This decision document has been prepared to explain the regulatory decisions reached under Directive 94-08 (Dir94-08) - Assessment Criteria for Determining Environmental Safety of Plants with Novel Traits, its companion document BIO2017-03 - The Biology of Brassica napus (L.) (Canola/Rapeseed) and Section 2.6 - Guidelines for the Assessment of Novel Feeds: Plant Sources, of Chapter 2 of the RG-1 Regulatory Guidance: Feed Registration Procedures and Labelling Standards.
The Canadian Food Inspection Agency (CFIA) – specifically the Plant Biosafety Office of the Plant Health and Biosecurity Directorate, the Plant and Biotechnology Risk Assessment Unit of the Plant Health Science Directorate and the Animal Feed Division of the Animal Health Directorate – has evaluated information submitted by Bayer CropScience Inc. This information concerns the male sterile and herbicide tolerant canola event MS11. CFIA has determined that this plant with novel traits (PNT) does not present altered environmental risk nor, as a novel feed, does it present livestock feed safety or nutrition concerns when compared to canola varieties currently grown and permitted to be used as livestock feed in Canada.
Taking into account these evaluations, unconfined release into the environment and use as livestock feed of canola event MS11 is therefore authorized by the Plant Biosafety Office of the Plant Health and Biosecurity Directorate and the Animal Feed Division of the Animal Health Directorate, respectively, as of January 30, 2018. Any canola lines derived from canola event MS11 may also be released into the environment and used as livestock feed, provided that:
- no inter-specific crosses are performed
- the intended uses are similar
- it is known based on characterization that these plants do not display any additional novel traits and are substantially equivalent to canola varieties that are currently grown and permitted to be used as livestock feed in Canada, in terms of their potential environmental impact and livestock feed safety and nutrition
- the novel genes are expressed at a level similar to that of the authorized line, and
- data used to establish the substantial equivalence of lines derived from canola event MS11 be made available to the CFIA upon request
Canola event MS11 is subject to the same phytosanitary import requirements as unmodified canola varieties. Canola event MS11 is required to meet the requirements of other jurisdictions, including but not limited to, the Food and Drugs Act and the Pest Control Products Act.
Please note that the livestock feed and environmental assessments of novel feeds and PNTs are critical steps in the potential commercialization of these plant types. Other requirements, such as the assessment of novel foods by Health Canada, have been addressed separately from this review.
January 30, 2018
This bulletin was created by the Canadian Food Inspection Agency. For further information, please contact the Plant Biosafety Office or the Animal Feed Division by visiting the contact page.
On this page
- Brief identification of the modified plant
- Background information
- Description of the novel traits
- Criteria for the environmental assessment
- Potential for canola event MS11 to become a weed of agriculture or be invasive of natural habitats
- Potential for gene flow from canola event MS11 to sexually compatible plants whose hybrid offspring may become more weedy or more invasive
- Potential for canola event MS11 to become a plant pest
- Potential impact of canola event MS11 or its gene products on non-target organisms, including humans
- Potential impact of canola event MS11 on biodiversity
- Criteria for the livestock feed assessment
- New information requirements
- Regulatory decision
I. Brief identification of the modified plant
Designation of the modified plant: Canola event MS11, OECD Unique Identifier BCS-BNØ012-7
Applicant: Bayer CropScience Inc.
Plant species: Canola (Brassica napus L.)
Novel traits: Male sterility, tolerance to glufosinate-ammonium herbicide
Trait introduction method: Agrobacterium-mediated transformation
Intended use of the modified plant: Canola event MS11 is intended to be used in hybrid seed production systems. Grain derived from MS11 will be used for traditional canola human food and livestock feed uses. Canola event MS11 is not intended to be grown outside the normal production area for canola in Canada.
II. Background information
Bayer CropScience Inc. has developed a canola event that is male sterile and tolerant to glufosinate-ammonium herbicide. Canola event MS11 was developed using recombinant deoxyribonucleic acid (DNA) technology, resulting in the introduction of the barnase, barstar and phosphinothricin acetyltransferase (bar) genes. The barnase and barstar genes are derived from a ubiquitous soil bacterium Bacillus amyloliquefaciens (B. amyloliquefaciens). The barnase protein is a ribonuclease and its expression results in ribonucleic acid degradation, cell disruption and cell death. In canola event MS11, the tissue specific expression of barnase interferes with viable pollen formation and therefore confers the male sterility trait. The barstar protein inhibits the barnase ribonuclease when both proteins co-occur in planta. In canola event MS11, the barstar protein has not been found to inhibit the function of the barnase, and is believed to only improve the transformation efficiency of Brassica napus (B. napus) tissues. The bar gene is derived from the soil bacterium Streptomyces hygroscopicus (S. hygroscopicus) and encodes a phosphinothricin acetyltransferase (PAT) enzyme. This enzyme inactivates the herbicide glufosinate-ammonium.
Bayer CropScience Inc. has provided information on: the identity of canola event MS11; a detailed description of the transformation method; and information on insert copy number and intactness, levels of protein expression in the plant and the role of the inserted sequences. The novel proteins were identified and characterized. Information 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.
Canola event MS11 was field-tested in the US at 4 locations in 2014, of which two shared similar environmental and agronomic conditions to the canola growing regions of Manitoba, Saskatchewan and Alberta, and were considered to be representative of major Canadian canola growing regions. Only one of the two Canadian-equivalent US locations was selected for statistical analysis, as the other site was excluded due to flooding. Canola event MS11 was also field tested at 6 locations in Canada in 2014. Canola event MS11 was compared to an unmodified control canola variety with a similar genetic background. Several reference canola varieties were also included in the field trials to establish ranges of comparative values that are typical of currently grown canola varieties.
Agronomic characteristics of canola event MS11, such as early stand count, seedling vigour, days to flowering (50% of plants in flower), days to 10% of flowers remaining, days to maturity, seed yield, plant height, final stand count, seedling vigor, sterile plants per plot, lodging resistance, and pod shattering, were compared to those of the unmodified control canola variety and to the ranges established from the reference canola varieties.
Nutritional components of canola event MS11 grain and forage, such as protein, fat, moisture, ash, amino acids, fatty acids, vitamins, minerals and anti-nutrients, were compared to those of the unmodified control canola variety and to the range established from the reference canola varieties.
The Plant and Biotechnology Risk Assessment (PBRA) Unit of the Plant Health Science Directorate, CFIA, has reviewed the above information, in light of the assessment criteria for determining environmental safety of PNTs, as described in Directive 94-08 (Dir94-08) - Assessment Criteria for Determining Environmental Safety of Plants with Novel Traits. The PBRA Unit has considered:
- the potential for canola event MS11 to become a weed of agriculture or to be invasive of natural habitats
- the potential for gene flow from canola event MS11 to sexually compatible plants whose hybrid offspring may become more weedy or more invasive
- the potential for canola event MS11 to become a plant pest
- the potential impact of canola event MS11 and its gene products on non-target organisms, including humans; and
- the potential impact of canola event MS11 on biodiversity
The Animal Feed Division (AFD) of CFIA has also reviewed the above information with respect to the assessment criteria for determining the safety and nutrition of livestock feed, as described in Section 2.6 - Guidelines for the Assessment of Novel Feeds: Plant Sources, of Chapter 2 of the RG-1 Regulatory Guidance: Feed Registration Procedures and Labelling Standards.
The AFD has considered both intended and unintended effects and similarities and differences between canola event MS11 and unmodified canola varieties relative to the safety and nutrition of feed ingredients derived from canola event MS11 for their intended purpose, including:
- the potential impact of canola event MS11 on livestock nutrition; and
- the potential impact of canola event MS11 on animal health and human safety, as it relates to the potential transfer of residues into foods of animal origin and worker/bystander exposure to the feed
The AFD has also considered whether feeds derived from canola event MS11 meet the definitions and requirements of feeds as listed in Schedule IV of the Feeds Regulations.
III. Description of the novel traits
1. Development method
Canola event MS11 was developed through Agrobacterium-mediated transformation of canola cells and contains the barnase, barstar genes from B. amyloliquefaciens and the bar gene from S. viridochromogenes as well as associated regulatory elements. Transformed cells were selected on the basis of tolerance to glufosinate-ammonium herbicide and regenerated to produce plants. Canola event MS11 was identified as a successful transformant and chosen for further development based on results of molecular analyses, herbicide tolerance trials and agronomic evaluations.
2. Male sterility
Expression of the barnase gene, which encodes the barnase ribonuclease, results in ribonucleic acid degradation, cell disruption and cell death in the absence of its inhibitor, barstar. When the barnase ribonuclease is expressed in developing anthers it prevents viable pollen from forming. This is the basis for the plant male sterility trait in canola event MS11. The barnase gene was isolated from the ubiquitous soil bacterium B. amyloliquefaciens. The barnase ribonuclease produced in canola event MS11 is expected to be identical to the native protein from B. amyloliquefaciens.
The expression of the barnase protein in canola event MS11 is driven by a promoter that restricts gene expression to the tapetum cells during anther development. Samples of canola tissues were collected from plants from three field trials. Tissues were collected from unsprayed plants and plants sprayed with glufosinate-ammonium herbicide. Barnase protein expression was below the lower limit of quantification in all tissue samples from canola event MS11.
The potential allergenicity and toxicity of the barnase protein were evaluated. The weight of evidence indicates that the barnase protein is unlikely to be allergenic, based on the following information. The source of the barnase gene, B. amyloliquefaciens, is not commonly associated with allergenicity. The barnase protein amino acid sequence lacks relevant similarities to known allergens. Finally, unlike many allergens, the amino-acid sequence of the barnase protein does not possess glycosylation sites. It was also concluded that the barnase protein is unlikely to be toxic to livestock and other non-target organisms because it lacks a mode of action that could be intrinsically toxic to livestock and other non-target organisms. Furthermore, the barnase protein amino acid sequence lacks relevant similarities to known toxins. For a more detailed discussion of the potential allergenicity and toxicity of the barnase protein, see Section V, part 2 Potential impact of canola Event MS11 on animal health and human safety as it relates to the potential transfer of residues into foods of animal origin and worker/bystander exposure to the feed.
Expression of the barstar gene, which encodes an inhibitor of the barnase ribonuclease, normally prevents cell death induced by the barnase ribonuclease. In canola event MS11 the expression of the barstar protein is claimed to improve the ability to transform B. napus tissues, although no empirical evidence was supplied to support this hypothesis. Furthermore, as evidenced by the male sterility phenotype of canola event MS11, the barstar protein in canola event MS11 does not interfere with the function of the barnase ribonuclease in canola event MS11. The barstar gene was isolated from the ubiquitous soil bacterium B. amyloliquefaciens. The barstar protein produced in canola event MS11 is expected to be identical to the native protein from B. amyloliquefaciens.
The expression of the barstar protein in canola event MS11 is driven by a constitutive promoter. Samples of canola tissues were collected from plants from three field trials. Tissues were collected from unsprayed plants and plants sprayed with glufosinate-ammonium herbicide. The mean barstar protein expression ranged from below the lower limit of quantification to 1.04 μg/g dry weight in all tissue samples from canola event MS11.
The potential allergenicity and toxicity of the barstar protein to non-target organisms were evaluated. The weight of evidence indicates that the barstar protein is unlikely to be allergenic, based on the following information. The source of the barstar gene, B. amyloliquefaciens, is not commonly associated with allergenicity. The barstar protein amino acid sequence lacks relevant similarities to known allergens. Finally, unlike many allergens, the amino-acid sequence of the barstar protein does not possess glycosylation sites. It was also concluded that the barstar protein is unlikely to be toxic to livestock and other non-target organisms because it lacks a mode of action to suggest that it is intrinsically toxic to livestock and other non-target organisms and because the barstar protein amino acid sequence lacks relevant similarities to known toxins. For a more detailed discussion of the potential allergenicity and toxicity of the barstar protein, see Section V, part 2 Potential impact of canola Event MS11 on animal health and human safety as it relates to the potential transfer of residues into foods of animal origin and worker/bystander exposure to the feed.
3. Tolerance to glufosinate-ammonium herbicide
In susceptible plants, glufosinate-ammonium herbicide inhibits the glutamine synthetase enzyme, resulting in the accumulation of lethal levels of ammonia in plant tissues. Canola event MS11 was developed to be tolerant to the herbicide glufosinate-ammonium herbicide by incorporation of the bar gene. The bar gene encodes a phosphinothricin acetyltransferase (PAT) enzyme, which is sometimes referred to as a bialaphos resistance (BAR) enzyme. The PAT protein acetylates the primary amino group of glufosinate-ammonium, inactivating the herbicide. Introduction of the bar gene into canola event MS11 confers commercial-level tolerance to the herbicide glufosinate-ammonium. The bar gene was derived from S. hygroscopiucus, a common saprophytic soil bacterium that is found worldwide, and the PAT protein produced in canola event MS11 is identical to the native enzyme.
The expression of the PAT protein in canola event MS11 is driven by a constitutive, green-tissue specific promoter. Samples of canola tissues were collected from plants from three field trials. Tissues were collected from unsprayed plants and plants sprayed with glufosinate-ammonium herbicide. The average PAT protein expression from unsprayed MS11 canola plants, as evaluated by ELISA, was as follows: 21.88 μg/g dry weight (dwt) in whole plant tissues, 0.17 μg/g dwt in root tissues, and 0.34 μg/g dwt in grain. Similar PAT protein levels were observed in the tissues of herbicide-treated MS11 canola plants.
To obtain sufficient quantities of PAT protein for assessment of environmental and feed safety, it was necessary to express the bar gene in an E. coli-production system. Equivalency was demonstrated between the canola event MS11 PAT protein and the E. coli-produced PAT protein by comparing their molecular weights, immunoreactivity, glycosylation status, N-terminal sequence analysis, MALDI-TOF mass spectrometry and functional activities. Based on the results, the proteins were found to be equivalent. Demonstration of equivalence between the PAT protein produced in E. coli and the PAT protein produced in canola event MS11 allows the PAT protein produced in E. coli to be used in studies to confirm the safety of the PAT protein produced in canola event MS11.
The potential allergenicity and toxicity of the PAT protein to non-target organisms were evaluated. The weight of evidence indicates that the PAT protein is unlikely to be allergenic, based on the following information. The source of the bar gene, is S. hygroscopiucus, is not commonly associated with allergenicity. The PAT protein amino acid sequence lacks relevant similarities to known allergens. Unlike many allergens, the E. coli-produced PAT protein was shown experimentally to be rapidly degraded in simulated gastric fluid and not to be heat stable. Finally, unlike many allergens, the amino-acid sequence of the PAT protein does not possess glycosylation sites. It was also concluded that the PAT protein is unlikely to be toxic to livestock and other non-target organisms because it lacks a mode of action to suggest that it is intrinsically toxic to livestock and other non-target organisms, because the PAT protein amino acid sequence lacks relevant similarities to known toxins, and because no adverse effects were observed when E. coli-produced PAT protein was ingested by mice at doses of approximately 2,000 mg/ kg bwt. For a more detailed discussion of the potential allergenicity and toxicity of the PAT protein, see Section V, part 2 Potential impact of canola Event MS11 on animal health and human safety as it relates to the potential transfer of residues into foods of animal origin and worker/bystander exposure to the feed.
4. Stable integration into the plant genome
Molecular characterization by Southern blot analysis demonstrated that canola event MS11 contains one intact copy of the gene cassettes containing the barnase, barstar and bar genes and their regulatory elements inserted at a single site in the canola genome. No additional elements, including intact or partial DNA fragments of the gene cassettes or backbone sequences from the plasmid vector, linked or unlinked to the intact insert, were detected in canola event MS11.
The stability of the insert within canola event MS11 was verified by Southern blot analysis and by detection of the barnase, barstar and PAT proteins over five generations. The inheritance pattern of the insert and the barnase, barstar and PAT protein expression trait across five segregating generations of canola event MS11 showed that the insert segregates according to Mendelian rules of inheritance for a single genetic locus.
IV. Criteria for the environmental assessment
1. Potential for canola event MS11 to become a weed of agriculture or be invasive of natural habitats
Canola (B. napus) possesses some of the characteristics that are common to weeds and invasive plants. It is an annual crop that may persist in unmanaged ecosystems without human intervention. There have been reports of B. napus becoming a weed of agriculture in North America and other parts of the world; however, it has not become an abundant or problematic weed in Canada, despite being cultivated in Canada for many years. B. napus plants can grow as volunteers in cultivated fields in the seasons following a B. napus crop, but they are usually eliminated by soil cultivation or the use of herbicides. According to the information provided by Bayer CropScience Inc., canola event MS11 was determined not to be significantly different from unmodified canola varieties in this respect.
CFIA evaluated data submitted by Bayer CropScience Inc. on the reproductive biology and life history traits of canola event MS11. As previously mentioned, canola event MS11 was field tested in the US at 4 locations in 2014, of which two shared similar environmental and agronomic conditions to the canola growing regions of Manitoba, Saskatchewan and Alberta and were considered to be representative of major Canadian canola growing regions. Only one of the two Canadian-equivalent US locations was selected for statistical analysis as the other site was excluded due to flooding. Canola event MS11 was also field tested at 6 locations in Canada in 2014. During the field trials, canola event MS11 was compared to the unmodified control canola variety. Reference canola varieties were also included in these trials to establish ranges of comparative values that are representative of currently grown canola varieties. Canola event MS11 plants were either sprayed with glufosinate-ammonium herbicide, or not sprayed. Canola event MS11 sprayed with glufosinate-ammonium herbicide was planted at double the seeding rate per plot compared to unsprayed canola event MS11and the unmodified control canola variety, since 50% of seedlings of canola event MS11 would not be tolerant to the herbicide application. Therefore, all plots had similar plant densities after the glufosinate-ammonium treatment. Phenotypic and agronomic traits were evaluated, covering a broad range of characteristics that encompass the entire life cycle of the canola plant. The traits included early stand count, seedling vigour, days to flowering (50% of plants in flower), days to 10% of flowers remaining, days to maturity, seed yield, plant height, final stand count, seedling vigor, sterile plants per plot, lodging resistance, and pod shattering. In the unsprayed trials, three statistically significant differences were observed between canola event MS11 and the unmodified control canola variety in the individual-site analyses (specifically, final stand count, early stand count, and days to 10% of flowers remaining), however there was no consistent trend in the data across locations that would indicate the differences were due to the genetic modification. In the sprayed trials, six statistically significant differences were observed between sprayed canola event MS11 and the unmodified control canola variety in the individual-site analyses (specifically, final stand count, early stand count, days to flowering, days to 10% of flowers remaining, days to maturity and yield), of which four traits showed a consistent trend across locations (specifically, early stand count, days to 10% of flowers remaining, days to maturity and yield). The mean values of days to 10% of flowers remaining, days to maturity and yield of sprayed canola event MS11 were within the reference range established from the reference canola varieties included in the same field trials. Since the values were within established ranges, the differences between sprayed canola event MS11 and the unmodified control canola variety were not considered to be biologically meaningful. The value of early stand count for sprayed canola event MS11 was higher and outside of the reference range established from the reference canola varieties. The higher early stand count was accounted for by the double seeding rate used in plots of canola event MS11 sprayed with glufosinate-ammonium compared to the unsprayed plots.
Bayer CropScience Inc. provided information on the dormancy and the germination of canola event MS11 under warm and cold temperature regimes. No significant difference was detected between canola event MS11 and the unmodified control canola variety. Therefore the introduction of the novel traits did not impact the germination of the canola seed and did not confer dormancy to the canola seed.
The susceptibility of canola event MS11 to various abiotic stressors was evaluated in the same locations as the agronomic characteristics studies. The stressors included heat, wind, lack of moisture and excessive moisture. No trend in increased or decreased susceptibility to these abiotic stressors was observed in canola event MS11 compared to the unmodified control canola variety.
The susceptibility of canola event MS11 to canola pests and pathogens was evaluated in the field at the same locations as the agronomic characteristic studies (further detail provided below in Section IV, part 3: Potential for canola event MS11 to become a plant pest). No trend in increase or decrease of susceptibility to pests or pathogens was observed in canola event MS11 compared to the unmodified control canola variety.
No competitive advantage was conferred to plants of canola event MS11 as a result of the introduced traits, other than that conferred by tolerance to glufosinate-ammonium herbicide. Tolerance to glufosinate-ammonium herbicide provides a competitive advantage only when this herbicide is used and will not, in and of itself, make a glufosinate-ammonium tolerant plant weedier or more invasive of natural habitats. Canola event MS11 plants growing as volunteers will not be controlled if glufosinate-ammonium herbicide is used as the only weed control tool. However, control of canola event MS11 as a volunteer weed in subsequent crops or in fallow ground can be achieved by the use of other classes of herbicides or by mechanical means.
The novel traits have no intended or observed effects on weediness or invasiveness. CFIA has therefore concluded that canola event MS11 has no altered weediness or invasiveness potential in Canada compared to currently grown canola varieties.
CFIA considers the changes in usual agronomic practices that may arise from volunteer plants with novel herbicide tolerances. Similarly, CFIA considers the potential that continued application of the same herbicide in subsequent rotations may lead to increased selection pressure for herbicide tolerant weed populations. To address these issues, an herbicide tolerance management plan which includes integrated weed management strategies should be implemented. These plans may include a recommendation to rotate or combine weed control products with alternate modes of action and to employ other weed control practices.
With respect to its unconfined release into the environment, cultivation of canola event MS11 is subject to herbicide tolerance management requirements. According to Bayer CropScience Inc., canola event MS11 is not intended to be cultivated as an individual event in Canada. Therefore, an herbicide tolerance management plan specific to this product is not required at this time. Before canola event MS11 is cultivated in Canada as an individual event or in combination with other canola events in pyramided products, Bayer CropScience Inc. must submit a herbicide tolerance management plan to CFIA.
2. Potential for gene flow from canola event MS11 to sexually compatible plants whose hybrid offspring may become more weedy or more invasive
Bayer CropScience Inc. provided information on floral morphology and pollen viability of canola event MS11. Canola event MS11 was compared to the unmodified control canola variety with a similar genetic background. No differences were detected between canola event MS11 and the unmodified control canola variety, except that the male sterile anthers of canola event MS11 were less developed in size, less intense in their yellow coloration and the pollen was non-viable. These flower and pollen characteristics are inherent to the male sterile trait of canola event MS11 and were expected. Canola event MS11 is male sterile despite the introduction of the barstar gene and therefore will not pollinate other plants, thereby limiting gene flow with sexually compatible plants in the Canadian environment. Hybrid canola plants derived from the pollination of canola event MS11by a restorer-of-fertility canola line may pollinate certain weedy relatives. Stable gene transfer from B. napus is most likely within Brassica crops such as B. juncea and B. rapa (see biology document BIO2017-03 - The Biology of Brassica napus L. (Canola/Rapeseed) for more information). Any hybrids resulting from outcrossing between B. rapa or B. juncea and canola event MS11 could be controlled by herbicides other than glufosinate or by mechanical means.
If glufosinate-tolerant individuals arose through interspecific or intergeneric hybridization, the novel trait would confer no competitive advantage to these plants unless challenged by glufosinate. This would only occur in managed ecosystems where glufosinate is used for weed control. Should glufosinate -tolerant individuals arise, they could be controlled using mechanical means or herbicides other than glufosinate. Hybrids, if they develop, could potentially result in the loss of glufosinate as a tool to control these species. This, however, can be mitigated by the use of sound crop management practices.
This information led CFIA to conclude that gene flow from canola event MS11 to related species in Canada is possible but would not result in increased weediness or invasiveness of the resulting progeny.
3. Potential for canola event MS11 to become a plant pest
Canola is not considered to be a plant pest in Canada and the male sterility and glufosinate-ammonium herbicide tolerance traits introduced into canola event MS11 are unrelated to plant pest potential (for example the potential for the plant to harbour new or increased populations of pathogens or pests).
The susceptibility of canola event MS11 to canola pests and pathogens was evaluated in the field at the same locations as the agronomic characteristic studies. The stressors observed included flea beetles, bertha armyworms, grasshoppers, diamond black moth, aphid, lygus bug, sclerotinia and blackleg. The evaluations of canola event MS11 did not show any increase or decrease in susceptibility to these pests or pathogens compared to the unmodified control canola variety.
Bayer CropScience Inc. quantitatively assessed arthropod abundance at two locations in Canada in 2015. One reference canola variety was also included in these field trials. A total of 18,940 arthropods were captured and identified at 4 different canola growth stages throughout the season. The pest insects observed included adults and nymphs of Onion thrip (Thrips tabaci), Flower thrip (Frankliniella tritici) and Thrips vulgatissimus; nymphs and adults of the aphid Lipaphis erysimi; larvae and pupae of Plutella xylostella; and nymphs of Macrosiphum. At one location, the statistical analysis showed a significant difference between canola event MS11 and the unmodified control canola variety and between canola event MS11 and the reference canola variety for the larvae of Plutella xylostella. Therefore, it is unlikely that this difference was caused by the genetic modification as it was not observed at the other location. No other statistically significant difference between canola event MS11 and the unmodified control canola variety was observed in terms of arthropod abundance.
CFIA has therefore concluded that canola event MS11 does not display any altered plant pest potential compared to currently grown canola varieties.
4. Potential impact of canola event MS11 on non-target organisms
The glufosinate-ammonium herbicide tolerance and male sterile traits introduced into canola event MS11 are unrelated to a potential impact on non-target organisms. The barnase and barstar proteins in MS11 are expected to be identical to those produced in MS8 and RF3 canola events. Furthermore, the PAT protein produced in canola event MS11 is identical to the native enzyme. Canola events MS8 and RF3 have been previously authorized for unconfined environmental release in Canada (DD1996-17). Detailed evaluation of the barnase, barstar and PAT proteins expressed in canola event MS11 led to the conclusion that none of these proteins displays any characteristic of a potential toxin or allergen (see Section V, part 2: Potential impact of canola Event MS11 on animal health and human safety as it relates to the potential transfer of residues into foods of animal origin and worker/bystander exposure to the feed). As the environmental safety of the barnase, barstar and PAT proteins have previously been established, no negative impacts resulting from exposure of organisms to the barnase, barstar and PAT proteins expressed in canola event MS11 are expected.
Compositional analyses showed that the levels of key nutrients and anti-nutrients in grain from canola event MS11 are comparable to those in grain from conventional canola varieties (see Section V, part 1: Potential impact of canola event MS11 on livestock nutrition). Therefore, it is very unlikely that the introduction of the novel traits may have caused unintended changes to the composition of canola event MS11 tissues that would negatively impact organisms interacting with canola event MS11.
Field evaluations of canola event MS11 did not indicate any increased or decreased resistance to insect pests or pathogens compared to the unmodified control canola variety (see Section IV, part 3: Potential for canola event MS11 to become a plant pest).
Quantitative assessment of the abundance of beneficial insects (larvae and pupae of Syrphids; larvae of Orius tristicolor) at two locations in Canada in 2015 did not show statistically significant difference between canola event MS11 and the unmodified control canola.
Collectively, these information elements indicate that the interactions between canola event MS11 and the populations of animals and microorganisms interacting with canola crops will be similar compared to currently grown canola varieties.
CFIA has therefore determined that the unconfined release of canola event MS11 in Canada will not result in altered impacts on non-target organisms, including humans, compared to currently grown canola varieties.
5. Potential impact of canola event MS11 on biodiversity
Canola event MS11 expresses no novel phenotypic characteristics that would extend its range beyond the current geographic range of canola production in Canada. Canola event MS11 is unlikely to cause adverse effects to non-target organisms and does not display increased weediness, invasiveness or plant pest potential.
Canola event MS11 and its male fertile progeny have tolerance to glufosinate-ammonium herbicides. Canola (B. napus) can outcross to B. rapa and B. juncea, and potentially to wild relatives, under natural conditions in Canada. However, the consequences of the transfer of the glufosinate-ammonium tolerance trait are minimal as the novel trait does not confer any selective advantage in the absence of the herbicide, and glufosinate-ammonium tolerant hybrids can be controlled by herbicides with other modes of action, or through mechanical means.
The use of the glufosinate-ammonium herbicide in cropping systems has the intended effect of reducing local weed populations within agro-ecosystems. This may result in a reduction in local weed species biodiversity, and may have effects on other trophic levels which utilize these weed species. It must be noted however that the goal of reduction in weed biodiversity in agricultural fields is not unique to the use of PNTs, canola event MS11 or the cultivation of canola. It is therefore unlikely that canola event MS11 will have any indirect effects on biodiversity, in comparison to the effects that would be expected from cultivation of currently grown canola varieties in Canada.
CFIA has concluded that the modified genes and corresponding novel traits do not confer to canola event MS11 any characteristic that would result in unintended environmental effects following unconfined release. CFIA has therefore concluded that the potential impact on biodiversity of canola event MS11 is unlikely to be different from that of the canola varieties that are currently grown in Canada.
V. Criteria for the livestock feed assessment
The AFD considered nutrient and anti-nutrient profiles; the safety of feed ingredients derived from canola event MS11, including the presence of gene products, residues and metabolites in terms of animal health and human safety as it relates to the potential transfer of residues into foods of animal origin and worker/bystander exposure to the feed; and whether feeds derived from canola event MS11 meet the definitions and requirements of feeds as listed in Schedule IV of the Feeds Regulations.
1. Potential impact of canola event MS11 on livestock nutrition
Nutrient and anti-nutrient composition
The nutritional equivalence of the canola event MS11 (unsprayed plants and sprayed plants treated with glufosinate-ammonium) to those of the unsprayed, unmodified control canola variety and six unsprayed reference canola varieties was determined from 9 field trials in the canola growing regions of Canada and US during the 2014 growing season. Grain samples were analyzed for moisture, ash, protein, crude fat, carbohydrates (by calculation), acid detergent fibre (ADF) and neutral detergent fibre (NDF). Grain samples were further analyzed for amino acids, fatty acids, vitamins, minerals, and anti-nutrients (phytic acid, total sinapine, insoluble and soluble tannins) as recommended by the OECD consensus document for new varieties of canola (OECD, 2011 - PDF (209 kb)). Composition data was analyzed statistically using a mixed model analysis of variance, and statistical differences among treatments were identified and assessed (P<0.05). The biological relevance of any statistically significant difference among canola varieties was assessed by comparing the observed mean values to the range of the values observed for the reference canola varieties grown in the trials.
No statistically significant differences were observed between grain from canola event MS11 (unsprayed) and the unmodified control canola variety for moisture, protein, fat, ash, carbohydrates, ADF and NDF. Statistically significant differences were found between grain from canola event MS11 (sprayed) and the unmodified control canola for moisture, protein, fat, ash, ADF and NDF. However, these differences were not biologically significant as all means were within the range of the values observed in the reference canola varieties grown in the trials.
No statistically significant differences were observed between grain from canola event MS11 (unsprayed) and the unmodified control canola variety for minerals. Statistically significant differences were found between grain from canola event MS11 (sprayed) and the unmodified control canola variety for phosphorus, magnesium, potassium, copper and zinc. However, these differences were not biologically significant as all means were within the range of the values observed in the reference canola varieties grown in the trials.
No statistically significant differences were observed between grain from canola event MS11 (unsprayed) and the unmodified control canola variety for vitamins. Statistically significant differences were found between grain from canola event MS11 (sprayed) and the unmodified control canola variety for gamma tocopherol and vitamin K. However, these differences were not biologically significant as all means were within the range of the values observed in the reference canola varieties grown in the trials.
No statistically significant differences were observed between grain from canola event MS11 (unsprayed) and the unmodified control canola variety for amino acids. Except for proline, alanine, cysteine, methionine, phenylalanine and lysine, no statistically significant differences were observed between grain from canola event MS11 (sprayed) and the unmodified control canola variety for amino acids. However, all means were within the range of the values observed in the reference canola varieties grown in the trials.
No statistically significant differences were observed between grain from canola event MS11 (unsprayed) and the unmodified control canola variety for fatty acids. Statistically significant differences were found for palmitic, stearic, oleic, arachidic, behenic and lignoceric acids between grain from canola event MS11 (sprayed) and the unmodified control canola variety. However, all means were within the range of the values observed in the reference canola varieties grown in the trials.
Except for gluconapin, progoitrin, total glucosinolates, and insoluble tannins no statistically significant differences were observed between grain from canola event MS11 (unsprayed) and the unmodified control canola variety for anti-nutrients. Statistically significant differences were observed for gluconapin, progoitrin, and total glucosinolates between grain from canola event MS11 (sprayed) and the unmodified control canola variety. However, all means were within the range of the values observed in the reference canola varieties grown in the trials.
Broiler performance
A total of 140 broiler chickens (Gallus gallus domesticus) were randomly allocated to three diet groups to compare feed consumption, growth variables, health and survival. Broiler chickens were fed nutritionally equivalent diets containing canola event MS11 and the unmodified control canola and an unmodified commercial reference variety over a 6-week period. Health, mortality, weight gain, diet conversion efficiencies and market dressed carcass, muscle (breast, thigh, leg and wing), and abdominal fat pad weights were evaluated. The performance and mortality data for birds fed canola event MS11 diets were not statistically different from birds on the diets containing the unmodified control canola variety and the reference canola variety over the entire trial. No statistically significant differences were observed between broilers fed canola event MS11 meal and those fed the unmodified control canola variety meal for body weight gain, feed intake, and feed conversion ratio. Carcass weights, weights of legs, thighs, wings and breasts were not statistically different for the diets containing canola event MS11 meal compared to those fed diets containing the unmodified control canola variety meal or reference canola variety meal.
Conclusion
It was concluded, based on the evidence provided by Bayer Crop Science Inc. that the nutritional composition of the canola event MS11 is similar to that of the conventional canola (B. napus) varieties. Feed ingredients derived from canola event MS11 are considered to meet present ingredient definitions for canola.
2. Potential impact of canola Event MS11 on animal health and human safety as it relates to the potential transfer of residues into foods of animal origin and worker/bystander exposure to the feed
Canola event MS11 produces the barnase protein which results in male sterility, the barstar protein that functions to inhibit barnase and restores fertility under normal circumstances, and the PAT protein which confers tolerance to glufosinate ammonium herbicide. In canola event MS11 the barstar protein did not significantly affect the barnase activity and did not restore fertility.
The assessment of canola event MS11 evaluated the impact of the following potential hazards relative to the safety of feed ingredients derived from this event:
- The novel protein barnase
- The novel protein barstar
- The novel protein PAT
Barnase protein
To obtain sufficient quantities of the barstar protein for assessment of environmental and feed safety, it was necessary to express the barnase gene in an E. coli production system. A weight-of evidence approach was used to assess the equivalence of canola event MS11-produced and the E. coli-produced barnase proteins. DNA sequence analysis confirmed that the barnase gene sequence expressed in E. coli and the gene sequence inserted in to canola event MS11 were identical. Furthermore, N-terminal sequence analysis and MALDI-TOF mass spectrometry of the E. coli-produced barnase protein confirmed that it shares the identical amino acid sequence to what would be expected of the plant-produced barnase protein. Additionally, data was provided to characterize the structure and function of the E.coli-produced barnase protein, such as molecular weight, immunoreactivity, lack of glycosylation, and enzymatic activity. The cumulative weight of evidence establishes that the E.coli-produced barnase protein is an acceptable surrogate for canola event MS11 barnase protein.
The potential allergenicity and toxicity of the barnase protein to livestock were evaluated. With respect to its potential allergenicity, no single experimental method yields decisive evidence, thus a weight-of-evidence approach was taken, taking into account information obtained with various test methods. The source of the barnase gene, B. amyloliquefaciens, is not known to produce allergens and a bioinformatics evaluation of the barnase protein amino acid sequence confirmed the lack of relevant similarities between the barnase protein and known allergens. E. coli-produced barnase protein safety studies indicated that, unlike many allergens, this protein is rapidly degraded in simulated gastric fluid and is not heat stable. Finally, unlike many allergens, E.coli-produced barnase protein was shown experimentally to be unglycosylated. Thus, the weight of evidence indicates that the barnase protein is unlikely to be allergenic.
In terms of its potential toxicity to livestock, the barnase protein lacks a mode of action to suggest that it is intrinsically toxic to livestock and a bioinformatics evaluation of the barnase protein amino acid sequence confirmed the lack of relevant similarities between the barnase protein and known toxins. In addition, E. coli-produced barnase protein safety studies indicated that no adverse effects were observed when the barnase protein was ingested by mice at doses of approximately 2000 mg/kg bw. This information indicates that the barnase protein is unlikely to be toxic to livestock.
The livestock exposure to the barnase protein is expected to be negligible as the barnase protein is expressed at very low levels in canola event MS11 and is rapidly degraded under conditions which simulate the mammalian digestive tract.
Barstar protein
To obtain sufficient quantities of barstar protein for the assessment of feed safety, it was necessary to express the barstar gene in an E. coli production system. A weight-of evidence approach was used to assess the equivalence of canola event MS11-produced and the E. coli-produced barstar proteins. DNA sequence analysis confirmed that the barstar gene sequence expressed in E. coli and the gene sequence inserted in to canola event MS11 were identical. Furthermore, N-terminal sequence analysis and MALDI-TOF mass spectrometry of the E. coli-produced barstar protein confirmed that it shares the identical amino acid sequence to what would be expected of the plant-produced barstar protein. Additionally, data was provided to characterize the structure and function of the E. coli- produced barstar protein, such as molecular weight, immunoreactivity, lack of glycosylation, and functional activity. The cumulative weight of evidence establishes that the E. coli-produced barstar protein is an acceptable surrogate for canola event MS11 barstar protein.
The potential allergenicity and toxicity of the barstar protein to livestock were evaluated. With respect to its potential allergenicity, no single experimental method yields decisive evidence, thus a weight-of-evidence approach was taken, taking into account information obtained with various test methods. The source of the barstar gene, B. amyloliquefaciens, is not known to produce allergens and a bioinformatics evaluation of the barstar protein amino acid sequence confirmed the lack of relevant similarities between the barstar protein and known allergens. E. coli-produced barstar protein safety studies indicated that, unlike many allergens, this protein is rapidly degraded in simulated gastric and intestinal fluid and is not heat stable. Finally, unlike many allergens, E. coli-produced barstar protein was shown experimentally to be unglycosylated. The weight of evidence indicates that the barstar protein is unlikely to be allergenic.
In terms of its potential toxicity to livestock, the barstar protein lacks a mode of action to suggest that it is intrinsically toxic to livestock and a bioinformatics evaluation of the barstar protein amino acid sequence confirmed the lack of relevant similarities between the barstar protein and known toxins. In addition, E. coli-produced barstar protein safety studies indicated that no adverse effects were observed when the barstar protein was ingested by mice at doses of approximately 2000 mg/kg bw. This information indicates that the barstar protein is unlikely to be toxic to livestock.
The livestock exposure to the barstar protein is expected to be negligible as the barstar protein is expressed at very low levels in canola event MS11 and is rapidly degraded under conditions which simulate the mammalian digestive tract.
PAT protein
To obtain sufficient quantities of PAT protein for the assessment of feed safety, it was necessary to express the bar gene in an E. coli production system. Equivalency was demonstrated between canola event MS11-produced PAT protein and the E. coli-produced PAT protein by comparing their molecular weight, immunoreactivity, N-terminal sequence analysis, glycosylation, and functional activity. Demonstration of equivalence between the PAT proteins produced in E. coli and in canola event MS11 allows the PAT protein produced in E. coli to be used in studies to confirm the safety of the PAT protein produced in canola event MS11.
The potential allergenicity and toxicity of the PAT protein to livestock were evaluated. With respect to its potential allergenicity, no single experimental method yields decisive evidence, thus a weight-of-evidence approach was taken, taking into account information obtained with various test methods. The source of the bar gene, S. hygroscopicus, is not known to produce allergens and a bioinformatics evaluation of the PAT protein amino acid sequence confirmed the lack of relevant similarities between the PAT protein and known allergens. E. coli-produced PAT protein safety studies indicated that, unlike many allergens, this protein is rapidly degraded in simulated gastric and intestinal fluid and is heat labile. Finally, unlike many allergens, E. coli-produced PAT protein was shown experimentally to not be glycosylated. The weight of evidence indicates that the PAT protein is unlikely to be allergenic.
In terms of its potential toxicity to livestock, the PAT protein lacks a mode of action to suggest that it is intrinsically toxic to livestock and a bioinformatics evaluation of the PAT protein amino acid sequence confirmed the lack of relevant similarities between the PAT protein and known toxins. In addition, E. coli-produced PAT protein safety studies indicated that no adverse effects were observed when the PAT protein was ingested by mice at doses of approximately 2000 mg/kg bw. This information indicates that the PAT protein is unlikely to be toxic to livestock.
The livestock exposure to the PAT protein is expected to be negligible as the PAT protein is expressed at very low levels in canola event MS11 and is rapidly degraded under conditions which simulate the mammalian digestive tract.
Conclusion
It was concluded, based on the evidence provided by Bayer Crop Science Inc.'s MS11, that the novel barnase protein-based male sterility trait, the barstar protein-based trait , and the PAT protein-based herbicide tolerance trait will not confer to canola event MS11 any characteristic that would raise concerns regarding the safety of canola event MS11. Feed ingredients derived from canola event MS11 are considered to meet present the ingredient definitions for canola.
VI. New information requirements
If at any time, Bayer CropScience Inc. becomes aware of any new information regarding risk to the environment livestock or human health, which could result from the unconfined environmental release or livestock feed use of canola event MS11 or lines derived from it, Bayer CropScience Inc. is required to immediately provide such information to CFIA. On the basis of such new information, CFIA will re-evaluate the potential impact of canola event MS11 on the environment, livestock and human health and may re-evaluate its decision with respect to the livestock feed use and unconfined environmental release authorizations of canola event MS11.
VII. Regulatory decision
Based on the review of the data and information submitted by Bayer CropScience Inc. and input from other relevant scientific sources, the Plant and Biotechnology Risk Assessment Unit of the Plant Health Science Directorate, CFIA, has concluded that the unconfined environmental release of canola event MS11 does not present altered environmental risk when compared to canola varieties that are currently grown in Canada.
Based on the review of the data and information submitted by Bayer CropScience Inc. and input from other relevant scientific sources, the Animal Feed Division of the Animal Health Directorate, CFIA, has concluded that the novel barnase protein-based male sterility, barstar protein and PAT protein-based herbicide tolerance traits will not confer to canola event MS11 any characteristic that would raise concerns regarding the safety or nutrition of canola event MS11. Livestock feeds derived from canola are currently listed in Schedule IV of the Feeds Regulations. Canola event MS11 has been found to be as safe as and as nutritious as currently and historically grown canola varieties. Canola event MS11 and its products are considered to meet present ingredient definitions.
Unconfined release into the environment and use as livestock feed of canola event MS11 is therefore authorized by the Plant Biosafety Office of the Plant Health and Biosecurity Directorate and the Animal Feed Division of the Animal Health Directorate, respectively, as of January 30, 2018. Any corn lines derived from canola event MS11 may also be released into the environment and used as livestock feed, provided that:
- no inter-specific crosses are performed
- the intended uses are similar
- it is known based on characterization that these plants do not display any additional novel traits and are substantially equivalent to canola varieties that are currently grown and permitted to be used as livestock feed in Canada, in terms of their potential environmental impact and livestock feed safety and nutrition
- the novel genes are expressed at levels similar to those in the authorized line and data used to establish the substantial equivalence of lines derived from canola event MS11 be made available to CFIA upon request
Canola event MS11 is subject to the same phytosanitary import requirements as unmodified canola varieties. Canola event MS11 is required to meet the requirements of other jurisdictions, including but not limited to, the Food and Drugs Act and the Pest Control Products Act.
Please refer to Health Canada's Decisions on Novel Foods for a description of the food safety assessment of canola event MS11. The food safety decisions are available on the Health Canada website.