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Decision Document: DD96-07:
Determination of Environmental Safety of Monsanto
Canada Inc.'s Roundup® Herbicide-Tolerant
Brassica napus Canola Line GT200

This Decision Document has been prepared to explain the regulatory decision reached under the guideline Dir94-08 Assessment Criteria for Determining Environmental Safety of Plants with Novel Traits and the companion document Dir94-09 The Biology of Brassica napus L. (Canola/Rapeseed).

The Canadian Food Inspection Agency (CFIA), specifically the Plant Biosafety Office of the Plant Health and Production Division, has evaluated information submitted by Monsanto Canada Inc. regarding the canola line GT200. This line has Roundup-Ready^TM genes that express novel tolerance to glyphosate, the active ingredient of Roundup® herbicide. The Department has determined that this plant does not present altered environmental interactions when compared to existing commercialized canola varieties in Canada. A feed safety and efficacy review has not been undertaken as this line will not be commercialized. No seed of GT200, its progeny and byproducts, will enter human and livestock food and feed chains without safety assessments by Health Canada and CFIA.

Unconfined release into the environment and other B. napus lines derived from it, but without the introduction of any other novel trait, is therefore considered safe.

Table of Contents

1. Brief Identification of the Plant with Novel Traits (PNT)
2. Background Information
3. Description of the Novel Glyphosate Tolerance
   1. The Roundup-Ready^TM Genes
   2. Development Method
   3. Stable Integration into the Plant's Genome
4. Assessment Criteria for Environmental Safety
   1. Potential of the GT200 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 Invasive
   3. Altered Plant Pest Potential
   4. Potential Impact on Non-Target Organisms
   5. Potential Impact on Biodiversity
5. Regulatory Decision

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

II. Background Information

Monsanto Canada Inc. has developed a Brassica napus canola line, GT200, derived from the variety Westar, which expresses their proprietary Roundup-Ready^TM genes. These confer novel tolerance to glyphosate, the active ingredient of Roundup® herbicide, which can control or suppress economically important weeds in canola production. Line GT200 is very closely related to line GT73, that was authorized for environmental release in 1995 (see DD95-02).

The development of GT200 was based on recombinant DNA technology, by the introduction into the variety Westar of a genetic construct containing two bacterial derived genes (the Roundup-Ready^TM genes). One gene imparts reduced sensitivity to Roundup® at the biochemical site of the herbicide's activity and the other expresses an enzyme that enables the plant to degrade glyphosate. The expression of both genes is directed to the chloroplasts, the site of the herbicide activity, by the addition of genetic coding sequences of a plant derived transit peptide. No antibiotic resistance marker genes were introduced into GT200.

GT200 has been field tested in Canada under confined conditions from 1992 to 1994 in Alberta, Saskatchewan, Manitoba and Ontario.

Monsanto has submitted information that includes data generated from CFIA-managed cooperative trials. This information described:

• the identity of GT200, including a detailed description of the modification method;
• the stability of the gene insertions;
• the role of these genes and their regulatory sequences in the donor organisms;
• the molecular characterization of the genes;
• the identification and characterization of the novel proteins, including levels of
• expression and their potential toxicity to livestock and non-target organisms.

Scientific references were listed to support information where available. In addition, Monsanto has provided information, data and recorded observations comparing GT200 to Westar or other unmodified canola counterparts. These comparisons have addressed characteristics that included seed production (yield), days to maturity, silique shattering and susceptibility to the fungal pathogen blackleg.

The Plant Biosafety Office of the Plant Health and Production Division, CFIA, has reviewed the above information, considering the Regulatory Directive Dir94-08, for determining environmental safety of plants with novel traits, which lists the following assessment criteria:

• potential of the PNT to become a weed of agriculture or be invasive of natural habitats,
• potential for gene flow to wild relatives whose hybrid offspring may become more weedy or more invasive,
• potential for the PNT to become a plant pest,
• potential affects of the PNT or its gene products on non-target species, including humans, and
• potential impact on biodiversity.

