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Decision Document 98-21:
Determination of the Safety of Monsanto Canada Inc.'s
Roundup® Herbicide-Tolerant Brassica rapa
Canola Lines ZSR500, ZSR502 and ZSR503

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, the companion document T-1-12-96 The Biology of Brassica rapa L. (Canola/ Rapeseed) and the guidelines Dir95-03 Guidelines for the Assessment of Livestock Feed from Plants with Novel Traits.

The Canadian Food Inspection Agency (CFIA), specifically the Plant Biosafety Office of the Plant Health and Production Division and the Feed Section of the Animal Health and Production Division, has evaluated information submitted by Monsanto Canada Inc. regarding the canola lines ZSR500, ZSR502 and ZSR503. These lines were obtained through inter- specific crosses with B. napus plants transformed with Roundup-ReadyTM genes that express tolerance to glyphosate, the active ingredient of Roundup® herbicide (please see Decision Document DD95-02). The Agency has determined that these plants do not present altered environmental interactions when compared to existing commercialized canola varieties in Western Canada, and are considered substantially equivalent to canola (B. rapa) currently approved as livestock feed.

Unconfined release into the environment of the B. rapa canola lines ZSR500, ZSR502 and ZSR503 is authorized in British Columbia, Alberta, Saskatchewan and Manitoba. Their use as livestock feed is also authorized. Also, any B. rapa lines derived from them, resulting from the same transformation event or transformed with the same genetic construct, may be considered substantially equivalent, provided it is known following thorough characterization that: no inter-specific crosses are performed; the intended use is similar; and these plants do not display any additional novel traits.

Please note that, while determining the environmental safety of plants with novel traits is a critical step in the commercialization of these plant types, other requirements still need to be addressed, such as Variety Registration (CFIA).

Table of Contents

1. Brief Identification of the Plants with Novel Traits (PNT's)
2. Background Information
3. Description of the Novel Glyphosate Tolerance
   1. The Roundup-ReadyTM Genes
   2. Development Method
   3. Stable Integration into the Plants' Genome
4. Assessment Criteria for Environmental Safety
   1. Potential of the PNT's to Become Weeds 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. Nutritional Assessment Criteria for Use as Livestock Feed
   1. Anti-Nutritional Factors
   2. Nutritional Composition of the PNT's
6. Regulatory Decision

I. Brief Identification of the Plants with Novel Traits (PNT's)

II. Background Information

Monsanto Canada Inc. and Zeneca Seeds have developed three Brassica rapa canola lines, ZSR500, ZSR502 and ZSR503, derived from inter-specific crosses with the Brassica napus line GT73, that expresses Roundup-ReadyTM genes. These genes confer novel tolerance to glyphosate, the active ingredient of Roundup® herbicide, which can control or suppress economically important weeds in canola production. The B. napus line GT73, also referred to as RT73, was authorized for environmental release in 1995 (please see decision document DD95-02).

One of the Roundup-ReadyTM genes 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.

Glyphosate tolerant B. rapa has been field tested in Canada under confined conditions in Saskatchewan in 1995 and in Alberta, Saskatchewan and Manitoba in 1996.

Monsanto has referred to the information submitted for the environmental release approval of B. napus line GT73. They have also submitted information on the identity of ZSR500, ZSR502 and ZSR503, the description of the modification method, and provided expression levels of the novel proteins.

Scientific references were listed to support information where available. In addition, Monsanto has provided information, data and recorded observations comparing ZSR500, ZSR502 and ZSR503 to unmodified Polish canola counterparts. These comparisons have addressed characteristics that included days to flower, days to maturity, seed production (yield), crop height, lodging, white rust tolerance, protein and oil content, and fatty acid composition.

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's to become weeds 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's to become plant pests,
• potential effects of the PNT's or their gene products on non-target species, including humans, and
• potential impact on biodiversity.

The Feed Section of the Animal Health and Production Division, CFIA, has also reviewed the above information in light of the assessment criteria for determining safety and efficacy of livestock feed, as described in Dir95-03. These include:

• potential impact to livestock, and
• potential impact to livestock nutrition.

III. Description of the Novel Glyphosate Tolerance

1. The Roundup-ReadyTM Genes

• Two genes were introduced, which in combination provided field level tolerance to glyphosate, the active ingredient in Roundup® herbicide.
• The first gene expresses a bacterial derived version from Agrobacteium tumefaciens strain CP4, of the plant 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS), an enzyme involved in the shikimate biochemical pathway for the production of the aromatic amino acids phenylalanine, tyrosine and tryptophan. Glyphosate inhibits the native plant EPSPS, thus blocking the shikimate pathway and halting the synthesis of these amino acids and leading to growth suppression or death of the plant. The CP4 EPSPS enzyme is highly insensitive to glyphosate and thus allows the aromatic amino acid needs of the plant.
• The second gene is derived from Achromobacter sp., a ubiquitous soil bacteria species which expresses an enzyme that degrades glyphosate by conversion to aminomethylphosphonic acid (AMPA) and glyoxylate, thereby deactivating the herbicidal effect. The coding sequence of this gene has been altered to enhance the efficiency of glyphosate degradation, compared to the original bacterial version.
• A plant-derived coding sequence expressing a chloroplast transit peptide was co- introduced with each of the Roundup-ReadyTM genes. This peptide facilitates the import of the newly translated Roundup-ReadyTM enzymes into the chloroplasts, the site of both the shikimate pathway and the site of action for glyphosate.
• Both genes associated with their transit peptide coding sequence, are linked to the same constitutive promoter. Expression of both Roundup-ReadyTM proteins in seed was quantified.
• The range of expression of both proteins in the seed was comparable to those of B. napus GT73. The average expression levels (in µg/mg f.w.) of the first protein ranged from 0.032 to 0.053 for ZSR500; from 0.014 to 0.053 for ZSR502; and from 0.025 to 0.043 for ZSR503. The average expression levels (in µg/mg f.w) of the second protein ranged from 0.064 to 0.124 for ZSR500; from 0.056 to 0.148 for ZSR502 and; from 0.061 to 0.135 for ZSR503.
• The Roundup-ReadyTM 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

• The Brassica napus line GT73 was developed by transforming the variety "Westar" 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-ReadyTM 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 GT73.
• GT73 was then crossed with Hysyn Polish canola varieties. The mid-season B. rapa canola lines ZSR500, ZSR502 and ZSR503 were derived from four parental populations. Since populations were combined for their development, these lines are referred to as synthetic lines.

3. Stable Integration into the Plants' Genomes

• Monsanto provided information on segregation and Southern blot analysis demonstrating that GT73 had a stable single genetic insert, consisting of single copies of the Roundup-ReadyTM genes (please see decision document DD95-02).
• Since Monsanto was able to cross GT73 with B. rapa and transfer the herbicide tolerance successfully, it is suggested that the genetic insert was located in the B. rapa portion (genome A) of the amphidiploid B. rapa/B. oleracea genome of B. napus.

IV. Assessment Criteria for Environmental Safety

1. Potential of the PNT's to Become Weeds of Agriculture or Be Invasive of Natural Habitats

The CFIA has evaluated the data and information submitted by Monsanto Canada Inc., on the reproductive and survival biology of the lines ZSR500, ZSR502 and ZSR503. From this, the CFIA has found that vegetative vigor, flowering and time to maturity and seed production were within the normal range of expression found in unmodified Polish canola lines. ZSR500, ZSR502 and ZSR503 have no specific added genes for cold tolerance or winter hibernation. They did not show any stress adaptation other than tolerance to glyphosate.

The biology of B. rapa, described in T-1-02-98, shows that this species normally is not invasive of unmanaged habitats in Canada. It occasionally is a weed of cultivated land, especially in the eastern prairies of Canada. According to the information and data provided by Monsanto, ZSR500, ZSR502 and ZSR503 were found to be no different from unmodified canola counterparts in this respect. The CFIA concurs that no competitive advantage was conferred by the insertion of the Roundup-ReadyTM 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 the CFIA to conclude that ZSR500, ZSR502 and ZSR503 have 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

Brassica rapa is an obligate outcrosser with other plants of the same species, including bird rape (a weedy B. rapa biotype), and potentially with plants of the species B. napus, B. juncea, B. carinata, B. nigra, Diplotaxis muralis, and Erucastrum gallicum (see T-1- 02-98). Since Polish rape is produced primarily in Western Canada, potential introgression of the herbicide tolerance is most likely to occur with B. napus, the other major canola species. The latter species is not considered as a weed in Canada and the B. napus line GT73 has already been authorized for environmental release in 1995.

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. rapa 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.

Although Polish rape is a minor crop in eastern provinces, there is a possibility for potential introgression of the herbicide tolerance from the cultivated to the wild B. rapa if such B. rapa varieties were to be grown in these regions. There is no species-specific barriers to the gene flow between cultivated and wild B. rapa. The latter is considered a weed of cultivated land in Eastern Canada.

The above considerations led the CFIA to conclude that gene flow from the lines ZSR500, ZSR502 and ZSR503 to other plant species is indeed possible, but would not result in increased weediness or invasiveness of these relatives. In Eastern Canada however, the movement of glyphosate tolerance into weedy B. rapa populations where Roundup® herbicide is used for weed control is undesirable and could potentially result in the loss of the use of this herbicide on other plant crops, such as glyphosate tolerant corn and soybean, and any of their potential benefits.

3. Altered Plant Pest Potential

The intended effects of the novel herbicide tolerance trait is unrelated to plant pest potential, and B. rapa is not a plant pest in Canada (see T-1-02-98). In addition, agronomic characteristics, and qualitative and quantitative composition of ZSR500, ZSR502 and ZSR503 were demonstrated by Monsanto to be within the range of values displayed by unmodified canola varieties. The 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. Seed protein profiles and fatty acid composition fall within the range of those of the unmodified counterparts. A search of the FAST amino-acid sequence database revealed no significant homology of the novel proteins with known toxins or allergens entered in that database.

Based on the above, the CFIA has determined that the unconfined release of the lines ZSR500, ZSR502 and ZSR503 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, the lines ZSR500, ZSR502 and ZSR503 have no novel phenotypic characteristics which would extend their 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.

The CFIA has therefore concluded that the potential impact on biodiversity of the lines ZSR500, ZSR502 and ZSR503 is equivalent to that of currently commercialized canola varieties.

V. Nutritional Assessment Criteria as Livestock Feed

1. Nutritional Composition of the PNT's

Analyses of the nutritional composition including protein, fat, fibre and ash were conducted on samples the three PNT lines. Parkland, which is a close parent of the lines, was used as the non PNT control. The nutritional composition of the derived lines varied with respect to each other and the control canola variety. Of the three lines, two had significantly lower protein than the control line, while line ZSR502 was similar. Oil composition of two of the lines was significantly lower than the Parkland control, however line ZSR500 was similar in oil content to the control. All three of the lines contained significantly higher fibre than the Parkland control. Line ZSR503 was lower in ash than the control the other two lines were similar. Of the three lines line ZSR503 was significantly different from the control for all proximates i.e. higher fibre and lower protein and ash. Despite the differences from the control Parkland variety all proximate values were well within the normal range for these components in canola and the variation likely derives from the fact that Parkland is only a closely related line.

2. Anti-Nutritional Factors

Erucic acid and glucosinolate content of the PNT lines was substantially equivalent to the levels determined for the non-transformed control. All values were below the prescribed maximum levels for these anti-nutritional factors in canola as set out in the Feeds Regulations.

VI. Regulatory Decision

Based on the review of data and information submitted by Monsanto Canada Inc., the Plant Biosafety Office of the Plant Health and Production Division, CFIA, has concluded that the Roundup-ReadyTM genes and their corresponding novel Roundup® herbicide tolerance do not present altered environmental interactions when compared to existing commercialized canola varieties in Western Canada, the primary production area for canola (B.rapa) in Canada. Outcrossing to related plants, would result in no ecological advantage in Western Canada.

This decision takes into consideration the presence of populations of wild bird rape (Brassica rapa) in Quebec, and the Atlantic provinces of Canada, and to a lesser extent in the Southern part of Ontario; and the undesirable movement of glyphosate herbicide tolerance into these weedy populations where Roundup® herbicide is used for weed control. This decision has been reached after consultation with plant breeders, weed scientists, and agronomists in academia and provincial government services.

Based on the review of submitted data, the Feed Section of the Plant Health and Production Division has concluded that the novel genes expressed by the canola lines ZSR500, ZSR502 and ZSR503 do not raise safety or nutritional concerns as livestock feed. As these lines have been assessed and found to be substantially equivalent to traditional B. rapa varieties with respect to safety, the canola lines ZSR500, ZSR502 and ZSR503 and their byproducts are approved for use as livestock feed ingredients in Canada.

If at any time, Monsanto becomes aware of any information regarding risk to the environment, or risk to animal or human health, that could result from release of these materials in Canada, or elsewhere, Monsanto must immediately provide such information to the CFIA. On the basis of such new information, the CFIA may re-evaluate the potential impact of the release and re-evaluate its decision.

Unconfined release into the environment of the B. rapa canola lines ZSR500, ZSR502 and ZSR503 is authorized in British Columbia, Alberta, Saskatchewan and Manitoba. Their use as livestock feed is also authorized. Also, any B. rapa lines derived from them, resulting from the same transformation event or transformed with the same genetic construct, may be considered subtantially equivalent, provided it is known following thorough characterization that: no inter-specific crosses are performed; the intended use is similar; and these plants do not display any additional novel traits.

Please note that while determining the environmental safety of plants with novel traits is a critical step in the commercialization of these plant types, other requirements may still need to be addressed, such as Variety Registration (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: