DD1998-27: Determination of the Safety of Calgene's BXN™ Cotton (Gossypium hirsutum L.)

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Issued: 1997-10

This Decision Document has been prepared to explain the regulatory decision reached under the guidelines Dir95-03 Guidelines for the Assessment of Plants with Novel Traits as Livestock Feed and Dir94-08 Assessment Criteria for Determining Environmental Safety of Plants with Novel Traits.

The Canadian Food Inspection Agency (CFIA), specifically the Feed Section of the Animal Health and Production Division, with input from the Plant Health Risk Assessment Unit, CFIA and advice from the Plant Biotechnology Office, CFIA, has evaluated information submitted by Calgene Inc. Coker 315 cotton plants (Gossypium hirsutum) were transformed with genes conferring resistance to the herbicide bromoxynil and resistance to kanamycin which was used as a selectable trait. The CFIA has determined that feed derived from these plants with novel traits should not pose a concern to environmental or livestock feed safety.

Feed use, as cottonseed and meal of the BXN™ cotton lines 10215, 10222 and 10224 is therefore authorized. Also, any Gossypium hirsutum lines derived from them, or resulting from the same transformation events, may be released, provided that: no inter-specific crosses are performed; the intended use is similar; and it is known following thorough characterization that these plants do not display any additional novel traits.

Table of Contents

  1. Brief Identification of the Plant with a Novel Trait (PNT)
  2. Background Information
  3. Description of the Novel Traits
    1. Tolerance to Bromoxynil Herbicide
    2. Kanamycin Resistance
    3. Development Method
    4. Stable Integration into the Plant's Genome
  4. Assessment Criteria for Environmental Safety
    1. Potential of the PNT to become a Weed of Agriculture or to be Invasive of Natural Habitats
    2. Potential for Gene Flow to Wild Relatives Whose Hybrid 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
  5. Nutritional Assessment Criteria for Use as Livestock Feed
    1. Nutritional Composition of the PNT
    2. Anti-nutritional Factors
  6. Regulatory Decision

I. Brief Identification of the Plant with a Novel Trait (PNT)

Designation(s) of the PNT: BXN™ cotton lines 10215, 10222, 10224

Applicant: Calgene Inc.

Plant Species: Cotton (Gossypium hirsutum L.).

Novel Traits: Tolerance to bromoxynil herbicide; kanamycin (antibiotic) resistance.

Trait Introduction Method: Agrobacterium tumefaciens-mediated transformation.

Proposed Use of PNT's: 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

Calgene Inc. has developed three cotton lines tolerant to bromoxynil herbicide. These cotton lines, referred to as BXN™ cotton (lines 10215, 10222, and 10224) in the present document express a novel tolerance to bromoxynil herbicide. This novel herbicide tolerance permits the use of bromoxynil in cotton production to control or suppress economically important weeds.

The development of the BXN™ cotton lines was accomplished with recombinant DNA technology, resulting in the insertion of two bacterial genes into the Coker 315 cotton variety. A gene expressing a nitrilase enzyme that enables the plant to transform bromoxynil into non- phytotoxic compounds was introduced, along with a gene conferring resistance to the antibiotic kanamycin. The antibiotic resistance gene is of no agricultural interest but the trait was used to select transformed from non-transformed plants during the development phase.

Field tests for the BXN™ cotton lines took place at 57 locations in 13 states in the United States. The lines have received complete approval for commercialization in the United States. Health Canada has determined that cottonseed oil derived from this cotton is substantially equivalent to that derived from currently commercialized cotton (August 1996.)

Calgene has provided data on the identity of the BXN™ cotton lines, a description of the modification method, data and information on the gene insertion sites, copy numbers and levels of expression in the plant, the role of the inserted genes and regulatory sequences in donor organisms, and full nucleotide sequences. The novel proteins were identified, characterized, and compared to the original bacterial proteins,. Relevant scientific publications were also supplied.

Agronomic characteristics such as yield, plant vigour, growth, morphology, germination and flowering, were compared to those of unmodified cotton counterparts. Stress adaptation was evaluated, including susceptibility to various pathogens. Processing qualities of cotton lint such as micronaire, length, strength and elongation were compared. The modified plant was determined to be not significantly different to the unmodified counterpart.

Data to support the suitability of cottonseed and cottonseed meal from the BXN™ cotton lines as livestock feed was provided. Proximate analyses, including crude protein, crude fat, crude fibre, and ash, were provided. The amino acid composition, and residual oil in the cottonseed meal of the modified and unmodified plants was also provided.

The Feed Section of the Plant Products Division, with input from the Plant Health Risk Division, CFIA and advice from the Plant Biotechnology Section has reviewed the above information. The following assessment criteria, as described in the regulatory directives Dir94- 08 and Dir95-03, were used to determine the suitability as livestock feed and the environmental safety of feed from these plants with novel traits:

  • potential impact on livestock
  • potential impact on livestock nutrition
  • potential impact of the PNTs or their gene products on non-target species, including humans
  • potential for the PNTs to become plant pests,
  • potential of the PNTs to become weeds of agriculture or to be invasive of natural habitats,
  • potential impact on biodiversity
  • potential for gene-flow to wild relatives whose hybrid offspring may become more weedy or more invasive

III. Description of the Novel Trait

1. Tolerance to Bromoxynil Herbicide

  • Bromoxynil is active against dicotyledonous plants by blocking electron flow during the light reaction of photosynthesis.
  • A nitrilase gene from Klebsiella pneumoniae subspecies ozaenae (BXN gene) has been introduced into the Coker 315 cotton variety to provide a field level of tolerance to bromoxynil herbicides.
  • The bacterial nitrilase enzyme catalyzes the breakdown of bromoxynil into non phytotoxic compounds.
  • The expression of the nitrilase gene is linked to a constitutive promoter which controls gene expression. The nitrilase gene is linked to a 3' region from an Agrobacterium gene which provides a termination sequence and polyadenylation site.
  • Nitrilase levels were measured by western blot in seeds (acid delinted seeds, kernels and hulls), processed cottonseed, and cotton leaf tissue. Control, untransformed Coker cotton, spiked with purified nitrilase enzyme, was used to quantify the expression of the enzyme in the transformed plants. The concentration of nitralase in seed tissue was near the limits of detection and lower than the spiked control which contained 3.5 ng of nitralase in 600 g of cottonseed protein from untransformed controls. The highest calculated concentration of nitralase in the unprocessed seed protein was 1.3 µg nitrilase/ g cottonseed (1.3 ppm). Nitrilase levels were calculated to be 0.6 µg nitrilase/ g total cottonseed meal protein (0.6 ppm). The maximum concentration of nitrilase enzyme in leaf tissue was calculated to be 20 µg/g total leaf protein.
  • The nitrilase protein showed no significant homology with any known toxins or allergens in the FastDB database. The nitrilase protein was also shown to have low stability under simulated gastric conditions. After 15 minutes in 0.1 units of bovine pancreatin all of the nitrilase was degraded to immunoactive peptides. By 8 hours no signal could be detected on a western blot. The enzyme which confers bromoxynil tolerance is a bacterial version of an enzyme that is ubiquitous in nature including monocot plants such as corn, wheat and barley, and therefore would not be expected to be toxic or allergenic.

2. Kanamycin Resistance

  • Kanamycin is an aminoglycosidic antibiotic that binds to bacterial ribosomes, thus disrupting normal protein synthesis and killing the bacterial cell.
  • The kanamycin-resistance gene, isolated from the bacterium E. coli, codes for an enzyme that phosphorylates kanamycin, thereby preventing it from binding to ribosomes and rendering the cells resistant.
  • The gene is linked to a constitutive promoter. Expression levels for the aminoglycoside 3'-phosphotransferase ii (APH(3') ii) enzyme were determined by western blot. A spiked control was used to determine the relative concentration of APH (3')ii in the samples. The maximum concentration of APH(3')ii was 6.6 µg/g total protein, 5.7 µg/g total protein and 80 µg/g total protein, in seeds, meal and leaves respectively.
  • This protein is ubiquitous in the environment. It degrades rapidly in vitro in simulated mammalian gastric and intestinal fluids.
  • The nucleotide sequence shows no significant DNA homology with known toxins or allergens

3. Development Method

  • Coker line 315 cotton was transformed using a disarmed non-pathogenic Agrobacterium tumefaciens vector. The vector contained the T-DNA region of an Agrobacterium plasmid from which virulence and plant disease-causing genes were removed, and genes coding for bromoxynil and kanamycin resistance were inserted. The T-DNA portion of the plasmid is known to insert randomly into the plant's genome and the insertion is usually stable, as was demonstrated in the BXN™ cotton.

4. Stable Integration into the Plant's Genome

  • Plants that were five generations removed from the original transformant were probed for the presence of the BXN gene on a southern blot, then reprobed for the kanamycin resistance gene. The hybridization patterns were shown to be identical to the original blots showing that no rearrangement had taken place and that the insertion was stable. Field trials demonstrated that the gene was inherited in a Mendelian manner.

IV. Assessment Criteria for Environmental Safety

1. Potential of the PNT to Become a Weed of Agriculture or be Invasive of Natural Habitats

Cotton (G. 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 considered a weed pest in the regions where it is grown. It is not grown in Canada as it is not adapted to environmental conditions found at these latitudes

BXN™ lines have no specific added genes for cold tolerance or winter hibernation; no overwintered plants are possible. No cultivation of these cotton lines in any location in Canada will occur.

The biology of Gossypium hirsutum, described in the submission, shows that unmodified plants of this species are not invasive of unmanaged habitats in Canada. According to the information provided by Calgene Inc. the BXN™ cotton lines were determined not to be different from their unmodified counterparts with regard to their environmental safety.