III. Description of the Novel Glyphosate Tolerance

1. The Roundup-Ready^TM Genes

• Two genes have been introduced into the variety Westar, which in combination provide field level tolerance to glyphosate, the active ingredient in Roundup® herbicide. The exact nature of these genes is considered confidential business information by Monsanto.
• The first gene expresses a bacterial derived version of a plant enzyme involved in the shikimate biochemical pathway for the production of the aromatic amino acids phenylalanine, tyrosine and tryptophan. The plant version of this enzyme, ubiquitous in nature, is sensitive to glyphosate - the herbicide disrupts this essential pathway, leading to growth suppression or death of the plant. However, the bacterial derived version of this enzyme is highly insensitive to glyphosate and fulfils the aromatic amino acid needs of the plant.
• The second gene, also a bacterial derived gene from a strain of a species ubiquitous in nature, expresses an enzyme that degrades glyphosate, thereby deactivating the herbicidal effect. In line GT73, the coding sequence of this gene had been altered to enhance the efficiency of glyphosate degradation, compared to the original bacterial version (see DD95-02). Line GT200 contains the original bacterial version.
• A plant-derived coding sequence expressing a chloroplast transit peptide was co-introduced with each of the Roundup-Ready^TM genes. This peptide facilitates the import of the newly translated Roundup-Ready^TM enzymes into the chloroplasts, the site of both the shikimate pathway and glyphosate mode-of-action.
• Both genes associated with their transit peptide coding sequence, are linked to the same constitutive promoter. Expression of both Roundup-Ready^TM proteins was quantified, and averaged 0.031 and 0.105 µg/mg (f.w.) in seeds, and 0.031 and 0.069 µg/mg (f.w.) in leaves.
• The Roundup-Ready^TM proteins showed no significant homology with any known toxins or allergens. The novel enzyme associated with the shikimate pathway, is a version of an enzyme that is ubiquitous in nature, and therefore would not be expected to be toxic or allergenic to non-target organisms. For the second novel enzyme, which degrades glyphosate, Monsanto describes experiments with a variety of substrates that show that it has a narrow substrate specificity, and appears not to affect any plant specific pathways. Both proteins are inactivated by heat, and by proteases in simulated mammalian gastric and intestinal fluids.

2. Development Method

Brassica napus variety Westar was transformed using a disarmed non-pathogenic Agrobacterium tumefaciens vector; the vector contained the transfer DNA (T-DNA) region of an A. tumefaciens plasmid from which virulence and disease causing genes were removed, and replaced with the Roundup-Ready^TM genes. The T-DNA portion of A. tumefaciens plasmids are generally known to insert randomly into the plant's genome and the insertion is usually stable, as was shown to be the case in GT200.

3. Stable Integration into the Plant's Genome

Monsanto has provided information on segregation and Southern blot analysis that demonstrates that GT200 has a single genetic insert, consisting of single copies of the Roundup-Ready^TM genes.

GT200 is several generations removed from the original transformant. Mendelian inheritance of the herbicide tolerance, and Southern and polymerase chain reaction (PCR) analyses of third-generation material field tested in 1992 show the stability of the introduced traits.

IV. Assessment Criteria for Environmental Safety

1. Potential of the GT200 to Become a Weed of Agriculture or Be Invasive of Natural Habitats

CFIA has evaluated the data and information submitted by Monsanto Canada Inc., on the reproductive and survival biology of GT200. From this, the Department has found that vegetative vigor, overwintering capacity, time to maturity, seed production and yield were within the normal range of expression found in unmodified Westar. GT200 has no specific added genes for cold tolerance or winter hibernation. No overwintered plants were observed by Monsanto's cooperators in post-harvest years of field trials. The number of volunteers in the years following a field trial was comparable between plots of GT200 and Westar. GT200 did not show any stress adaptation other than its tolerance to glyphosate. Tests for resistance to the fungal pathogen Leptosphaeria maculans (blackleg) showed that GT200's susceptibility falls within the ranges currently displayed by Westar.

The biology of B. napus, described in Dir94-09, shows that this species normally is not invasive of unmanaged habitats in Canada. According to the information and data provided by Monsanto, GT200 was found to be no different from unmodified canola counterparts in this respect. CFIA concurs that no competitive advantage was conferred by the insertion of the Roundup-Ready^TM genes, other than tolerance to Roundup® herbicide. Glyphosate is commonly used for chemical fallow production and resistant canola volunteer plants will not be controlled. Still, they can be managed by growers using alternative herbicides with different modes of action.

The above considerations, together with the fact that the novel traits have no intended effect on weediness or invasiveness, have led CFIA to conclude that GT200 has no altered weed or invasiveness potential compared to currently commercialized canola varieties.

Note: A longer term concern, if there is general adoption of several different crop and specific herbicide weed management systems, is the potential development of crop volunteers with a combination of novel resistances to different herbicides. This could result in the loss of the use of these herbicides and any of their potential benefits. Therefore, agricultural extension personnel, in both the private and public sectors, should promote careful management practices for growers who use these herbicide-tolerant crops, to minimize the development of multiple resistance.

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

B. napus plants are known to outcross up to 30% with other plants of the same species, and potentially with plants of the species B. rapa, B. juncea, B. carinata, B. nigra, Diplotaxis muralis, Raphanus raphanistrum, and Erucastrum gallicum (see Dir94-09). Studies show that potential introgression of the herbicide tolerance is most likely to occur with B. rapa, the other major canola species, which occasionally is a weed of cultivated land, especially in the eastern prairies of Canada.

If glyphosate tolerant individuals did arise through interspecific or intergeneric hybridization, the tolerance would not confer any competitive advantage to these plants unless challenged by Roundup® herbicide. This would only occur in managed ecosystems where Roundup® is applied for broad spectrum weed control, or in plant varieties developed to exhibit Roundup® tolerance and in which Roundup® is used to control weeds. As with glyphosate tolerant B. napus volunteers, these individuals, should they arise, would be controlled using other available chemical means. Hybrids, if they developed, could potentially result in the loss of Roundup® as a tool to control these species. This however, can be avoided by the use of sound crop management practices.

The above considerations led CFIA to conclude that gene flow from GT200 to relatives is indeed possible, but would not result in increased weediness or invasiveness of these relatives.

3. Altered Plant Pest Potential

The intended effects of the novel herbicide tolerance trait is unrelated to plant pest potential, and B. napus is not a plant pest in Canada (see Dir94-09). In addition, agronomic characteristics, and qualitative and quantitative composition of GT200 were demonstrated by Monsanto to be within the range of values displayed by unmodified canola varieties. CFIA concurs with the conclusion that plant pest potential has not been inadvertently altered.

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, has led to the conclusion that these do not result in altered toxic or allergenic properties. The enzymes are rapidly inactivated in mammalian stomach and intestinal fluids by enzymatic degradation and pH-mediated proteolysis. Raw seeds of GT200 were shown to be substantially equivalent to current canola varieties for their content of glucosinolates and erucic acid. Seed protein profiles, amino acid and fatty acid compositions fall within the range of those of the unmodified counterparts. A search of the FAST amino-acid sequence database revealed no significant homology with known toxins or allergens entered in that database.

Based on the above, CFIA has determined that the unconfined release of GT200 will not result in altered impacts on interacting organisms, including humans, compared to current canola varieties.

5. Potential Impact on Biodiversity

The introduced genes were determined to be safe to non-target organisms. In addition, GT200 has no novel phenotypic characteristics which would extend its use beyond the current geographic range of canola production in Canada. Since outcross species are only found in disturbed habitats transfer of the novel herbicide tolerance would not impact unmanaged environments.

CFIA has therefore concluded that the potential impact on biodiversity of GT200 is equivalent to that of currently commercialized canola varieties.

V. Regulatory Decision

Based on the review of Monsanto Canada Inc.'s data and information submitted to the Plant Biosafety Office of the Plant Health and Production Division, CFIA concludes that the Roundup-Ready^TM genes and their corresponding novel Roundup® herbicide tolerance do not in themselves confer any intended ecological advantage to the plant or to its relatives, should gene flow occur. In addition, this novel herbicide tolerance does not alter the characteristics of GT200 with regards to the assessment criteria for environmental safety. Thorough comparisons of GT200 and unmodified B. napus canola showed no unexpected effects.

Unconfined release into the environment and other B. napus lines derived from it, but without the introduction of any other novel trait, is therefore considered safe.

GT200 is an experimental line. A feed safety and efficacy review has not been undertaken as this line will not be commercialized. Seed of GT200, its progeny and byproducts, are therefore not authorized to be fed to livestock at this time. No seed of GT200 nor its progeny will enter human or livestock food and feed chains without prior safety assessments by Health Canada and CFIA.

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: