DD2003-45: Determination of the Safety of Monsanto's Insect Resistant Bollgard II™ Cotton (Gossypium hirsutum L.)

Issued: 2003-06

This Decision Document has been prepared to explain the regulatory decision reached under the guideline Dir95-03 Guidelines for the Assessment of Livestock Feed from Plants with Novel Traits and based on the environmental criteria in 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 CFIA, with advice from the Plant Biosafety Office of the CFIA has evaluated information submitted by Monsanto Canada Inc. regarding Bollgard II, an insect resistant cotton. CFIA has determined that feed derived from this plant with a novel trait 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 the Bollgard II cotton is therefore authorized as of June 27, 2003. Bollgard II™ cotton event 15985 and any cotton varieties derived from it may be imported and/or released, provided no inter-specific crosses are performed, provided the intended use is similar and provided 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 potential environmental impact and livestock feed safety.

Bollgard II cotton is subject to the same phytosanitary import requirements as its unmodified counterparts.


Table of Contents

I. Brief Identification of Plant with Novel Traits (PNT)

II. Background Information

III. Description of the Novel Traits

  1. Development Method
  2. Resistance to Lepidopteran pests of Cotton
  3. Greek Letter - Beta-D-glucuronidase (GUS)
  4. Stable Integration into the Plant's Genome

IV. Criteria for the Environmental Assessment

  1. Potential to Become a Weed of Agriculture or 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
  5. Potential Impact on Biodiversity
  6. Potential for Development of Lepidopteran Resistance to the Bollgard II™ Cotton

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 Plant with Novel Traits (PNT)

Designation(s) of the PNT: Bollgard II™ cotton event 15985, OECD identifier MON-15985-7

Applicant: Monsanto Canada Inc.

Plant Species: Cotton (Gossypium hirsutum L.)

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

Trait Introduction Method: Microprojectile bombardment of plant cells.

Proposed Use of the 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

Monsanto Canada Inc. developed a cotton line resistant to certain lepidopteran pests. The cotton line, designated as Bollgard II™, was developed to provide a method to control yield losses from insect feeding damage caused by certain lepidopteran pests.

Bollgard II™ cotton line was developed using recombinant DNA technology, resulting in the introduction of a bacterial gene conferring lepidopteran resistance and a marker gene. These genes were introduced into the previously approved genetically modified Bollgard™ cotton. Bollgard™ cotton contains the cry1Ac gene which conveys resistance to certain lepidopteran pests and the nptII gene which confers 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. Please see decision document 96-14 "Determination of Environmental Safety of BollgardInsect Resistant Cotton (Gossypium hirsutum L.)" for a summary of the safety review of Bollgard™ cotton.

Monsanto Canada Inc. has provided data on the identity of the Bollgard II cotton event 15985, 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. The expression of the genes in Bollgard cotton and Bollgard II™ was compared. 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.

The Bollgard II™ cotton line was field tested in eight locations in the United States under confined research field trial conditions in 1998.

Agronomic characteristics of Bollgard II cotton line such as onset of flowering, susceptibilities to various cotton pests and pathogens were compared to those of unmodified cotton counterparts.

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

The Feed Section, 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 feed from this plant with novel traits:

  • potential impact of Bollgard II cotton on livestock nutrition,
  • potential impact of Bollgard II cotton on livestock and workers/by-standers,
  • potential of Bollgard II cotton to become a weed of agriculture or be invasive of natural habitats,
  • potential for gene flow from Bollgard II cotton to wild relatives whose hybrid offspring may become more weedy or more invasive,
  • potential of Bollgard II cotton to become a plant pest,
  • potential impact of Bollgard II cotton or their gene products on non-target species, including humans
  • potential impact of Bollgard II cotton on biodiversity, and
  • potential for development of Lepidopteran resistance to the Bollgard II™ cotton.

III. Description of the Novel Traits

1. Development Method

Bollgard™ cotton, also known as variety DP50B, which contains the cry1Ac gene and the nptII selectable marker gene was retransformed to introduce a second insect control gene, cry2Ab and the uidA marker gene. This variety was retransformed by particle acceleration technology with a gel purified linear DNA fragment containing the cry2Ab and the uidA coding regions. Transformed cells were identified using the uidA marker gene and cotton plants were regenerated. An appropriate event was selected and conventionally bred to create the Bollgard II commercial variety.

2. Resistance to Lepidopteran pests of Cotton

Bacillus thuringiensis var. kurstaki is a common gram-positive soil-borne bacterium. Bacillus thuringiensis var. kurstaki present in commercially available microbial pest control products contains both the cry2Aa and cry2Ab genes. The cry2Aa gene is expressed by Bacillus thuringiensis var. kurstaki, however, the cry2Ab gene is not expressed in these commercial products due to an inefficient promoter. As such the Cry2Ab protein is not expressed in soil bacteria or commercially available microbial formulations. The coding region of the Cry2Ab protein is similar to the Cry2Aa protein, sharing 88% amino acid sequence similarity.

Cry2Ab 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, pink bollworm and army worm.

The cry2Ab gene expressed in Bollgard II™ cotton is linked to a constitutive promoter, (i.e., results in expression in all cotton tissues). Cry2Ab protein expression was determined from plants grown in various locations across the USA. Mean Cry2Ab expression in Bollgard II™ was 23.8 µg /g fresh weight and 43.2 µg /g fresh weight in leaf and seed tissue respectively. In the whole plant the Cry2Ab protein averaged 8.8 µg /g fresh weight. Expression varied over the growing season, with the levels peaking mid season, then declining. Cry2Ab was below the level of quantitation (0.25 µg /g fresh weight) in pollen.

The new insert did not affect the protein expression of Cry1Ac or NPTII as compared to the parental line.

Unlike typical allergens, Cry2Ab protein is present at low levels (<0.004% dry weight in seeds) in the plant. Protein allergens are normally resistant to digestion unlike the Cry2Ab protein which was shown to degrade readily in simulated gastric fluid (digested within 15s), in simulated intestinal fluid incubations Cry2Ab was digested to the trypsin resistant core within 1 minute as was expected. Also unlike known allergens the Cry2Ab protein was susceptible to heat processing and was deactivated in cottonseed meal after 25 minutes at 121 °C and 30 psi, used to simulate the heat processing step used commercially to produce cottonseed meal.

The amino acid sequence of Cry2Ab was provided. A search for amino acid sequence similarity between the Cry2Ab protein and known allergens, using a database assembled from the public domain databases GenBank, EMBL, Pir and SwissProt, revealed no significant amino acid sequence homologies (based on sequence identity of 8 or more contiguous amino acids). A search of a similarly constructed database of known toxins indicated no amino acid sequence homologies between known toxins and the Cry2Ab protein, with the exception of homologies to other known Cry proteins found in Bacillus thuringiensis and related species.

Due to the low levels of Cry2Ab protein expressed in the cotton plant it was necessary to produce Cry2Ab protein by bacterial fermentation to obtain sufficient quantities to conduct some of the safety studies (acute oral mouse toxicity study, 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.

Monsanto Canada Inc. has provided to the CFIA a method for detection and identification of cotton containing the Cry2Ab protein.

3. Greek Letter - Beta-D-glucuronidase (GUS)

The development of plant varieties containing useful new traits depends on a means to select for the rare transformed cells containing the gene(s) of interest from those plants that fail to take up or maintain the added DNA. As a way to do this marker genes are used to identify the cells to be carried forward to the regeneration process. In this case Greek Letter - Beta-D-glucuronidase (GUS) was employed.

The uidA coding sequence encodes for the production of Greek Letter - Beta-D-glucuronidase (GUS) which allows for the selection of cells that also contain the cry2Ab gene. GUS is an exohydrolase that catalyzes the hydrolysis of a range of the Greek Letter - Beta-glucuronidases into their corresponding acids and the aglycones including the artificial substrate p-nitrophenyl-Greek Letter - Beta-D-glucuronide. Hydrolysis of this compound releases a blue dye that functions as a visual aid in the plant transformation process. GUS is naturally present in plants, animals and microorganisms.

The uidA gene was isolated from E. coli strain K12. The uidA sequence is 99.7% homologous to that of naturally present in human gut, with the 0.2% non-homology due to addition of a restriction site at the beginning of the sequence for plant transformation purposes and an one amino acid change in the plant inserted DNA.

The uidA gene is linked to a constitutive promoter (i.e., results in expression in all cotton tissues). Mean GUS protein expression was determined from plants grown in various locations across the USA. The expression of the GUS protein in Bollgard II™ cotton was 106 µg/g fresh weight in leaf, 58.8 µg/g fresh weight in seed.

Unlike typical allergens, GUS protein is present at low levels (<0.007% dry weight in seeds) in the plant. Protein allergens are normally resistant to digestion and heat processing unlike the GUS protein which was shown to degrade readily in simulated gastric fluid (digested within 15s), in simulated intestinal fluid incubations 91% of the initial GUS enzymatic activity dissipated after 120 minutes. Also, unlike known allergens the GUS protein is deactivated in cottonseed meal after 25 minutes at 121°C and 30 psi to used to simulate the heat processing step used commercially to produce cottonseed meal.

The amino acid sequence of GUS was provided. A search for amino acid sequence similarity between the GUS protein and known allergens, using a database assembled from the public domain databases GenBank, EMBL, Pir and SwissProt, revealed no significant amino acid sequence homologies (based on sequence identity of 8 or more contiguous amino acids). A search of a similarly constructed database of known toxins indicated no amino acid sequence homologies between known toxins and the GUS protein.

Due to the low levels of GUS protein expressed in the cotton plant it was necessary to produce GUS protein by bacterial fermentation to obtain sufficient quantities of the protein to conduct some of the safety studies (acute oral mouse toxicity study, 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 and immunological reactivity. The plant expressed GUS protein differs by a single amino acid substitution relative to the GUS protein produced by E. coli fermentation. Based on the information provided this single amino acid difference does not change the 3-D structure of the protein and was not deemed to affect the suitability of the bacterially produced GUS for use in safety studies.

4. Stable Integration into the Plant's Genome

Southern blot analysis of Bollgard II cotton event 15985 indicated that there is one site of integration of the introduced DNA which includes a single copy of the cry2Ab and uidA genes. The data demonstrates that the cry2Ab coding region and associated promoter and terminator sequences are intact. The data demonstrates that the uidA coding region and its terminator are intact, however, a section of the 5' end of the promoter is not present.

Southern blot analysis over 5 generations R1, R2, R3, R4 and BC3F2 probed with the cry2Ab coding region demonstrated the stability of the DNA insert. Segregation analysis was performed on three stages in the breeding process, designated as the R1, R2, BC1F1, and BC2F2. The backcross data is consistent with that of a single locus that segregates according to Mendelian genetics.

Southern blot analysis and Mendelian segregation data provides evidence of the stable inheritance of the genetic elements introduced into the Bollgard II™ cotton event 15985.


IV. Criteria for the Environmental Assessment

Bollgard II™ cotton event 15985 will not be grown in Canada and will only be imported as human food or livestock feed.

1. Potential to Become a Weed of Agriculture or be 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. Bollgard II™ cotton event 15985 has not been modified to have altered cold-tolerance and information supplied by Monsanto indicates that the reproductive and survival biology of Bollgard II™ cotton is unchanged compared to unmodified counterparts.

CFIA has concluded that Bollgard II cotton is unlikely to become a weed of agriculture or invasive of natural habitats.

2. Potential for Gene Flow to Wild Relatives Whose Hybrid 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 species of cotton (G. barbadense and G. tomentosum) are found only in tropical and sub-tropical regions.

The CFIA has therefore determined that gene flow from Bollgard II™ cotton 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 Bollgard II cotton event 15985 are similar to those described for currently commercialized cotton varieties.

The CFIA has therefore determined that Bollgard II cotton does not present a plant pest concern.

4. Potential Impact on Non-target Organisms

Studies were conducted to determine whether six species of beneficial insects (ladybird beetle, adult and larval honey bees, Collembola, green lacewing, parasitic wasp and earthworm) are susceptible to the Cry2Ab protein. No adverse effects were observed at the maximum environmental concentration to which these organisms would be exposed. In most of the studies, the no observed effect concentration (NOEC) exceeded the maximum predicted environmental concentration by 10- to over 100- fold.

Information was also supplied to the CFIA about studies conducted on two other non-target organisms. Bobwhite Quail and Channel Catfish fed Bollgard II™ cottonseed at 10 and 20 per cent of their diets, respectively, exhibited no mortality and no adverse effects on survival, growth or behaviour.

The potential for Cry proteins to effect non-target lepidopterans is well known. No species of endangered or threatened lepidopterans are known to feed on cotton. Risk to non-target lepidopterans is therefore expected to be negligible.

