DD1998-25: Determination of Environmental Safety of Rhône Poulenc's Oxynil Herbicide-Tolerant Brassica napus Canola Line Westar Oxy-235
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This Decision Document has been prepared to explain the regulatory decision reached under the guidelines Dir94-08 Assessment Criteria for Determining Environmental Safety of Plants with Novel Traits and its companion document Dir94-09 The Biology of Brassica napus L. (canola/rapeseed) and the guidelines Dir95-03 Guidelines for the Assessment of Plants with Novel Traits as Livestock Feed.
The Canadian Food Inspection Agency (CFIA), specifically the Plant Biotechnology Office and the Feed Section of the CFIA, has evaluated information submitted by Rhône Poulenc Inc. regarding the canola line Oxy-235. This line has a gene that confers tolerance to ioxynil and bromoxynil. The CFIA has determined that this plant with novel traits does not present altered environmental interactions or pose concerns for the safety of livestock consuming feed derived from the PNT, when compared to currently commercialized canola varieties in Canada.
Unconfined release into the environment and use as livestock feed of the canola line Oxy-235 is therefore authorized as of February 18, 1997. All other canola lines resulting from the same transformation event and all their descendants are also approved, provided no inter-specific crosses are performed, provided the intended use is similar, provided it is known following thorough characterization that these plants do not display any additional novel traits and that the resulting lines are substantially equivalent to currently grown canola, in terms of their potential environmental impact and livestock feed safety.
Table of Contents
- Brief Identification of the Plants with Novel Traits (PNT's)
- Background Information
- Description of the Novel Bromoxynil Tolerance
- The Bromoxynil Tolerance Gene
- Development Method
- Stable Integration into the Plants' Genome
- Assessment Criteria for Environmental Safety
- Potential of the Oxy-235 line 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 Invasive
- Altered Plant Pest Potential
- Potential Impact on Non-Target Organisms
- Potential Impact on Biodiversity
- Nutritional Assessment Criteria for Use as Livestock Feed
- Nutritional Composition of the PNT
- Anti-Nutritional Factors
- Regulatory Decision
I. Brief Identification of the Plants with Novel Traits (PNT's)
Designation(s) of the PNT: Westar Oxy-235, Oxy-235
Applicant: Rhône Poulenc Canada Inc.
Plant Species: Brassica napus canola
Novel Traits: Novel tolerance to bromoxynil and ioxynil, expressed by the novel gene
Trait Introduction Method: Agrobacterium tumefaciens-mediated transformation
Proposed Use of PNT: Production of canola type B. napus varieties
II. Background Information
Rhône Poulenc Canada Inc. has developed a Brassica napus canola line, Oxy-235, derived from the variety Westar, which expresses their proprietary gene. This gene confers novel tolerance to bromoxynil and ioxynil herbicides, which can control or suppress economically important weeds in canola production.
The development of Oxy-235 was based on recombinant DNA technology with the introduction of a genetic construct containing one gene from a soil bacterium into the variety Westar. The gene expresses an enzyme that enables the plant to transform bromoxynil and ioxynil into non-phytotoxic compounds. No antibiotic resistance marker genes were introduced into Oxy-235
Oxy-235 has been field tested in Canada under confined conditions from 1992 to 1996 in Saskatchewan and Manitoba.
Rhône Poulenc has submitted information that includes agronomic and quality data. This information described:
- the identity of Oxy-235, 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 provided to support information where available. In addition, Rhône Poulenc has provided information, data and recorded observations comparing Oxy-235 to the variety Westar and 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 (Leptosphaeria maculans).
The Plant Biotechnology Office of the CFIA, has reviewed the above information, in accordance with the following assessment criteria for determining environmental safety of plants with novel traits as described in the regulatory directive Dir94-08:
- potential of the PNT to become a 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 to become a plant pests
- potential effects of the PNT or its gene product on non-target species, including humans
- potential impact on biodiversity
The Feed Section of the Animal Health and Production Division, CFIA, has also reviewed the above information with respect to the assessment criteria for determining safety and efficacy of livestock feed, as described in Dir95-03. The following have been considered:
- potential impact to livestock
- potential impact to livestock nutrition
III. Description of the Novel Bromoxynil Tolerance
1. The Bromoxynil Tolerance Gene
- The oxynil family of herbicides is active against dicotyledonous plants by blocking electron flow during the light reaction of photosynthesis.
- One gene from the bacteria Klebsiella pneumoniae subspecies ozaenae has been introduced into the canola variety Westar providing a field level of tolerance to oxynil herbicides.
- The gene codes for a bacterial enzyme, nitrilase, which hydrolyses ioxynil and bromoxynil into non-phytotoxic compounds.
- The expression of the nitrilase gene is linked to a promoter and an enhancer which control gene expression. The nitrilase gene is linked to a polyadenylation signal from an Agrobacterium gene.
- The gene is expressed at a level of 1 microgram nitrilase per milligram of total protein in leaves and approximately 100-fold less in seeds.
- The nitrilase protein showed no significant homology with any known toxins or allergens in the Genebank and Swiss-Pro databases. The nitrilase protein was also shown to have low stability under simulated gastric conditions. The enzyme which confers oxynil 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. 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 oxynil tolerance gene. 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 Oxy-235.
3. Stable Integration into the Plant's Genome
Rhône Poulenc has provided information on segregation and from Southern blot analysis that demonstrates that Oxy-235 has a single genetic insert, consisting of a single copy of the oxynil tolerance gene.
Oxy-235 is several generations removed from the original transformant. Mendelian inheritance of the herbicide tolerance, and Southern analyses of third-generation material show the stability of the introduced traits.
IV. Assessment Criteria for Environmental Safety
1. Potential of Oxy-235 to Become a Weed of Agriculture or Be Invasive of Natural Habitats
The CFIA has evaluated the data and information submitted by Rhône Poulenc Canada Inc. on the reproductive and survival biology of Oxy-235. From this, it was determined that vegetative vigor, overwintering capacity, time to maturity, seed production and yield were within the normal range of expression found in unmodified canola. Oxy-235 has no specific added genes for cold tolerance or winter hibernation. No overwintered plants were observed by Rhône Poulenc's cooperators in post-harvest years of field trials. Oxy-235 did not show any stress adaptation other than its tolerance to oxynil herbicides. Visual observations for resistance to the fungal pathogen Leptosphaeria maculans (blackleg) suggests that Oxy-235 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 Rhône Poulenc, Oxy-235 was 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 oxynil tolerance genes, other than tolerance to oxynil herbicides.
Note: Ioxynil and bromoxynil are commonly used on cereal crops which may be grown in rotation with canola, and resistant canola volunteer plants will not be controlled by these herbicides. Nevertheless, they can be managed with herbicides having different modes of action such as 2,4-D and MCPA. The efficacy of these and other herbicides in controlling Oxy-235 was demonstrated by the applicant.
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 Oxy-235 has no altered weed or invasiveness potential compared to currently commercialized canola varieties.
Note: A longer term concern, should there be general adoption of several different crop/specific herbicide weed management systems, is the potential development of crop volunteers with a combination of novel tolerances 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 tolerance.
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 gene is most likely to occur with B. rapa, the other major canola species in Canada which is occasionally a weed of cultivated land, especially in the eastern provinces.
Should oxynil tolerant individuals arise through interspecific or intergeneric hybridization, the tolerance would not confer any competitive advantage to these plants unless challenged by oxynil herbicides. This would occur in managed ecosystems where oxynils are applied for broad spectrum weed control, or in crop plants which exhibit oxynil tolerance such as cereals, and in which oxynils are used to control weeds.
As with oxynil tolerant B. napus volunteers, these individuals, should they arise, could be controlled using other available chemical means. Oxynil tolerant hybrids, if they developed, could potentially result in the loss of oxynils as a tool to control these species. This however, can be avoided by the use of sound crop management practices including both crop and herbicide rotations.
The above considerations led CFIA to conclude that gene flow from Oxy-235 to relatives of B. napus is 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 Oxy-235 were demonstrated by Rhône Poulenc 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 gene and resulting enzyme, 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 enzyme is rapidly inactivated in mammalian stomach and intestinal fluids by enzymatic degradation and pH-mediated proteolysis. Raw seeds of Oxy-235 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 Swiss-Pro amino-acid sequence database revealed no significant homology with known toxins or allergens entered in that database.
Based on the above, the CFIA has determined that the unconfined release of Oxy-235 will not result in altered impacts on interacting organisms, including humans, when compared to current commercialized canola varieties.
5. Potential Impact on Biodiversity
The introduced genes and their gene products were determined to be safe to non-target organisms. In addition, Oxy-235 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 have an impact on unmanaged environments.
CFIA has therefore concluded that the potential impact on biodiversity of Oxy-235 is no different from that of currently commercialized canola varieties.
V. Nutritional Assessment Criteria For Use as Livestock Feed
1. Nutritional Composition of the PNT
Analyses of the nutritional composition including protein oil and fatty acid profile were conducted on samples of Oxy-235 and control Westar lines. Proximate analysis was conducted on samples from four elite lines and their respective PNT lines. Overall nutritional composition of the transformed lines was shown to be substantially equivalent to non-transformed canola. There were no significant differences in crude protein, crude fat, fatty acid profile, crude fibre or ash content between transformed lines and their respective controls.
2. Anti-Nutritional Factors
The erucic acid and glucosinolate content of Oxy-235 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 Rhône Poulenc Inc.'s data and information submitted, the Plant Biotechnology Office of the CFIA concludes that the oxynil tolerance gene, the novel gene product and the associated novel oxynil herbicide tolerance trait do not in themselves confer any intended ecological advantage to Oxy-235 or to its relatives, should gene flow occur. In addition, this novel herbicide tolerance does not alter the characteristics of Oxy-235 with regards to the assessment criteria for environmental safety. Thorough comparisons of Oxy- 235 with unmodified B. napus canola showed no unexpected effects.
Unconfined release into the environment and use as livestock feed of the canola line Oxy-235 is therefore authorized. Any other lines and intraspecific hybrids resulting from the same transformation event, and all their descendants, are also approved, provided no inter-specific crosses are performed, provided the intended use is similar, provided it is known following thorough characterization that these plants do not display any additional novel traits and that the resulting lines are substantially equivalent to currently grown canola, in terms of their potential environmental impact and livestock feed safety.
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 Health and Production Division
Canadian Food Inspection Agency
59 Camelot Drive
Nepean, Ontario, K1A 0Y9
Tel: (613) 225-2342
Fax: (613) 228-6629
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