterial fermentation to obtain sufficient quantities to conduct some of the safety studies (e.g. acute oral toxicity study in mice, simulated gastric fluid digestion studies). The bacterial produced protein was compared to the plant produced protein and shown to be of similar molecular weight and immunological reactivity. Neither the plant or bacterially expressed PAT is glycosylated.

4. Stable Integration into the Plant's Genome

Plants that were four and seven generations removed from the original transformant (BC3F1 and BC3F4) were probed for the presence of the synthetic cry1A(c) and pat gene on a southern blot. Southern blot analysis of cotton event 3006-210-23 indicated that there is one site of integration of the introduced DNA which includes a single copy of the synthetic cry1A(c) and PAT gene. The data demonstrates that the coding region for both the synthetic cry1A(c) and pat gene and associated promoter and terminator sequences are intact.

Southern blot analysis showed stability of the synthetic cry1A(c) and pat gene within a generation (BC3F2). Data presented also demonstrated that the genes segregated according to Mendelian inheritance over several generations (BC3F1, BC2F1, BC1F2 and BC3F2)

IV. Criteria for the Environmental Assessment

Lines derived from cotton event 3006-210-23 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 3006-210-23 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 3006-210-23 has not been modified to have altered cold-tolerance and information supplied by Dow AgroSciences indicates that the reproductive and survival biology of cotton event 3006-210-23 is unchanged compared to unmodified counterparts.

CFIA has concluded that Cry1Ac cotton event 3006-210-23 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 cotton event 3006-210-23 to wild relatives in Canada is not possible.

3. Altered Plant Pest Potential

The intended effects of the novel trait is unrelated to plant pest potential, and cotton is not a plant pest in Canada. In addition, agronomic characteristics of cotton event 3006-210-23 are similar to those described for currently commercialized cotton varieties.

The CFIA has therefore determined that cotton event 3006-210-23 does not present a plant pest concern.

4. Potential Impact on Non-Target Organisms

As cotton event 3006-210-23 will not be grown in Canada. In the event that cotton event 3006-210-23 seed was accidentally released into the environment, any resulting plants would not be expected to set seed. Therefore, exposure to the Cry1Ac protein is expected to be minimal to non-existent.

Studies were conducted to determine whether four species of beneficial insects (ladybird beetle, honey bees, green lacewing, parasitic wasp), two species of soil invertebrates (earthworms and Collembola), the aquatic invertebrate Daphnia magna, birds (Northern bobwhite quail), fish (Rainbow trouts) and mammals (mice) are susceptible to the Cry1Ac protein. No adverse effects were observed at doses than exceeded the maximum predicted environmental concentration by 4- to over 100- fold.

The potential for Cry proteins to effect non-target lepidopterans is well known. Incidental exposure of a non-target butterfly to Cry1Ac may occur if pollen is present on host plants and is consumed. Studies were conducted on first instars Monarch butterfly (Danaus plexippus L.). The dietary concentration resulting in 50% growth reduction relative to controls exceeds the estimated environmental concentration by over 10 fold. As cotton event 3006-210-23 will not be grown in Canada, risk to non-target lepidopterans is therefore expected to be negligible.

Based on the above, CFIA has determined that the use of cotton event 3006-210-23, 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 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. Cry1Ac cotton event 3006-210-23 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 Cry1Ac cotton event 3006-210-23 does not present any adverse impacts on biodiversity in Canada.

6. Potential for Development of Lepidopteran Resistance to the cotton event 3006-210-23

Since no lines derived from cotton event 3006-210-23 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 cottonseed may develop resistance to the insecticidal protein, however none of these insects are pests of cultivated crops, nor are they normally controlled with Bt pesticides.

V. Criteria for the Livestock Feed Assessment

1. Potential Impact on Livestock Nutrition

Nutritional Composition and Anti-Nutritional Factors

The nutrient/antinutrient analysis was conducted to demonstrate the equivalence of cotton event 3006-210-23 to conventional cottonseed. Along with cotton event 3006-210-23 (Cry1Ac) and the control variety, a second novel cotton event, 281-24-236 (Cry1F), as well as the stacked event 281-24-236/3006-210-23 (designated as WideStrike® cotton) were included in the same trial. Statistical analysis of the data considered all four of these treatments. The applicant provided compositional data from a study conducted using cottonseed from six sites in the United States in 2003. At each site (AZ, MS, 2 sites in TX, NC, CA) plots were arranged in a randomized block design containing 3 blocks, each containing the four treatments (Control, cotton event 281-24-236, cotton event 3006-210-23 and the stacked event WideStrike® cotton). Seed compositional analysis included proximates, acid detergent fibre, neutral detergent fibre, minerals Ca, P, Mg, K, Zn, Cu, Fe, Mn, amino acids, fatty acids, and vitamins. Differences observed included one amino acid (Trp) and one minor fatty acid (palmitoleic). These differences were not consistent across locations. No other significant treatment effects on composition were observed.

Seed from the same trial outlined above, from control, cotton event 281-24-236, cotton event 3006-210-23 and the stacked event WideStrike® cotton was analysed for cyclopropenoid fatty acids (sterculic, malvalic, dihydrosterculic), aflatoxins, total gossypol and free gossypol. There were no differences among varieties in the cyclopropenoid fatty acids. Total aflatoxins were all below 20 parts per billion in the control and transgenic varieties. For total gossypol, cotton event 3006-210-23 and the stacked event WideStrike® cotton were not different from the control, while cotton event 281-24-236 values were significantly lower than the control line. Gossypol levels were within the expected range reported from the literature. No differences in per cent free gossypol among the treatments were observed.

Information on nutritional composition of seed, oil and meal from a 2001 trial at the same locations was also submitted. This data was not amenable to statistical analysis, but provided some support the compositional data. Additionally, a 42-day feeding trial with broiler chickens fed one of 4 diets containing 10% cottonseed meal derived from the stacked event WideStrike® cotton, 2 different commercial lines, and a control non-transgenic line related to the stacked event WideStrike® cotton, further supported the equivalence of the stacked event WideStrike® cotton to other cottonseed meal used in livestock feed.

Based on the information submitted, the applicant has demonstrated that cotton event 281-24-236, cotton event 3006-210-23 and the stacked event WideStrike® cotton are equivalent to cotton varieties currently used as livestock feed.

2. Potential Impact on Livestock and Workers/By-standers

  • The history of use and literature suggest that the bacterial B.t. Cry proteins are not toxic to humans and other vertebrates. The low mammalian toxicity of B.t. microbial insecticide mixtures containing Cry proteins has been demonstrated over the last 40 years.
  • PAT is a highly substrate specific enzyme that has been well defined. Exposure to PAT protein is not new. The pat gene is isolated from Streptomyces viridochromogenes a common soil bacterium. The pat gene is present in the environment with no known adverse effects on humans and animals. In addition, PAT from the pat gene has been expressed in various crops authorized in Canada.
  • Cotton expressed Cry1A(c) and PAT proteins have been demonstrated to be rapidly digested under simulated gastric fluid conditions and feed processing conditions. These proteins have shown no sequence similarities with known allergens or toxins.
  • Based on a chicken feeding study utilizing cottonseed (protein expression level of 0.62 ppm), no adverse effects were demonstrated. Additionally, mouse acute oral toxicity studies with the Cry 1Ac protein, the microbial PAT protein, and the Cry 1F/Cry 1Ac microbial protein were conducted which showed no treatment related adverse effects demonstrated at 350 to 700 mg/kg, 5000 mg/kg, and 350 to 375 mg/kg respectively.
  • Cotton is not known for the production of endogenous allergens and the transformation event which produced cotton event 3006-210-23 would not be expected to induce their synthesis.

Based on the expected exposure levels and the results of the above tests, the CFIA concludes that, the introduced genes are unlikely to be novel toxins or allergens.

VI. New Information Requirements

If at any time, Dow AgroSciences becomes aware of any information regarding risk to the environment, or risk to human or animal health that could result from release of these materials in Canada, or elsewhere, Dow AgroSciences will immediately provide such information to the CFIA. On the basis of such new information, the CFIA will re-evaluate the potential impact of the proposed use and will re-evaluate its decision with respect to the livestock feed authorization of cotton event 3006-210-23.

VII. Regulatory Decision

Based on the review of data and information submitted by Dow AgroSciences, including comparisons of cotton event 3006-210-23 with the unmodified parental counterparts, the Animal Feed Division, 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 event 3006-210-23. 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 3006-210-23 has been assessed and found to be as safe and as nutritious as traditional cotton varieties. Cotton event 3006-210-23 and its products are considered to meet the present ingredient definitions and are approved for use as livestock feed ingredients in Canada. Cotton event 3006-210-23 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 3006-210-23 is therefore authorized as of July 28, 2005. Cotton event 3006-210-23 and any other cotton lines derived from it may be imported and/or released, provided no inter-specific crosses are performed, provided the intended uses are similar, and 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.

Cotton event 3006-210-23 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 3006-210-23. The food safety decisions are available at the following Health Canada web site: http://www.hc-sc.gc.ca/fn-an/gmf-agm/appro/index-eng.php

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
Telephone: 613-225-2342

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