Decision Document DD2011-87:
Determination of the Safety of Bayer CropScience Inc.'s TwinLink (T304-40 X GHB119) cotton (Gossypium hirsutum) and Cotton Events T304-40 and GHB119

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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 Labeling Standards, 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 Health Directorate, with advice from the Plant and Biotechnology Risk Assessment (PBRA) Unit of the Plant Health Science Directorate, has evaluated information submitted by Bayer CropScience Inc. regarding TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119, which are resistant to certain lepidopteran insects and tolerant to glufosinate ammonium herbicide. The CFIA has determined that feed derived from these modified plants 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 TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119 is therefore authorized by the Animal Feed Division of the Animal Health Directorate as of December 15, 2011. TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119 and any cotton lines derived from them may be used as livestock feed provided that:

  1. no inter-specific crosses are performed;
  2. the intended use(s) are similar;
  3. 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
  4. the novel genes are expressed at a level similar to that of the authorized lines.

TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119 are subject to the same phytosanitary import requirements as their unmodified counterparts.

Please note, that the livestock feed safety of novel feeds is a critical step 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
Telephone: 613-225-2342

Table of Contents

  1. Brief Identification of the Modified Plant
  2. Background Information
  3. Description of the Novel Trait
    1. Development Methods
    2. Resistance to Lepidopteran Insects
    3. Glufosinate-Ammonium Herbicide Tolerance
    4. Stable Integration into the Plant Genome
  4. Criteria for the Environmental Assessment
    1. Potential of TwinLink (T304-40 X GHB119) Cotton and Cotton Events T304-40 and GHB119 to Become Weeds of Agriculture or be Invasive of Natural Habitats
    2. Potential for Gene Flow from TwinLink (T304-40 X GHB119) Cotton and Cotton Events T304-40 and GHB119 to Wild Relatives Whose Hybrid Offspring May Become More Weedy or More Invasive
    3. Altered Plant Pest Potential of TwinLink (T304-40 X GHB119) Cotton and Cotton Events T304-40 and GHB119
    4. Potential Impact of TwinLink (T304-40 X GHB119) Cotton and Cotton Events T304-40 and GHB119 on Non-Target Organisms
    5. Potential Impact of TwinLink (T304-40 X GHB119) Cotton and Cotton Events T304-40 and GHB119 on Biodiversity
    6. Potential for Development of Insect Resistance to TwinLink (T304-40 X GHB119) Cotton and Cotton Events T304-40 and GHB119
  5. Criteria for the Livestock Feed Assessment
    1. Potential Impact of TwinLink (T304-40 X GHB119) Cotton and Cotton Events T304-40 and GHB119 on Livestock Nutrition
    2. Potential Impact of TwinLink (T304-40 X GHB119) Cotton and Cotton Events T304-40 and GHB119 on Livestock and Workers/Bystanders
  6. New Information Requirements
  7. Regulatory Decision

I. Brief Identification of the Modified Plant

Designation of the Modified Plant: Cotton event GHB119 has the OECD designation BCS-GHØØ5-8. Cotton event T304-40 has the OECD designation BCS-GHØØ4-7. T304-40 and GHB119 were conventionally bred to create TwinLink (T304-40 X GHB119) cotton.
Applicant: Bayer CropScience Inc.
Plant Species: Cotton (Gossypium hirsutum)
Novel Traits: Cotton event GHB119 is resistant to lepidopteran pests such as larvae of tobacco budworm (Heliothus virescens) and cotton bollworm (Helicoverpa zea) and tolerant to glufosinate ammonium herbicide. Cotton event T304-40 is resistant to lepidopteran pests such as larvae of tobacco budworm, cotton bollworm, and fall armyworm (Spodoptera frugiperda), and tolerant to glufosinate ammonium herbicide. TwinLink (T304-40 X GHB119) cotton possesses all the traits of the parental lines.
Trait Introduction Methods: Agrobacterium-mediated transformation for cotton events T304-40 and GHB119 and conventional breeding of cotton events T304-40 and GHB119 for TwinLink (T304-40 X GHB119) cotton
Proposed Use of the Modified Plants: 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 Inc. developed, through the use of recombinant DNA techniques, cotton events resistant to a number of lepidopteran pests of cotton and tolerant to tolerant to glufosinate ammonium herbicide. The cotton events, designated as T304-40 and GHB119, were conventionally bred to produce TwinLink (T304-40 X GHB119) cotton. These cottons were developed to control yield losses from insect feeding damage caused by lepidopteran larvae. The target lepidopteran pests include the tobacco budworm, cotton bollworm, and fall army worm.

Cotton event GHB119 was developed using recombinant DNA technology, resulting in the introduction of the cry2Ae gene of Bacillus thuringiensis, which encodes the Cry2Ae protein, conferring resistance to lepidopteran larvae such as bollworm, tobacco budworm, and fall armyworm, and the bar gene of Streptomyces hygroscopicus, which encodes the phosphinothricin N-acetyltransferase (PAT) protein, conferring tolerance to glufosinate ammonium herbicide. Cotton event T304-40 was developed using recombinant DNA technology, resulting in the introduction of the cry1Ab gene of B. thuringiensis, which encodes the Cry1Ab protein, conferring resistance to lepidopteran larvae such as bollworm and tobacco budworm, and the bar gene. TwinLink (T304-40 X GHB119) cotton is the result of a conventional cross between T304-40 and GHB119 transgenic cotton events. TwinLink (T304-40 X GHB119) cotton expresses the Cry1Ab and the Cry2Ae insecticidal proteins, as well as the PAT protein.

Bayer CropScience Inc. has provided data on the identities of TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119, a description of the transformation methods, data and information on the gene insertion sites gene copy number, and levels of Cry2Ae, Cry1Ab, and PAT proteins in the plants and the roles of the inserted genes and regulatory sequences. The Cry2Ae, Cry1Ab, and PAT proteins from TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119 were shown to be equivalent to the proteins produced in bacterial expression systems developed to produce the proteins. The microbially-produced reference proteins were used to generate sufficient quantities of pure protein for safety studies. 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.

Agronomic and phenotypic evaluations were conducted on TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119 and non-transgenic control cotton lines grown at seven locations throughout the U.S. cotton belt in 2007 and 2008. 22 agronomic parameters were evaluated in TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119, including plant height, days to bloom, % open bolls, height-to-node ratios and yield.

Nutritional components of TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119 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 directive Dir94-08 were used to determine the safety and efficacy as livestock feed and the environmental safety of this novel feed:

  • potential impact of TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119 on livestock nutrition;
  • potential impact of TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119 on livestock and workers/bystanders;
  • potential of TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119 to become a weed or be invasive of natural habitats;
  • potential for gene flow from TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119 to wild relatives whose hybrid offspring may become more weedy or more invasive;
  • potential of TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119 to become a plant pest;
  • potential impact of TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119 or their gene products on non-target species, including humans;
  • potential impact of TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119 on biodiversity; and
  • potential for development of insect resistance to TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119.

Bayer CropScience Inc. has provided to the CFIA methods for detection and identification of TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119.

III. Description of the Novel Traits

1. Development Method

The cry2Ae and bar genes were introduced in cotton event GHB119 and the cry1Ab and bar genes were introduced into cotton event T304-40 via Agrobacterium-mediated transformation of cells from cotton varieties Coker 312 and Coker 315, respectively. Transformed cells were selected for by growing in the presence of glufosinate ammonium. Cotton events T304-40 and GHB119 were identified as successful transformants and were chosen for further development as cultivated lines. TwinLink (T304-40 X GHB119) cotton is the result of a conventional cross between T304-40 and GHB119 transgenic events.

2. Resistance to Lepidopteran Insects

B. thuringiensis is a common gram-positive soil-borne bacterium. There have been many reports of B. thuringiensis subtypes producing, in their spore forming stage, insecticidal protein crystals, including the delta-endotoxins Cry1Ab and Cry2Ae. Cry1Ab and Cry2Ae display activity against certain lepidopteran insects. The Cry1Ab protein has been shown to be non-toxic to humans, other vertebrates and beneficial insects, and B. thuringiensis subsp. kurstaki-based foliar insecticides have been registered for over 30 years in Canada and have a long history of safe use.

Unlike typical allergens, the Cry1Ab and Cry2Ae proteins produced in TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119 are present at low levels in cottonseed (maximum 1.14 µg/g dwt Cry1Ab and 0.63 µg/g dwt Cry2Ae), are not stable when digested by simulated gastric fluid, and are not glycosylated.

The amino acid sequences of the Cry1Ab and Cry2Ae proteins were compared to several protein sequence databases and were shown to share no significant structural similarity with known toxins or allergens. Acute mouse studies reported no deleterious side effects when animals were administered Cry1Ab or Cry2Ae proteins by oral gavage at 2000 mg protein/kg body weight.

Due to the low levels of Cry1Ab and Cry2Ae proteins expressed in TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119 it was necessary to produce these proteins by bacterial fermentation in Escherichia coli and B. thuringiensis, respectively, 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, and heat stability). The microbial produced proteins were compared to their respective plant produced proteins and were shown to be equivalent on the basis of some or all of the following criteria: molecular weight, immunological reactivity, peptide mapping profile, glycosylation status, and N-terminal sequence.

3. Glufosinate-Ammonium Herbicide Tolerance

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 bar gene engineered TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119 encodes the enzyme PAT. This enzyme detoxifies phosphinothricin by acetylation into an inactive compound. The bar gene was originally isolated from S. hygroscopicus, an aerobic soil bacterium. The PAT enzyme is therefore naturally occurring in the soil. More generally, acetyltransferase enzymes are ubiquitous in nature.

Unlike typical allergens, the PAT protein produced in TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119 is present at low levels in cottonseed (maximum 3.1 µg/g dwt), is not stable when digested by simulated gastric fluid, and is not glycosylated.

The amino acid sequence of the PAT protein was compared to several protein sequence databases and was shown to share no significant structural similarity with known toxins or allergens. Mouse studies reported no deleterious side effects when animals were administered PAT intravenously at 10 mg/kg body weight.

Due to the low levels of PAT protein expressed in TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119 it was necessary to produce this protein by bacterial fermentation in E. coli to obtain sufficient quantities to conduct some of the safety studies (e.g., acute oral mouse toxicity study and gastric and intestinal fluid digestion studies). The E. coli produced protein was compared to its respective plant produced proteins and was shown to be equivalent on the basis of some or all of the following criteria: molecular weight, immunological reactivity, peptide mapping profile, glycosylation status, and N-terminal sequence.

4. Stable Integration into the Plant Genome

Molecular characterization by Southern blot analysis demonstrated that cotton events T304-40 and GHB119 each contain a single DNA insertion. The GHB119 insertion consists of one intact copy of the cry2Ae-bar gene cassettes. No additional elements, including intact or partial DNA fragments of the cry2Ae-bar cassettes or backbone sequences from the plasmid vector, linked or unlinked to the intact insert, were detected. The T304-40 insertion consists of one nearly complete copy of the cry1Ab-bar gene cassettes flanked by an inverted incomplete copy of the cry1Ab gene cassette and one additional copy of the cry1Ab terminator. No backbone sequences were detected. The presence of additional DNA sequences associated with the cry1Ab-bar gene cassettes in T304-40 has 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 insertions across several generations. The inheritance pattern of the cry1Ab-pat and the cry2Ae-pat inserts showed that these insertions segregate according to Mendelian rules of inheritance for a single genetic locus.

IV. Criteria for the Environmental Assessment

Lines derived from TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119 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 TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119 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 events T304-40 and GHB119 have not been modified to have altered cold-tolerance and information supplied by Bayer CropScience indicates that the reproductive and survival biology of TwinLink (T304-40 X GHB119) cotton is unchanged compared to unmodified counterparts.

CFIA has concluded that TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119 are unlikely to become weeds 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 honey bees, 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 TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119 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 TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119 are similar to those described for currently commercialized cotton varieties.

The CFIA has therefore determined that TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119 do not present a plant pest concern.

4. Potential Impact on Non-Target Organisms

The Cry1Ab protein has a history of safe use in corn in Canada. The Cry1Ab protein shows no amino acid homology to known protein toxins other than other Cry proteins and is rapidly degraded under conditions that simulate mammalian digestion. The Cry1Ab protein produces no adverse effects at high dose in acute oral rodent toxicity studies and is safe to non-target organisms such as fish, birds and mammals. The Cry1Ab protein expressed in TwinLink (T304-40 X GHB119) cotton and cotton event T304-40 is unlikely to have a direct or indirect effect on arthropod populations, with the exception of sensitive lepidopteran larvae feeding on cotton.

Similarly, the Cry2Ae protein shows no amino acid homology to known protein toxins other than other Cry proteins. No adverse effects were observed in an acute oral toxicity study where Cry2Ae was administered to mice at 2000 mg/kg body weight. In addition, Cry2Ae has 86% identity with the Cry2Ab protein has a history of safe feed and food use in Canada. The Cry2Ae protein expressed in TwinLink (T304-40 X GHB119) cotton and cotton event GHB119 is unlikely to have a direct or indirect effect on arthropod populations, with the exception of sensitive lepidopteran larvae feeding on cotton.

Feeding studies confirmed that beneficial insects (spotted lady beetle, honey bee) and a soil invertebrate (springtail) are not adversely affected by exposure to Cry2Ae or TwinLink (T304-40 X GHB119) cotton.

Seed content analysis determined that levels of proximates (moisture, fat, protein, fibre, ash), amino acids, fatty acids, minerals, gossypol and cyclopropenoid fatty acids of TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119 all fall within accepted ranges for cotton.

TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119 will not be grown in Canada. In the event that seed from TwinLink (T304-40 X GHB119) cotton or cotton events T304-40 and GHB119 was accidentally released into the environment, any resulting plants would not be expected to set seed. Therefore, exposure of non-target organisms to the Cry1Ab and Cry2Ae proteins from TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119 is expected to be minimal to non-existent.

Based on the above, the CFIA has determined that the use of TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119 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 T304-40 and cotton event GHB119 have not been modified to have altered cold-tolerance, and therefore these events and TwinLink (T304-40 X GHB119) cotton are not expected to enter or survive in managed or unmanaged ecosystems. In addition, there is no evidence to suggest that TwinLink (T304-40 X GHB119) cotton or cotton events T304-40 and GHB119 would cause negative impacts on interacting organisms.

The CFIA has therefore concluded that TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119 do not present any adverse impacts on biodiversity in Canada.

6. Potential for Development of Lepidopteran 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 these lines is not required.

V. Criteria for the Livestock Feed Assessment

1. Potential Impact of Cotton Event TwinLink, GHB119, and T304-40 on Livestock Nutrition

TwinLink (T304-40 X GHB119) cotton

Nutritional Composition

Compositional data of TwinLink (T304-40 X GHB119) cotton was compared to that of its non-transgenic control Coker 315 cotton from seven replicated field trials in typical cotton producing areas in the US, during the 2007 growing season. Cottonseed samples were collected from replicated plots at each site and analysed for proximates, acid detergent fibre (ADF), neutral detergent fibre (NDF), amino acids, fatty acids, minerals and alpha-tocopherol (vitamin E). There were no statistically significant differences between TwinLink (T304-40 X GHB119) cotton and the control cotton at majority of the sites for crude protein, fat, ash, ADF and NDF. All mean values from both the transgenic and control cotton were within literature ranges. Statistically significant differences were observed between TwinLink (T304-40 X GHB119) cotton and the control cotton for myristic, behenic, stearic, oleic and linoleic, palmitoleic, linolenic and arachidic acids at some sites. All fatty acids of the transgenic and control cotton were however within literature values. Except for cystine and lysine, no statistically significant differences were observed between TwinLink (T304-40 X GHB119) cotton and the control cotton for the other amino acids. The mean values for cystine and lysine were within literature values. There were no statistically significant differences between TwinLink (T304-40 X GHB119) cotton and the control cotton for phosphorus, potassium and zinc. Significant effects were observed for magnesium, calcium, iron and alpha-tocopherol (Vitamin E) at some sites, however, means for both TwinLink (T304-40 X GHB119) cotton and the control cotton were within literature values for commercial cotton varieties.

Anti-nutrients

Phytic acid, total gossypol, free gossypol, and cyclopropenoid fatty acids (malvalic, sterulic and dihydrosterulic acids) were analyzed in TwinLink (T304-40 X GHB119) cottonseeds and compared to those of cottonseeds from its non-transgenic control, Coker 315. Except for free gossypol, significant differences between TwinLink (T304-40 X GHB119) cotton and the control were observed at few sites for the other anti-nutrients. However, all mean values for both TwinLink (T304-40 X GHB119) cotton and the control cotton were within literature values for commercial cotton varieties.

GH119 Cotton Event

Nutritional Composition

Compositional data of cotton event GHB119 was compared to that of its non-transgenic control Coker 312 from six replicated field trials in typical cotton producing areas in the US during the 2006 growing season. Cottonseed samples were collected from replicated plots at each site and analyzed for proximates, ADF, NDF, amino acids, fatty acids, minerals and tocopherols. There were no statistically significant differences between cotton event GHB119 and the control cotton for crude fat, ADF and NDF. Significant differences between cotton event GH119 and the control cotton were observed for ash and protein at some sites, however, all mean values were within the literature ranges. Significant differences were observed between cotton event GHB119 and the control cotton at some sites for the fatty acids (except behenic acid), however, the mean values were within commercial ranges. Significant differences were observed between cotton event GHB119 and the control cotton for most of the amino acids, however, all mean values for both transgenic and control were within the commercial ranges. No significant differences were observed between transgenic and control for magnesium, phosphorus, potassium, zinc, while significant differences were found for calcium, iron and the tocopherols at some sites. All mean values were within the commercial ranges.

Anti-nutrients

Phytic acid, total gossypol, free gossypol, and cyclopropenoid fatty acids (malvalic, sterulic and dihydrosterulic acids) were analysed in GHB119 cottonseeds and compared to those in cottonseeds from its non-transgenic control, Coker 312. Except for phytic acid, significant differences between transgenic and the control were observed at some sites for the other analytes. However, all mean values for both transgenic and control were within literature values for commercial cotton varieties.

T304-40 Cotton Event

Nutritional Composition

Compositional data of cotton event T304-40 was compared to that of its non-transgenic control Coker 315 from eight replicated field trials in typical cotton producing areas in the US, during the 2007 growing season. Cottonseed samples were collected from replicated plots at each site and analyzed for proximates, ADF, NDF, amino acids, fatty acids, minerals and alpha- tocopherols. No statistically significant differences were observed between cotton event T304-40 and the control cotton for crude fat and ADF. Statistically significant differences were found between cotton event T304-40 and the control cotton for ash, protein and NDF, however, all mean values were within literature ranges. Except for potassium, significant differences were observed for the minerals and alpha-tocopherol at some sites, but the mean values were within the range for commercial varieties. Except for cystine, palmitic, arachidic and lignoceric acids, significant differences were observed between cotton event T304-40 and the control cotton for amino acids and fatty acids at some sites, but the means for both the trangenic and control cotton were within literature ranges.

Anti-nutrients

Phytic acid, total gossypol, free gossypol, and cyclopropenoid fatty acids (malvalic, sterulic and dihydrosterulic acids) from T304-40 cottonseeds were compared to those of cottonseeds from the non-transgenic control, Coker 315. Except for total gossypol and malvalic acid, statistical significant differences were observed between transgenic and non-transgenic cotton for free gossypol, sterulic acid, dihydrosterulic acid and phytic acid at some sites. However, all means for both transgenic and control cottonseeds were within the literature ranges for commercial cotton varieties.

The evidence provided by Bayer CropScience supports the conclusion that the nutritional composition of Twin Link (GHB119 x T304-40); the parental lines T304-40 and GHB119 are substantially equivalent to the conventional cotton varieties.

2. Potential Impact of Cotton Event TwinLink, GHB119, and T304-40 on Livestock and Workers/ Bystanders

The Cry1Ab, Cry2Ae, and PAT proteins expressed in TwinLink (T304-40 X GHB119) cotton and cotton events TGH119 and T304-40 do not possess homology with any known allergens or toxins. The Cry1Ab and Cry2Ae proteins are heat labile, and all three proteins are rapidly degraded under conditions similar to those encountered in the gastrointestinal tract. This information, in addition to familiarity with Cry1Ab and PAT proteins, suggests that none of three proteins considered are likely to be allergenic or toxic to humans or animals outside of the expected lepidopterans species.

No adverse effects from the proteins protein were observed in acute oral or intravenous toxicity studies in mice using levels over 100 times higher than the highest predicted livestock dose level per kg body weight, assuming consumption of cotton leaves and cotton seed as 100% of the forage and grain factions of the diet, respectively. Additional supporting information for Cry2Ae was provided to show that it was similar to other Cry2 proteins previously assessed by the CFIA.

The evidence provided by Bayer CropScience Inc. supports the conclusion that the potential impacts on livestock and workers/bystanders of TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119 are equivalent to those of currently commercialized cotton lines.

VI. New Information Requirements

If at any time, Bayer CropScience Inc. becomes aware of any information regarding risk to the environment, including risk to human or animal health, which could result from release of TwinLink (T304-40 X GHB119) cotton, cotton events T304-40 or cotton event GHB119 materials in Canada or elsewhere, Bayer CropScience 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 TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119 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 TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119.

VII. Regulatory Decision

Based on the review of data and information submitted by Bayer CropScience Inc., including comparisons of TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119 with the unmodified parental counterparts, the Animal Feed Division of the Animal Health Directorate, CFIA, has concluded that the novel gene and their corresponding trait does not confer to the plants any characteristic that would raise any concerns regarding the safety or nutritional composition of cotton line TwinLink, GHB119, and T304-40. 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. TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119 have been assessed and found to be as safe and as nutritious as traditional cotton varieties. TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119 and its products are considered to meet the present ingredient definitions and are approved for use as livestock feed ingredients in Canada. TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119 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 TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119 are therefore authorized as of December 15, 2011. TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119 and any other cotton lines derived from them may be used as livestock feed provided 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.

TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119 are subject to the same phytosanitary import requirements as their unmodified counterparts.

Please refer to Health Canada's Decisions on Novel Foods for a description of the food safety assessment of TwinLink (T304-40 X GHB119) cotton and cotton events T304-40 and GHB119.

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