2. Potential for Gene Flow to Wild Relatives Whose Hybrid Offspring May Become More Weedy or More Invasive

The pollen is heavy and sticky and cross pollination usually occurs in the presence of insect pollinators (bumble bees and honey bees). Cotton is normally self pollinating and the range of natural crossing among cotton plants is very limited.

Wild species of cotton (G. barbadense and G. tomentosum) occur only in arid parts of the tropics and sub-tropics. There are no wild relatives in Canada that can naturally hybridize with G. hirsutum. The CFIA therefore concludes that gene flow from BXN™ lines to cotton relatives is not possible in Canada.

3. Altered Plant Pest Potential

The intended effects of both novel traits are unrelated to plant pest potential, and cotton is not a plant pest in Canada. In addition, agronomic characteristics of BXN™ lines are similar to those described for currently commercialized cotton varieties.

The CFIA has therefore determined that the BXN™ lines do not display any altered plant pest potential.

4. Potential Impact on Non-Target Organisms

The detailed characterization of the novel genes and resulting enzymes, as briefly summarized in Part iii of the present document, led to the conclusion that these do not result in altered toxic or allergenic properties. The nitrilase and APH(3')ii enzymes are rapidly inactivated in simulated gastric fluids by enzymatic degradation. Raw seeds of the BXN™ lines were shown to be substantially equivalent to conventional cotton varieties for their content of antinutritional factors. Seed protein content, amino acid, fibre, oil and fatty acid compositions fall within the range of those of the unmodified counterparts.

Based on the above, the CFIA has determined that the use of BXN™ lines in livestock feeds will not result in altered impacts on interacting organisms, including humans, when compared to current commercialized cotton varieties.

5. Potential Impact on Biodiversity

BXN™ lines have no novel phenotypic characteristics which would extend their use beyond the current geographic range of cotton production into Canada. Since cotton does not outcross to wild relatives in Canada, there will be no transfer of novel traits to unmanaged environments. The CFIA has determined that the BXN™ cotton lines will have no impact on biodiversity.

V. Nutritional Assessment Criteria for Use as Livestock Feed

1. Nutritional Composition of the PNT

Cottonseed contains a high number of anti-nutritional factors and raw seed is unsuitable for monogastric animals. Processing serves to lower the levels of endogenous toxins and renders the cottonseed meal suitable for livestock feed. Gossypol and cyclopropenoid fatty acids are naturally present in cotton and are considered to be undesirable, anti-nutritional compounds of concern for food and feed safety. Levels of gossypol and cyclopropenoid fatty acids measured in BXN™ cotton were similar to parental varieties and within the range for cultivated cotton. Gossypol content in the control Coker 315 line and the BXN™ lines was approximately 1% of the whole cottonseed. The natural range is from 0.002-6.64% of whole cottonseed. The average percent of cyclopropenoid fatty acids in cottonseed oil in the BXN cotton lines was 0.71. This value was the same in the controls.

2. Anti-Nutritional Factors

Proximate analysis of seed from the three BXN™ lines and three commercial varieties grown in four U.S. locations was conducted. While there were significant differences in protein and fat among the four locations, there was no effect of line, and no interaction between the locations and the lines, indicating that there were no differences between the BXN™ and control cotton lines.

Seed from the three BXN™ lines was shown to be substantially equivalent to the control commercial varieties in nutrient composition.

VI. Regulatory Decision

Based on the review of data and information submitted by Calgene Inc. and thorough comparisons of the BXN™ cotton lines with unmodified cotton counterparts, the Feed Section of the Plant Products Division has concluded that the novel genes and their corresponding traits do not in themselves raise any concerns regarding the safety or nutritional composition of the BXN™ lines. Cottonseed and cottonseed meal are currently listed in Schedule iv of the Feeds Regulations and are, therefore, approved for use in livestock feeds in Canada. As BXN™ cotton has been assessed and found to be substantially equivalent to traditional cotton varieties, with respect to safety and nutritional quality, BXN™ lines and their products are considered to meet the present ingredient definitions and are approved for use as livestock feed ingredients in Canada. None of the BXN™ cotton lines will be grown in Canada nor can the seed overwinter, therefore the release of the feed (meal or seed) into the environment would have no intended or unintended effect.

Feed use, as cottonseed and meal of the BXN™ cotton lines 10215, 10222 and 10224 is therefore authorized. Also, any Gossypium hirsutum lines derived from them, or resulting from the same transformation events, may be released, provided that: no inter-specific crosses are performed; the intended use is similar; and it is known following thorough characterization that these plants do not display any additional novel traits.

This bulletin is published by the Plant Health and Production Division. For further information, please contact the Plant Biosafety Office or the Feed Section at:

Plant Biosafety Office
Plant Health and Production Division
Plant Products Directorate
59 Camelot Drive, Nepean
Ontario, K1A 0Y9

Feed Section
Animal Health and Production Division
Animal Products Directorate
59 Camelot Drive, Nepean
Ontario, K1A 0Y9

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