As Bollgard II™ cotton will not be grown in Canada, exposure to the Cry protein is expected to be minimal to non-existent. In the event that Bollgard II™ cotton 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 Bollgard II™ cotton event 15985, 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.

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. Bollgard II cotton event 15985 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 Bollgard II cotton does not present any adverse impacts on biodiversity in Canada.

6. Potential for Development of Lepidopteran Resistance to Bollgard IICotton

Since Bollgard II cotton event 15985 will not 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 in Canada, 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

Cottonseed from Bollgard II cotton event 15985 was compared with parental line DP50B in a replicated experiment (4 sites). Cottonseed from each line from four additional sites was also analysed (single rep per site). Composite oil and meal samples by line were obtained from the eight sites for these three lines in addition to line DP50 (non-transgenic control). Eight reference samples of commercial cottonseed, and three reference samples each of oil and meal from commercial lines were also used.

Various analyses on cotton seed, meal, and oil included: protein, fat, fibre, ash, amino acids, fatty acids, minerals (Ca, Cu, Fe, Mg, Mn, P, K, Na, Zn), gossypol, aflatoxins, vitamin E.

These analyses showed that there were no differences proximate composition, amino acids, minerals, vitamin E, gossypol or aflatoxins in line 15985 compared with DP50B. There were statistical differences in the levels of the fatty acids myristic, stearic, linoleic, and the cyclopropenoid fatty acids malvalic, sterculic, dihydrosterculic. The levels of these fatty acids were within the reference ranges.

The applicant concluded that of the 48 statistical comparisons of seed components, there were six instances where 15985 was significantly different from the parental control. In all of these instances, the level of the component measured was within the range observed in the reference samples. As well, differences observed were not consistent over all sites. There were no differences in content of gossypol or aflatoxins. In the oil and meal samples, nutrient and gossypol levels were within reference ranges.

A dairy cattle feeding trial was conducted, in which cattle were fed a diet containing 10% cotton seed in a typical total mixed ration containing DB50 vs Bollgard II, Bollgard, and Roundup Ready cottonseed. There were no differences in animal performance including feed intake, milk yield or composition, or cow body condition. As well, no transgenic DNA or plant protein was detected in milk samples.

The applicant showed that 15985 is equivalent in composition and in terms of antinutritional factors to cottonseed currently on the market.

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. Cotton expressed Cry2Ab and GUS 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 various feeding studies utilizing cottonseed meal (catfish, northern bobwhite quail and lactating dairy cattle) no adverse affects have been demonstrated. Additionally, mouse acute oral toxicity test with the Cry2Ab protein and the GUS protein were conducted, with no treatment related adverse effects demonstrated at 69 mg/kg GUS and 1450 mg/kg Cry2Ab, the highest doses tested.

Cotton is not known for the production of endogenous allergens and the transformation event which produced Bollgard II cotton event 15985 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, Monsanto Canada Inc. becomes aware of any information regarding risk to the environment, including the development of lepidopteran resistance, or risk to human or animal health that could result from release of these materials in Canada, or elsewhere, Monsanto Canada 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 the proposed feed use and environmental release and will re-evaluate its decision with respect to the livestock feed use and environmental release authorizations of Bollgard II cotton event 15985.


VII. Regulatory Decision

Based on the review of data and information submitted by Monsanto Canada Inc., including comparisons of Bollgard II™ cotton event 15985 with the unmodified parental counterparts, the Feed Section, CFIA, has concluded that the novel genes and their corresponding traits do not confer to these plants any characteristic that would raise any concerns regarding the safety or nutritional composition of Bollgard II cotton event 15985. 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. Bollgard II cotton has been assessed and found to be as safe and as nutritious as traditional cotton varieties. Bollgard II™ cotton and its products are considered to meet the present ingredient definitions and are approved for use as livestock feed ingredients in Canada. Bollgard II™ cotton 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 the Bollgard IIcotton event 15985 therefore authorized as of June 27, 2003. Bollgard IIcotton and any other cotton varieties derived from it may be imported and/or released, provided no inter-specific crosses are performed, provided the intended use is similar, and provided it is known, following thorough characterization that these plants do not display any additional novel traits and are substantially equivalent to currently grown cotton, in terms of their livestock feed safety and environmental impact.

Bollgard II cotton 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 the Bollgard II™ cotton event 15985. 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