Decision Document DD2020-127 Determination of the Safety of Nuseed Canada Inc.'s Canola (Brassica napus L.) Event NS-B5ØØ27-4

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 Program (AFP) of the Animal Health Directorate – has evaluated information submitted by Nuseed Canada Inc. This information concerns canola event NS-B5ØØ27-4, which synthesizes omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs) and has tolerance to glufosinate-ammonium (GA) herbicide.

The CFIA has determined that canola event NS-B5ØØ27-4 does not present altered risk when compared to canola varieties currently grown and permitted to be used as livestock feed in Canada, subject to the conditions described in Section 7 – Regulatory Decision.

Regarding the feed authorization, the CFIA has authorized the use of canola event NS-B5ØØ27-4 oil as a source of omega-3 LC-PUFAs for fish feeds only. Furthermore, only pre-pressed solvent extracted defatted canola meal has been authorized for use as livestock feed. Conditions on the feed authorization are described in Section 7 - Regulatory Decision. Given that the canola oil for this event was approved for fish feed only, a new definition for omega-3 long chain polyunsaturated fatty acids canola oil was added to Schedule IV of the Feeds Regulations.

Taking into account the CFIA's environment and livestock feed evaluations, unconfined release into the environment of canola event NS-B5ØØ27-4 is therefore authorized by the Plant Biosafety Office of the Plant Health and Biosecurity Directorate as of July 28, 2020. Likewise, livestock feed use is authorized, with the specific use restrictions stated above, by the Animal Feed Program of the Animal Health Directorate as of July 28, 2020.

Any canola lines derived from canola event NS-B5ØØ27-4 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, and meet the conditions of the authorizations
• it is known based on characterization that these plants are substantially equivalent to the authorized line, and do not display any additional novel traits, and
• the novel genes are expressed at levels similar to those of the authorized line

With respect to its unconfined release into the environment, an appropriate herbicide tolerance management plan should be implemented. Before canola event NS-B5ØØ27-4 is cultivated in Canada as an individual event or in combination with other canola events in stacked/pyramided products, Nuseed Canada Inc. must submit a herbicide tolerance management plan to the CFIA.

Canola event NS-B5ØØ27-4 is subject to the same phytosanitary import requirements as unmodified canola varieties. Canola event NS-B5ØØ27-4 is required to meet the requirements of other jurisdictions, including but not limited to, the Food & Drugs Act and the Pest Control Products Act.

Please note that the livestock feed and environmental assessments of novel feeds and plants with novel traits s 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.

July 28, 2020

This bulletin was created by the Canadian Food Inspection Agency. For further information, please contact the Plant Biosafety Office or the Animal Feed Program by visiting the contact page.

On this page

• 1. Brief identification of the modified plant
• 2. Background information
• 3. Description of the novel traits
  • 3.1 Development method
  • 3.2 Synthesis of omega-3 long chain polyunsaturated fatty acids
  • 3.3 Tolerance to glufosinate-ammonium herbicide
  • 3.4 Stable integration into the plant genome
• 4. Criteria for the environmental assessment
  • 4.1 Potential for canola event NS-B5ØØ27-4 to become a weed of agriculture or be invasive of natural habitats
  • 4.2 Potential for gene flow from canola event NS-B5ØØ27-4 to sexually compatible plants whose hybrid offspring may become more weedy or more invasive
  • 4.3 Potential for canola event NS-B5ØØ27-4 to become a plant pest
  • 4.4 Potential impact of canola event NS-B5ØØ27-4 and its gene products on non-target organisms, including humans
  • 4.5 Potential impact of canola event NS-B5ØØ27-4 on biodiversity
• 5. Criteria for the livestock feed assessment
  • 5.1 Potential impact of canola event NS-B5ØØ27-4 on livestock nutrition
  • 5.2 Potential Impact of canola event NS-B5ØØ27-4 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
• 6. New information requirements
• 7. Regulatory decision

1. Brief identification of the modified plant

Designation of the modified plant: Canola Event NS-B5ØØ27-4 (OECD unique identifier NS-B5ØØ27-4)

Applicant: Nuseed Canada Inc.

Plant species: Canola (Brassica napus L.)

Novel traits: Synthesis of omega-3 long chain polyunsaturated fatty acids; tolerance to glufosinate-ammonium herbicide.

Trait introduction method: Agrobacterium-mediated transformation

Intended use of the modified plant: Canola event NS-B5ØØ27-4 is intended for cultivation within the United States and eventually Canada, and for processing either in the United States or Canada. Nuseed Canada Inc. intends to implement an identity preservation system at every step of production and handling. Canola event NS-B5ØØ27-4 oil is intended for use in human food and fish feeds, as a source of omega-3 long-chain polyunsaturated fatty acids. Only pre-pressed solvent-extracted defatted canola meal is intended for use as livestock feed. As Nuseed did not seek an authorization for other uses, no authorization has been granted for use of whole grain and forage derived from the canola event NS-B5ØØ27-4 as livestock feed at this time.

2. Background information

Nuseed has developed canola event NS-B5ØØ27-4, which produces omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs), primarily docosahexaenoic acid (DHA), and which is tolerant to glufosinate-ammonium (GA) herbicides.

Canola event NS-B5ØØ27-4 was developed by Nuseed using recombinant deoxyribonucleic acid (DNA) technology, resulting in the introduction of 8 gene expression cassettes encoding 5 fatty acid desaturase proteins, 2 fatty acid elongase proteins and 1 gene encoding a phosphinothricin-N-acetyltransferase (PAT) protein.

The 5 fatty acid desaturase and 2 elongase proteins are as follows:

• delta-6 desaturase from Micromonas pusilla (Micpu-Δ6D)
• delta-5 elongase from Pyramimonas cordata (Pyrco-Δ5E)
• delta-5 desaturase from Pavlova salina (Pavsa-Δ5D)
• omega-3 desaturase from Pichia pastoris (Picpa-ω3D)
• delta-4 desaturase from Pavlova salina (Pavsa-Δ4D)
• delta-12 desaturase from Lachancea kluyveri (Lackl-Δ12D)
• delta-6 elongase from Pyramimonas cordata (Pyrco-Δ6E)

The pat gene is derived from the soil bacterium Streptomyces viridochromogenes, and encodes the PAT protein, which confers tolerance to glufosinate-ammonium herbicides.

Nuseed Canada Inc. provided information on the identity of canola event NS-B5ØØ27-4, and a detailed description of the introduced genetic elements and new proteins encoded by these genetic elements. Nuseed Canada Inc. also provided information about how canola event NS-B5ØØ27-4 compares to other canola varieties in terms of its agronomic characteristics and environmental safety, and its nutrition and safety as an animal feed.

The Plant and Biotechnology Risk Assessment (PBRA) Unit of the Plant Health Science Directorate, CFIA, has reviewed the above information according to the assessment criteria for determining environmental safety of plants with novel traits 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 NS-B5ØØ27-4 to become a weed of agriculture or to be invasive of natural habitats;
• the potential for gene flow from canola event NS-B5ØØ27-4 to sexually compatible plants whose hybrid offspring may become more weedy or more invasive;
• the potential for canola event NS-B5ØØ27-4 to become a plant pest;
• the potential impact of canola event NS-B5ØØ27-4 and its gene products on non-target organisms, including humans; and
• the potential impact of canola event NS-B5ØØ27-4 on biodiversity.

The Animal Feed Program (AFP) of the 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 AFP has considered both intended and unintended effects and similarities and differences between canola event NS-B5ØØ27-4, and unmodified parental canola variety relative to the safety and efficacy of feed ingredients derived from canola event NS-B5ØØ27-4, for its intended purpose, including:

• the potential impact of canola event NS-B5ØØ27-4 on livestock nutrition; and
• the potential impact of canola event NS-B5ØØ27-4 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 AFP has also considered whether feeds derived from canola event NS-B5ØØ27-4 meet the definitions and requirements of feeds as listed in Schedule IV of the Feeds Regulations.

3. Description of the novel traits

3.1 Development method

Canola event NS-B5ØØ27-4 was developed through Agrobacterium tumefaciens-mediated transformation of Brassica napus cv. AV Jade cotyledonary petioles with a plasmid vector that included a single T-DNA. The T-DNA contained 8 gene cassettes including 5 fatty acid desaturases and 2 fatty acid elongases under control of seed-specific promoters, and an expression cassette encoding a PAT protein under control of a constitutive promoter. Transformed explants were selected on the basis of tolerance to glufosinate-ammonium and were regenerated to produce plants. Canola event NS-B5ØØ27-4 was identified as a successful transformant based on molecular analyses, fatty acid profiles, herbicide tolerance and agronomic evaluations.

3.2 Synthesis of omega-3 long chain polyunsaturated fatty acids

Canola event NS-B5ØØ27-4 was developed to synthesize omega-3 LC-PUFAs, including EPA and DHA. There were 7 integral membrane proteins (5 desaturases and 2 elongases) introduced into canola event NS-B5ØØ27-4. Unmodified canola produces primarily oleic and linoleic fatty acids in seeds through enzymes involved in de novo fatty acid synthesis, elongation, and desaturation. Expression of the omega-3 LC-PUFA synthesis genes in canola event NS-B5ØØ27-4 allows for production of DHA and its biosynthetic intermediate EPA from endogenous fatty acids.

The genes used for omega-3 LC-PUFA synthesis in canola event NS-B5ØØ27-4 were synthesized based on the sequences identified and characterized from the source organisms. The DNA sequence of each gene was modified to optimize the translation rate in canola.

Expression of the omega-3 LC-PUFA synthesis proteins in canola event NS-B5ØØ27-4 is driven by seed-specific promoters. Samples of canola tissues were collected from plants in 3 field trials in Australia. Tissues were collected from unsprayed plants. The tissues analyzed were whole plant during leaf development (BBCH 15); whole plant during stem elongation (BBCH 35); flowers, roots and whole plant excluding the flowers and roots at 50% full flowering (BBCH 65); pods at the developing seed stage (BBCH 79) and mature seed at senescence (BBCH 90). A liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS) quantification method was used to determine the amount of Micpu-Δ6D, Pyrco-Δ5E, Pavsa-Δ5D, Picpa-ω3D, Pavsa-Δ4D, Lackl-Δ12D and Pyrco-Δ6E proteins present in the tissue samples.

All of the 7 proteins in the introduced omega-3 LC-PUFA synthesis pathway were detected in immature and/or mature seeds of canola event NS-B5ØØ27-4. In all other tissues analyzed, the level of expression for all introduced omega-3 LC-PUFA proteins, in micrograms per gram fresh weight (μg/g fw), was below the limit of detection (<LOD, 0.004-0.012 µg/g fw).

The expression of the omega-3 LC-PUFA synthesis proteins in mature and immature seeds from unsprayed plants, were as follows:

The potential allergenicity and toxicity of the introduced omega-3 LC-PUFA synthesis proteins to livestock and non-target organisms was evaluated. The weight of evidence indicates that these proteins are unlikely to be allergenic, based on the following information:

• the source of the introduced omega-3 LC-PUFA synthesis genes (Micromonas pusilla, Pyramimonas cordata, Pavlova salina, Pichia pastoris, and Lachancea kluyveri) are not commonly associated with allergenicity
• bioinformatic evaluations of the amino acid sequences of the introduced omega-3 LC-PUFA synthesis proteins confirmed the lack of relevant similarities to known allergens

Taken together, this information implies that these proteins are unlikely to be allergenic.

It was also concluded that the omega-3 LC-PUFA synthesis proteins introduced in canola event NS-B5ØØ27-4 are unlikely to be toxic to livestock and non-target organisms because:

• they lack a mode of action to suggest that they are intrinsically toxic to livestock or non-target organisms
• the amino acid sequences of the omega-3 LC-PUFA synthesis proteins lack relevant similarities to known toxins

For a more detailed discussion of the potential allergenicity and toxicity of the introduced omega-3 LC-PUFA synthesis proteins, see Section 5.2: Potential impact of canola event NS-B5ØØ27-4 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.

3.3 Tolerance to glufosinate-ammonium herbicides

Glufosinate-ammonium inhibits the plant enzyme glutamine synthetase, an enzyme that converts glutamate and ammonia to glutamine. This results in reduced glutamine synthesis and accumulation of lethal levels of ammonia in susceptible plants. Ammonia is produced by plants as a result of normal metabolic processes, but elevated levels of ammonia can interfere with essential plant processes like photosynthesis, leading to plant death.

Canola event NS-B5ØØ27-4 was developed to be tolerant of glufosinate ammonium herbicides by introduction of the pat gene from Streptomyces viridochromogenes, a gram-positive soil bacterium. The pat gene encodes the PAT protein, which acetylates the primary amino group of glufosinate ammonium, rendering the herbicide inactive. The PAT protein produced in canola event NS-B5ØØ27-4 is identical to the native enzyme in terms of its amino acid sequence and confers commercial-level tolerance to glufosinate ammonium herbicides.

Expression of the PAT protein in canola event NS-B5ØØ27-4 is driven by a constitutive promoter. Samples of canola tissues were collected from plants in 3 field trials in Australia. Tissues were collected from unsprayed plants. The average protein expression levels of the PAT protein in micrograms per gram fresh weight (μg/g fw) from unsprayed plants, as evaluated by a quantitative LC-MRM-MS method, were as follows:

• 0.046 μg/g fw in whole plant at true leaves stage (BBCH 15)
• 0.11 μg/g fw in whole plant at visibly extended internodes stage (BBCH 35)
• 0.30 μg/g fw in flowers at 50% full flowering stage (BBCH 65)
• 0.17 μg/g fw in roots at 50% full flowering stage (BBCH 65)
• 0.13 μg/g fw in whole plant excluding the roots and flowers at 50% full flowering stage (BBCH 65)
• 0.72 – 0.97 μg/g fw in immature seed (BBCH 79), and
• 0.08 – 0.13 μg/g fw in mature seed at senescence (BBCH 90).

The potential allergenicity and toxicity of the PAT protein to livestock and 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 pat gene, Streptomyces viridochromogenes, is not commonly associated with allergenicity
• the PAT protein amino acid sequence lacks relevant similarities to known allergens

It was concluded that the PAT protein is unlikely to be toxic to livestock and non-target organisms because:

• it lacks a mode of action to suggest that it is intrinsically toxic to livestock or non-target organisms
• the PAT protein amino acid sequence lacks relevant similarities to known toxins

For a more detailed discussion of the potential allergenicity and toxicity of the modified PAT protein, see Section 5.2: Potential impact of canola event NS-B5ØØ27-4 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.

3.4 Stable integration into the plant genome

Molecular characterization by Illumina-based sequencing and Sanger sequencing demonstrated that canola event NS-B5ØØ27-4 contains 2 T-DNA integration sites, 1 in the A02 chromosome and 1 in the A05 chromosome.

The A02 chromosome locus contained a partial T-DNA insert comprising 4 gene cassettes for the Micpu-Δ6D, Pyrco-Δ5E, Pavsa-Δ5D and Picpa-ω3D proteins, with intact nucleotide sequences. The gene cassettes for the following proteins were absent in this partial insert: Pavsa-Δ4D, Lackl-Δ12D, Pyrco-Δ6E and PAT.

The A05 chromosome locus contained a second T-DNA insert comprising a duplicated set of 8 gene cassettes. The 2 sets of 8 genes were linked by 156 bp of palindromic left border (LB) sequences and flanked by 40 bp of right border (RB) sequence upstream and 42 bp of RB sequence downstream, which formed a palindromic structure with RB-LB:LB-RB orientation. The 2 sets of 8 genes included the respective complete expression cassettes with intact nucleotide sequences.

The presence of both T-DNA insertion loci was necessary to achieve higher expression levels of omega-3 long chain polyunsaturated fatty acids, primarily DHA. Thus, a transgenic line containing both T-DNA inserts was selected as the elite canola event NS-B5ØØ27-4. No backbone sequences from the plasmid vector, linked or unlinked to the intact inserts, were detected in canola event NS-B5ØØ27-4. No deleterious effects were observed at a phenotypic level as a result of the insertion locations in the A02 and the A05 chromosomes.

The stability of the inserts within canola event NS-B5ØØ27-4 was verified by Illumina-based sequencing and Kompetitive allele-specific (KASP) assay over 4 generations. The levels of omega-3 LC-PUFAs demonstrated the stability of the phenotypic trait over 4 generations. The inheritance pattern of both T-DNA inserts across 4 segregating generations of canola event NS-B5ØØ27-4 showed that, as expected, the 2 inserts segregated independently according to Mendelian rules of inheritance for 2 unlinked genetic loci.

4. Criteria for the environmental assessment

4.1 Potential for canola event NS-B5ØØ27-4 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 Nuseed Canada Inc., canola event NS-B5ØØ27-4 was determined not to be significantly different from unmodified canola varieties in this respect.

CFIA evaluated data submitted by Nuseed Canada Inc. on the reproductive biology and life history traits of canola event NS-B5ØØ27-4. Canola event NS-B5ØØ27-4 was tested at 3 locations in Canada and 3 locations in the US in 2017. It was determined that the 3 locations in Canada and 1 location in the US share 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. During the field trials, canola event NS-B5ØØ27-4 was compared to an unmodified control canola variety with the same genetic background. Reference canola varieties were also included in these trials to establish ranges of comparative values that are representative of currently grown canola varieties in Canada. Phenotypic and agronomic traits were evaluated, covering a broad range of characteristics that encompass the entire life cycle of the canola plant. The traits evaluated in the field trials included plant emergence, plant vigor, flowering time (50%), flowering end time, flowering duration, plant height, plant lodging, seed shattering, grain moisture and grain yield. The other 2 US locations were not representative of major Canadian growing regions, but the results from these locations were consistent with the results from the Canadian-equivalent locations. The results showed no biologically meaningful differences between canola event NS-B5ØØ27-4 and the unmodified control canola variety, and support a conclusion of phenotypic and agronomic equivalence to currently grown canola varieties.

Nuseed Canada Inc. evaluated the seed germination of canola event NS-B5ØØ27-4 under several temperature regimes. The germination rates of canola event NS-B5ØØ27-4 seeds were statistically lower than those of the unmodified control canola variety seeds at cold and warm temperatures. The trend toward reduced germination is likely a consequence of the modified fatty acid profile of the seed of canola event NS-B5ØØ27-4. A secondary dormancy test was performed to confirm that reduced germination of canola event NS-B5ØØ27-4 is not associated with increased seed secondary dormancy. The results showed that the secondary dormancy of canola event NS-B5ØØ27-4 seed was not increased and that the lower seed germination rates observed for canola event NS-B5ØØ27-4 was due to reduced seed viability. Reduced seed viability is not associated with increased weediness potential as it does not confer a competitive advantage to the canola plant.

The response of canola event NS-B5ØØ27-4 to abiotic stressors was evaluated in the field at 8 locations in Canada and 5 locations in the US from 2016 to 2018. The abiotic stressors included excessive rainfall, drought, heat, wind, cold/wet weather and hail. Observations were collected weekly during the growing season. No differences were observed between canola event NS-B5ØØ27-4 and the unmodified control canola variety for their responses to the abiotic stressors.

The susceptibility of canola event NS-B5ØØ27-4 to a range of canola pests and pathogens was evaluated in the field at 8 locations in Canada and 5 locations in the US from 2016 to 2018 (see Section 4.3: Potential for canola event NS-B5ØØ27-4 to become a plant pest). No consistent trend in decreased and increased susceptibility to pests or pathogens was observed in canola event NS-B5ØØ27-4 compared to the unmodified control canola variety.

No competitive advantage was conferred to plants of canola event NS-B5ØØ27-4, other than that conferred by tolerance to glufosinate ammonium herbicides, as the reproductive characteristics, growth characteristics and tolerance to abiotic and biotic stressors of canola event NS-B5ØØ27-4 were comparable to those of the unmodified control canola variety. Tolerance to glufosinate ammonium herbicides provides a competitive advantage only when these herbicides are used and will not, in itself, make a glufosinate-tolerant plant weedier or more invasive of natural habitats. Canola event NS-B5ØØ27-4 plants growing as volunteers will not be controlled if glufosinate ammonim is used as the only weed control tool. However, control of canola event NS-B5ØØ27-4 as a volunteer weed in subsequent crops or in fallow ground can be achieved using other classes of herbicides or mechanical means.

The novel traits have no intended or observed effects on weediness or invasiveness. CFIA has therefore concluded that canola event NS-B5ØØ27-4 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 that 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.

Nuseed Canada Inc. has submitted an herbicide tolerance management plan to CFIA, which was determined to be satisfactory when evaluated by the PBRA Unit. Nuseed Canada Inc. will make this herbicide tolerance management plan readily available to growers and agriculture extension personnel, in both private and public sectors, to promote careful management practices for canola event NS-B5ØØ27-4. Nuseed Canada Inc. will provide an efficient mechanism for growers to report agronomic problems to the company, which will facilitate the ongoing monitoring of canola event NS-B5ØØ27-4. Nuseed Canada Inc. will monitor grower implementation to determine the effectiveness of the herbicide tolerance management plan and make any changes to the plan as appropriate.

4.2 Potential for gene flow from canola event NS-B5ØØ27-4 to sexually compatible plants whose hybrid offspring may become more weedy or more invasive

Successful interspecific and intergeneric crosses between B. napus and some sexually compatible species have been reported in the scientific literature (see Biology Document BIO2017-03 - The Biology of Brassica napus L. (Canola/Rapeseed) for more information). However, many of these crosses have required extensive human intervention and the rates of natural hybridization between B. napus and weedy relatives resulting in fertile offspring appear to be very low. Sinapis arvensis is considered the worst of the weedy relatives of B. napus in Western Canada. Hybrids between both species can be produced under field conditions, however at very low frequency. Additionally, backcrossing of the hybrids to S. arvensis failed to produce viable progeny. Therefore, the likelihood of introgression of traits from B. napus to S. arvensis appears to be very low. In crosses with other sexually compatible wild related species (i.e. Raphanus raphanistrum and Erucastrum gallicum), no viable hybrid seed was produced. Stable gene transfer from B. napus is most likely with Brassica crops such as B. juncea and B. rapa.

The modified fatty acid profile introduced in seeds of canola event NS-B5ØØ27-4 is unrelated to weediness characteristics. Field trials confirmed that the reproductive characteristics, growth characteristics and tolerance to abiotic and biotic stressors of canola event NS-B5ØØ27-4 are comparable to those of the unmodified control canola variety (see Section 4.1: Potential for canola event NS-B5ØØ27-4 to become a weed of agriculture or be invasive of natural habitats).

Similarly, it is anticipated that the modified fatty acid profile would confer no competitive advantage to hybrids arising through outcrossing with canola event NS-B5ØØ27-4.

The glufosinate tolerance trait in canola event NS-B5ØØ27-4 would confer no competitive advantage to any hybrids resulting from outcrossing with canola event NS-B5ØØ27-4, unless challenged by a glufosinate-ammonium herbicide. This would only occur in managed ecosystems where a glufosinate herbicide is used for weed control. As with glufosinate-tolerant canola event NS-B5ØØ27-4 volunteers, these herbicide tolerant plants, should they arise, could be controlled using herbicides other than glufosinate-ammonium or by mechanical means. Hybrids, if they developed, could potentially result in the loss of glufosinate as a tool to control these species. This, however, can be avoided by the use of sound crop management practices. Nuseed Canada Inc.'s herbicide tolerance management plan for canola event NS-B5ØØ27-4 contains recommendations to minimize and manage outcrossing to sexually compatible species.

This information led CFIA to conclude that gene flow from canola event NS-B5ØØ27-4 to sexually compatible plants in Canada is possible, but would not result in increased weediness or invasiveness of the resulting progeny.

4.3 Potential for canola event NS-B5ØØ27-4 to become a plant pest

Canola is not considered to be a plant pest in Canada and the glufosinate tolerance trait introduced into canola event NS-B5ØØ27-4 is unrelated to plant pest potential (that is, the potential for the plant to harbour new or increased populations of pathogens or pests). However, considering the known roles of fatty acids in plant-pathogen and plant-pest interactions, Nuseed Canada Inc. submitted studies to examine the response of canola event NS-B5ØØ27-4 to plant pathogens and pests.

The susceptibility of canola event NS-B5ØØ27-4 to canola pests and pathogens was evaluated in the field at 8 locations in Canada and 5 locations in the US from 2016 to 2018. The pests observed included:

• aphids
• armyworms
• beet webworms
• cabbage worms
• corn rootworms
• cutworms
• diamondback moths
• flea beetles
• loopers
• lygus bugs
• painted lady larvae
• red turnip beetles
• seedpod weevils
• stinkbugs
• swede midges

The pathogens observed included:

• Alternaria
• anthracnose
• aster yellows
• blackleg
• downy mildew
• root rot complex
• Sclerotinia

Field observations did not indicate differences in the response of canola event NS-B5ØØ27-4 to these pest insects and diseases when compared to the unmodified control canola variety.

CFIA has therefore concluded that canola event NS-B5ØØ27-4 does not display altered plant pest potential compared to currently grown canola varieties.

4.4 Potential impact of canola event NS-B5ØØ27-4 and its gene products on non-target organisms, including humans

The CFIA evaluated the potential impacts of the novel traits expressed by canola event NS-B5ØØ27-4 (that is, modified fatty acid profile and tolerance to glufosinate) and the proteins that confer the novel traits on organisms interacting with canola.

The glufosinate tolerance trait introduced into canola event NS-B5ØØ27-4 is unrelated to a potential impact on non-target organisms.

Detailed characterization of the novel proteins produced in canola event NS-B5ØØ27-4 (Micpu-Δ6D, Pyrco-Δ5E, Pavsa-Δ5D, Picpa-ω3D, Pavsa-Δ4D, Lackl-Δ12D, Pyrco-Δ6E and PAT) led to the conclusion that none of these proteins displayed any characteristics of a potential toxin or allergen (see Section 5.2: Potential Impact of Canola Event NS-B5ØØ27-4 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).

Long-chain polyunsaturated fatty acids, like DHA and EPA, have many functions in both aquatic and terrestrial organisms. Cultivating canola event NS-B5ØØ27-4 will expose some organisms to levels of long-chain polyunsaturated fatty acids that they do not normally encounter. The main potential route of exposure is via seed consumption because the novel long-chain polyunsaturated fatty acids are only produced in the seed. Indirect exposure via the consumption of prey that has fed on canola event NS-B5ØØ27-4 seed has been shown to be negligible. Hixson et al. 2016^Footnote 1 studied the effect of DHA and EPA in the diet of a canola pest, cabbage white butterfly (Pieris rapae). The authors concluded that the presence of EPA and DHA in diets of larval P. rapae may alter adult mass and wing morphology. No effect was seen on other parameters measured, including developmental phenology, larval or pupal weight, food consumption and larval mortality. Therefore, the novel proteins produced in canola event NS-B5ØØ27-4 are unlikely to have a direct or indirect effect on arthropods in Canada, with the possible exception of limited impacts on pest species feeding on canola seed.

Field observations did not indicate differences in the response of canola event NS-B5ØØ27-4 to pest insects and diseases when compared to the unmodified control canola variety (see Section 4.3: Potential for canola event NS-B5ØØ27-4 to become a plant pest).

Collectively, these information elements indicate that the interactions between canola event NS-B5ØØ27-4 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 NS-B5ØØ27-4 in Canada will not result in altered impacts on non-target organisms, including humans, compared to currently grown canola varieties.

4.5 Potential impact of canola event NS-B5ØØ27-4 on biodiversity

Canola event NS-B5ØØ27-4 displays no novel phenotypic characteristics that would extend its range beyond the current geographic range of canola production in Canada. The novel traits expressed by canola event NS-B5ØØ27-4 have been determined to be unlikely to cause adverse effects on non-target organisms and canola event NS-B5ØØ27-4 does not display increased weediness, invasiveness or plant pest potential. Canola 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 novel traits to a hybrid progeny are minimal. The novel herbicide tolerance trait does not confer any selective advantage in the absence of the herbicide, and glufosinate-tolerant hybrids can be controlled by herbicides with other modes of action, or through mechanical means. The transfer of omega-3 LC-PUFA synthesis proteins and their corresponding products is not expected to confer a selective advantage or modify the interactions between the hybrid progeny and organisms interacting with these plants.

It is therefore unlikely that canola event NS-B5ØØ27-4 will have any direct effects on biodiversity, in comparison to the effects that would be expected from the cultivation of the canola varieties that are currently grown in Canada.

Canola event NS-B5ØØ27-4 is tolerant of glufosinate ammonium herbicides. The use of glufosinate-ammonium 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. However, it must be noted that the goal of reduction in weed biodiversity in agricultural fields is not unique to the use of plants with novel traits, canola event NS-B5ØØ27-4 or the cultivation of canola. It is therefore unlikely that canola event NS-B5ØØ27-4 will have any indirect effects on biodiversity, in comparison to the effects that would be expected from cultivation of currently grown canola varieties.

CFIA has concluded that the introduced genes and their corresponding novel traits do not confer to canola event NS-B5ØØ27-4 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 NS-B5ØØ27-4 is unlikely to be different from that of the canola varieties that are currently grown in Canada.

5. Criteria for the Livestock Feed Assessment

The Animal Feed Program (AFP) considered the safety and efficacy of feed ingredients derived from canola event NS-B5ØØ27-4, including nutrient and anti-nutrient profiles; 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 NS-B5ØØ27-4 meet the definitions and requirements of feeds as listed in Schedule IV of the Feeds Regulations.

5.1 Potential Impact of Canola Event NS-B5ØØ27-4 on Livestock Nutrition

5.1.1 Nutrient and anti-nutrient composition

The nutritional equivalence of canola event NS-B5ØØ27-4 to its unmodified control canola variety (AV Jade) and 7 reference canola varieties was assessed from 5 replicated field sites at 8 locations in the major canola growing regions of Australia in 2015. Canola grain samples harvested from all field plots were used for compositional data analysis. Grain samples from canola event NS-B5ØØ27-4 and the harvested unmodified control canola variety were also processed into meal and oil. Nutrients analyzed in the grain, meal and oil included the following: proximate (protein, crude fat, moisture, ash), carbohydrates (calculated), crude fibre (CF), acid detergent fibre (ADF), neutral detergent fibre (NDF), total dietary fibre (TDF), amino acids, fatty acids, minerals, vitamins, phytosterols, glucosinolates and anti-nutrients (phytic acid, tannins, sinapine, and coumaric and ferulic acids) as per the guidelines of the Organization for Economic Co-operation and Development (OECD, 2011). Analysis of variance was used for statistical examination of the compositional data and comparison of means was conducted at a 95% confidence level (P<0.05). Statistically significant differences observed between canola event NS-B5ØØ27-4 and the unmodified control canola variety were assessed within the range of the reference canola varieties included in the trials. Mean values were also compared to the range of values generated from the Agriculture and Food Systems Institute (AFSI) Crop Composition Database (formerly known as ILSI), and peer-reviewed scientific literature and OECD consensus documents (2011) to provide context for the comparative analyses and assess the broader biological relevance of the results.

5.1.1.1 Grain

No statistically significant differences were observed between canola event NS-B5ØØ27-4 and its unmodified control variety for protein, CF, NDF, ADF and amino acids (histidine, arginine, tryptophan, cysteine, glutamic acid, isoleucine, leucine, phenylalanine, serine and valine). Statistically significant differences were observed between canola event NS-B5ØØ27-4 and its unmodified control variety for ash, crude fat, carbohydrates and amino acids (aspartic acid, glycine, lysine, methionine, proline, threonine, tyrosine and alanine). However, the means of these nutrients in canola event NS-B5ØØ27-4 were within the ranges of the reference canola varieties and were also within the range of values in peer-reviewed literature and AFSI Crop Composition database. Statistically significant differences were observed for all vitamins analyzed, except delta and gamma tocopherols, and folic acid. Calcium, iron, potassium and zinc were statistically significantly different in canola event NS-B5ØØ27-4 compared to the unmodified control canola variety. However, all means were within the range of natural variation of the reference canola varieties, peer-reviewed literature values and AFSI Crop Composition database. Therefore, the observed compositional differences were not considered biologically relevant.

Total glucosinolate content in canola event NS-B5ØØ27-4 grain was 11.87 µmol/g compared to 12.09 µmol/g in the unmodified control variety. The total glucosinolate content of canola event NS-B5ØØ27-4 therefore met the canola quality standards of 4 - 26.8 µmol/g (OECD, 2011). Statistically significant differences were observed between canola event NS-B5ØØ27-4 and its unmodified control canola variety for brassicasterol, campesterol, sitosterol, stigmasterol and total phytosterols. Apart from sinapine, the anti-nutrients ferulic acid and phytic acid were not statistically significantly different from canola event NS-B5ØØ27-4 compared to its unmodified control canola varieties. However, all means of the phytosterols and anti-nutrients were within the natural variation of the reference canola varieties, peer-reviewed literature values and/or AFSI Crop Composition database.

5.1.1.2 Endogenous fatty acids not impacted by novel trait

No statistically significant differences were observed between canola event NS-B5ØØ27-4 and the unmodified canola variety for the following fatty acids: myristic (C14:0), palmitoleic (C16:1, n-7), stearic (C18:0) and lignoceric (C24:0) acids. Statistically significant differences were observed for total fatty acids, total trans fatty acids, and the following fatty acids: palmitic (C16:0), margaric (C17:0), margaroleic (C17:1), cis-vaccenic (C18:1, n-7), arachidic (C20:0), gondoic (C20:1, n-9), eicosadienoic (C20:2, n-6), behenic (C22:0), and nervonic (C24:1) acids. However, all mean values were within the range of natural variation of the reference canola varieties, peer-reviewed literature values and AFSI Crop Composition database.

5.1.1.3 Endogenous fatty acids impacted by novel trait

The following endogenous fatty acids were significantly different in canola event NS-B5ØØ27-4 compared to unmodified control variety: cis-7-hexadecenoic (C16:1, n-9), oleic (C18:1, n-9), linoleic (C18:2, n-6) and alpha-linolenic (C18:3, n-3, ALA) acids. These fatty acids were also outside the range of the reference varieties, values in peer reviewed literature or AFSI Crop Composition database, and therefore considered biologically relevant due to the genetic modification in canola event NS-B5ØØ27-4.

For the introduced fatty acids (not present in conventional canola), the following were detected in canola event NS-B5ØØ27-4: gamma-linolenic (C18:3, n-6), stearidonic (C18:4, n-3), eicosatetraenoic (C20:4, n-3), eicosapentaenoic (C20:5, n-3), docosatetraenoic (C22:4, n-3), docosapentaenoic (C22:5, n-3) and docosahexaenoic (C22:6, n-3) acids. The total n-3 LC-PUFAs in canola event NS-B5ØØ27-4 was approximately 12%, with DHA and EPA comprising 8.38% and 0.5%, respectively.

5.1.1.4 Meal (Crude and solvent extracted)

Protein, fat, ash, calcium, phosphorus, amino acids, tocopherols and anti-nutrients values in canola event NS-B5ØØ27-4 meals were comparable to the unmodified control variety. The oil content in the crude meal was 21% for the unmodified control variety and 16% in canola event NS-B5ØØ27-4; while it was 0.7% and 0.4%, respectively, in the solvent extracted meal. The crude meal content of EPA and DHA in canola event NS-B5ØØ27-4 was 0.1% and 1.3%, respectively. The total glucosinolates in crude meal were 15.55 µmol/g and 16.04 µmol/g for the unmodified control canola variety and for canola event NS-B5ØØ27-4, respectively. The total glucosinolate content for solvent extracted meal was 19.6 µmol/g and 18.05 µmol/g for the unmodified control variety and canola event NS-B5ØØ27-4, respectively. These levels were below the limits (30 µmol/g) defined for conventional canola meal (Schedule IV, Part I of the Feeds Regulations).

5.1.1.5 Fish studies with canola event NS-B5ØØ27-4 oil

The use of oil from canola event NS-B5ØØ27-4 in diets of Atlantic salmon both in the pre- and post-smolt life stages was evaluated in feeding trials. Salmon that were fed increasing levels of canola event NS-B5ØØ27-4 oil, had similar growth rates and feed intake as the controls. Increasing the inclusion of canola event NS-B5ØØ27-4 oil in diets led to increased fillet levels of ALA, EPA, DHA, n3:n6 fatty acid ratio, and improved skin and fillet color. Studies showed that NS-B5ØØ27-4 oil was an adequate alternative to fish oil in diets of Atlantic salmon. A second study examined the use of canola event NS-B5ØØ27-4 oil in Atlantic juvenile salmon in fresh water at 2 temperatures and 2 levels of dietary omega-3 fatty acids. No differences in growth were observed between the dietary groups, however, fish reared in higher temperatures had higher growth rates. It was concluded that omega-3 canola oils were a safe and effective replacement for fish oil in juvenile salmon. A third study examined the effects of canola event NS-B5ØØ27-4 oil on growth, fatty acid composition of different organs and tissues, and health of Atlantic salmon in sea water using 3 different inclusion rates (25%, 50% and 100% replacement of conventional canola oil). No statistically significant differences were observed between treatment groups for mortality, growth and feed intake of the fish. The apparent retention of EPA increased with increased dietary inclusion of canola event NS-B5ØØ27-4 oil. Results showed that the flesh color intensity was significantly higher in salmon fed NS-B5ØØ27-4 canola oils.

5.1.1.6 Broiler study with canola NS-B5ØØ27-4 meal

Growth and development of chickens fed diets containing canola event NS-B5ØØ27-4 meal was compared to a diet containing the unmodified control canola meal and another control diet containing corn/soybean meal. Canola seeds from canola event NS-B5ØØ27-4 and the unmodified control variety were pre-pressed, solvent extracted and fed at 10% inclusion rates over a 42-day period. Day-old broiler chicks were randomly assigned to 6 replicate pens (12 chicks per pen) for each dietary treatment. Diet and water was provided ad-libitum. There were no significant differences among the 3 treatments with regards to growth performance and feed efficiencies (P>0.05) of the chickens. Both the defatted canola event NS-B5ØØ27-4 meal and the unmodified control canola meal supported normal feed intake and growth rates in broiler chickens. There were no significant differences in the fatty acid profile of thigh or breast meat among treatments (P>0.05). Both sources of canola meal were safe to the broiler chickens, with no adverse effects observed.

5.1.1.7 Conclusions

It was concluded based on the scientific evidence provided by Nuseed Canada Inc. that the nutritional composition of canola event NS-B5ØØ27-4 is different from conventional canola with regards to the fatty acid profile only. The levels of oleic, linoleic and linolenic acids were significantly altered and new omega-3 long-chain polyunsaturated fatty acids were introduced in canola event NS-B5ØØ27-4. Canola event NS-B5ØØ27-4 has increased levels of omega-3 LC-PUFAs, including EPA (eicosapentaenoic acid, 20:5, n-3) and DHA (docosahexaenoic acid, 22:6, n-3), which are absent in conventional canola. Canola event NS-B5ØØ27-4 oil for aquaculture, meets the definition of omega-3 LC-PUFA canola oil already described in Part 1, Schedule IV of the Feeds Regulations, therefore no new ingredient definition was created for Nuseed Canada Inc's omega-3 oil. The pre-pressed solvent extracted defatted meal for all livestock would be comparable to conventional canola meal and therefore no new ingredient definition was created.

5.2 Potential Impact of Canola Event NS-B5ØØ27-4 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 NS-B5ØØ27-4 synthesizes omega-3 LC-PUFAs including EPA and DHA due to the insertion of genes encoding fatty acid desaturase and elongase proteins and is tolerant of glufosinate ammonium due to the production of the protein phosphinothricin N-acetyltransferase (PAT). A weight-of-evidence approach was used to evaluate the risk to livestock consuming feed ingredients from canola event NS-B5ØØ27-4, humans consuming foods of animal origin derived from those livestock, and workers/bystanders exposed to the feed ingredients from this event due to the following significant changes:

• novel protein delta-12 desaturase from Lachancea kluyveri (Lackl-Δ12D)
• novel protein omega-3 desaturase from Pichia pastoris (Picpa-ω3D)
• novel protein delta-6 elongase from Micromonas pusilla (Micpu-Δ6D)
• novel protein delta-6 desaturase from Pyramimonas cordata (Pyrco-Δ6E)
• novel protein delta-5 desaturase from Pavlova salina (Pavsa-Δ5D)
• novel protein delta-5 elongase from Pyramimonas cordata (Pyrco-Δ5E)
• novel protein delta-4 desaturase from Pavlova salina (Pavsa-Δ4D)
• modified protein phosphinothricin N-acetyltransferase from Streptomyces viridochromogenes (PAT)
• EPA/DHA levels in foods of animal origin

5.2.1 Delta-12 desaturase protein (Lackl-Δ12D)

The potential allergenicity and toxicity of the Lackl-Δ12D protein were evaluated. With respect to its potential allergenicity, the source of the Lackl-Δ12D gene, the yeast Lachancea kluyveri, is not known to produce allergens and a bioinformatics evaluation of the Lackl-Δ12D protein amino acid sequences confirmed the lack of relevant similarities between the Lackl-Δ12D protein and known allergens. Unlike many allergens, canola event NS-B5ØØ27-4 produced Lackl-Δ12D protein was shown experimentally to be rapidly degraded in simulated gastric and intestinal fluid and was shown in silico to be unglycosylated. The weight of evidence thus indicates that the Lackl-Δ12D protein is unlikely to cause an allergenic concern.

In terms of the potential toxicity, the Lackl-Δ12D protein lacks a mode of action to suggest that it is intrinsically toxic and a bioinformatics evaluation of the Lackl-Δ12D protein amino acid sequences confirmed the lack of relevant similarities between the Lackl-Δ12D protein and known toxins. The exposure to the Lackl-Δ12D protein is expected to be extremely low as the Lackl-Δ12D protein is expressed at very low level in canola event NS-B5ØØ27-4 and is degraded under conditions which simulate the mammalian digestive tract. The weight of evidence thus indicates that the Lackl-Δ12D protein is unlikely to cause a toxic concern.

Therefore, the Lackl-Δ12D protein in canola event NS-B5ØØ27-4 is unlikely to pose a risk to livestock, humans and workers/bystanders.

5.2.2 Omega-3 desaturase protein (Picpa-ω3D)

The potential allergenicity and toxicity of the Picpa-ω3D protein were evaluated. With respect to its potential allergenicity, the source of the Picpa-ω3D gene, the yeast Pichia pastoris, is not known to produce allergens and a bioinformatics analysis of the Picpa-ω3D protein amino acid sequences confirmed the lack of relevant similarities between the Picpa-ω3D protein and known allergens. Unlike many allergens, canola event NS-B5ØØ27-4 produced Picpa-ω3D protein was shown experimentally to be degraded in simulated gastric and intestinal fluid, and was shown in silico to be unglycosylated. The weight of evidence thus indicates that the Picpa-ω3D protein is unlikely to cause an allergenic concern.

In terms of the potential toxicity, the Picpa-ω3D protein lacks a mode of action to suggest that it is intrinsically toxic and a bioinformatics analysis of the Picpa-ω3D protein amino acid sequences confirmed the lack of relevant similarities between the Picpa-ω3D protein and known toxins. The exposure to the Picpa-ω3D protein is expected to be extremely low as the Picpa-ω3D protein is expressed at very low level in canola event NS-B5ØØ27-4 and is degraded under conditions that simulate the mammalian digestive tract. The weight of evidence thus indicates that the Picpa-ω3D protein is unlikely to cause a toxic concern.

Therefore, the Picpa-ω3D protein in canola event NS-B5ØØ27-4 is unlikely to pose a risk to livestock, humans and workers/bystanders.

5.2.3 Delta-6 elongase protein (Micpu-Δ6D)

The potential allergenicity and toxicity of the Micpu-Δ6D protein were evaluated. With respect to its potential allergenicity, the source of the Micpu-Δ6D gene, the microalgae Micromonas pusilla, is not known to produce allergens. A bioinformatics analysis of the Micpu-Δ6D protein amino acid sequences confirmed the lack of relevant similarities between the Micpu-Δ6D protein and known allergens. Unlike many allergens, canola event NS-B5ØØ27-4 produced Micpu-Δ6D protein was shown experimentally to be degraded in simulated gastric and intestinal fluid, and was shown in silico to be unglycosylated. The weight of evidence thus indicates that the Micpu-Δ6D protein is unlikely to cause an allergenic concern.

In terms of the potential toxicity, the Micpu-Δ6D protein lacks a mode of action to suggest that it is intrinsically toxic and a bioinformatics analysis of the Micpu-Δ6D protein amino acid sequences confirmed the lack of relevant similarities between the Micpu-Δ6D protein and known toxins. The exposure to the Micpu-Δ6D protein is expected to be extremely low as the Micpu-Δ6D protein is expressed at very low levels in canola event NS-B5ØØ27-4 and is degraded under conditions that simulate the mammalian digestive tract. The weight of evidence thus indicates that the Micpu-Δ6D protein is unlikely to cause a toxic concern.

Therefore, the Micpu-Δ6D protein in canola event NS-B5ØØ27-4 is unlikely to pose a risk to livestock, humans, and workers/bystanders.

5.2.4 Delta-6 desaturase protein (Pyrco-Δ6E)

The potential allergenicity and toxicity of the Pyrco-Δ6E protein were evaluated. With respect to its potential allergenicity, the source of the Pyrco-Δ6E gene, the microalgae Pyramimonas cordata, is not known to produce allergens. A bioinformatics analysis of the Pyrco-Δ6E protein amino acid sequences confirmed the lack of relevant similarities between the Pyrco-Δ6E protein and known allergens. Unlike many allergens, canola event NS-B5ØØ27-4 produced Pyrco-Δ6E protein was shown experimentally to be degraded in simulated gastric and intestinal fluid, and was shown in silico to be unglycosylated. The weight of evidence thus indicates that the Pyrco-Δ6E protein is unlikely to cause an allergenic concern.

In terms of the potential toxicity, the Pyrco-Δ6E protein lacks a mode of action to suggest that it is intrinsically toxic. A bioinformatics evaluation of the Pyrco-Δ6E protein amino acid sequences confirmed the lack of relevant similarities between the Pyrco-Δ6E protein and known toxins. The exposure to the Pyrco-Δ6E protein is expected to be extremely low as this protein is expressed at very low level in canola event NS-B5ØØ27-4 and is degraded under conditions that simulate the mammalian digestive tract. The weight of evidence thus indicates that the Pyrco-Δ6E protein is unlikely to cause a toxic concern.

Therefore, the Pyrco-Δ6E protein in canola event NS-B5ØØ27-4 is unlikely to pose a risk to livestock, humans and and workers/bystanders.

5.2.5 Delta-5 desaturase protein (Pavsa-Δ5D)

The potential allergenicity and toxicity of the Pavsa-Δ5D protein were evaluated. With respect to its potential allergenicity, the source of the Pavsa-Δ5D gene, the microalgae Pavlova salina, is not known to produce allergens and a bioinformatics evaluation of the Pavsa-Δ5D protein amino acid sequences confirmed the lack of relevant similarities between the Pavsa-Δ5D protein and known allergens. Unlike many allergens, canola event NS-B5ØØ27-4 produced Pavsa-Δ5D protein was shown experimentally to be degraded in simulated gastric and intestinal fluid, and was shown in silico to be unglycosylated. The weight of evidence thus indicates that the Pavsa-Δ5D protein is unlikely to cause an allergenic concern.

In terms of the potential toxicity, the Pavsa-Δ5D protein lacks a mode of action to suggest that it is intrinsically toxic and a bioinformatics evaluation of the Pavsa-Δ5D protein amino acid sequences confirmed the lack of relevant similarities between the Pavsa-Δ5D protein and known toxins. The exposure to the Pavsa-Δ5D protein is expected to be extremely low as the Pavsa-Δ5D protein is expressed at very low levels in canola event NS-B5ØØ27-4 and is degraded under conditions that simulate the mammalian digestive tract. The weight of evidence thus indicates that the Pavsa-Δ5D protein is unlikely to cause a toxic concern.

Therefore, the Pavsa-Δ5D protein in canola event NS-B5ØØ27-4 is unlikely to pose a risk to livestock, humans, and workers/bystanders.

5.2.6 Delta-5 elongase protein (Pyrco-Δ5E)

The potential allergenicity and toxicity of the Pyrco-Δ5E protein were evaluated. With respect to its potential allergenicity, the source of the Pyrco-Δ5E gene, the microalgae Pyramimonas cordata, is not known to produce allergens and a bioinformatics evaluation of the Pyrco-Δ5E protein amino acid sequences confirmed the lack of relevant similarities between the Pyrco-Δ5E and known allergens. Unlike many allergens, canola event NS-B5ØØ27-4 produced Pyrco-Δ5E protein was shown experimentally to be degraded in simulated gastric and intestinal fluid, and was shown in silico to be unglycosylated. The weight of evidence thus indicates that the Pyrco-Δ5E protein is unlikely to cause an allergenic concern.

In terms of the potential toxicity, the Pyrco-Δ5E protein lacks a mode of action to suggest that it is intrinsically toxic and a bioinformatics evaluation of the Pyrco-Δ5E protein amino acid sequences confirmed the lack of relevant similarities between the Pyrco-Δ5E protein and known toxins. The exposure to the Pyrco-Δ5E protein is expected to be extremely low as the Pyrco-Δ5E protein is expressed at very low levels in canola event NS-B5ØØ27-4 and is degraded under conditions that simulate the mammalian digestive tract. The weight of evidence thus indicates that the Pyrco-Δ5E protein is unlikely to cause a toxic concern.

Therefore, the Pyrco-Δ5E protein in canola event NS-B5ØØ27-4 is unlikely to pose a risk to livestock, humans, and workers/bystanders.

5.2.7 Delta-4 desaturase protein (Pavsa-Δ4D)

The potential allergenicity and toxicity of the Pavsa-Δ4D protein were evaluated. With respect to its potential allergenicity, the source of the Pavsa-Δ4D gene, the microalgae Pavlova salina, is not known to produce allergens. A bioinformatics evaluation of the Pavsa-Δ4D protein amino acid sequences confirmed the lack of relevant similarities between the Pavsa-Δ4D protein and known allergens. Unlike many allergens, canola event NS-B5ØØ27-4 produced Pavsa-Δ4D protein was shown experimentally to be degraded in simulated gastric and intestinal fluid, and was shown in silico to be unglycosylated. The weight of evidence thus indicates that the Pavsa-Δ4D protein is unlikely to cause an allergenic concern.

In terms of the potential toxicity, the Pavsa-Δ4D protein lacks a mode of action to suggest that it is intrinsically toxic. A bioinformatics evaluation of the Pavsa-Δ4D protein amino acid sequences confirmed the lack of relevant similarities between the Pavsa-Δ4D protein and known toxins. The exposure to the Pavsa-Δ4D protein is expected to be extremely low as this protein is expressed at very low levels in canola event NS-B5ØØ27-4 and is degraded under conditions that simulate the mammalian digestive tract. The weight of evidence thus indicates that the Pavsa-Δ4D protein is unlikely to cause a toxic concern.

Therefore, the Pavsa-Δ4D protein in canola event NS-B5ØØ27-4 is unlikely to pose a risk to livestock, humans, and workers/bystanders.

5.2.8 Phosphinothricin N-acetyltransferase protein (PAT)

The potential allergenicity and toxicity of PAT protein were evaluated. With respect to its potential allergenicity, the source of the pat gene, Streptomyces viridochromogenes, is not known to produce allergens and there is a history of safe use of the host organism and a history of safe exposure to the PAT. A bioinformatics analysis of the PAT protein amino acid sequence confirmed the lack of relevant similarities between the PAT protein and known allergens. The weight of evidence thus indicates that the PAT protein is unlikely to cause an allergenic concern.

In terms of the potential toxicity, the PAT protein lacks a mode of action to suggest that it is intrinsically toxic. There is a history of safe use for donor organism and source organism as well as safe exposure to the PAT. A bioinformatics analysis of the PAT protein amino acid sequences confirmed the lack of relevant similarities between the PAT protein and known toxins. The exposure to the PAT protein is expected to be extremely low as the PAT protein is expressed at very low level in canola event NS-B5ØØ27-4. The weight of evidence thus indicates that the PAT protein is unlikely to cause a toxic concern.

Therefore, the PAT in canola event NS-B5ØØ27-4 is unlikely to pose a risk to livestock, humans, and workers/bystanders.

5.2.9 EPA/DHA levels in foods of animal origin

The safety of EPA/DHA levels in foods of animal origin, following application of canola event NS-B5ØØ27-4 oil in fish feeds, was also evaluated as part of the feed safety assessment.

It was determined that canola event NS-B5ØØ27-4 oil, when used in fish feed, would not present a safety concern to humans via the potential transfer of EPA/DHA into foods of animal origin, when comparing the estimated exposure to EPA/DHA derived from the farmed and wild Atlantic salmonid fish.

5.2.10 Conclusion

It was concluded, based on the evidence provided by Nuseed Canada Inc., that the production of the 5 different fatty acid desaturases, and 2 elongases and the modified protein phosphinothricin N-acetyltransferase (PAT) protein, in canola event NS-B5ØØ27-4 are unlikely to pose a risk to livestock, humans and workers/by-standers. Therefore, pre-press solvent extracted defatted meal (approximately 1% oil) from canola event NS-B5ØØ27-4 seed is considered as safe as meal from conventional canola species currently available in the Canadian market. Additionally, the use of canola event NS-B5ØØ27-4 oil in fish feeds would not present a safety concern to humans via the potential transfer of EPA/DHA into foods of animal origin.

The safety and efficacy of the levels of omega-3 LC PUFAs in foods of animal origin such as milk, eggs and meat by feeding canola event NS-B5ØØ27-4 oil to livestock species other than fish were not assessed at this time. As such, Nuseed Canada Inc.is required to use the identity preservation system to ensure that the oil derived from canola event NS-B5ØØ27-4 seeds are used in fish feeds only and not fed to other livestock species.

Furthermore, the recycling or repurposing of the oil derived from canola event NS-B5ØØ27-4 intended for human consumption (for example, spent omega-3 canola oil obtained from human usages) is not authorized for use in livestock species other than fish.

Additionally, no authorization has been granted for use of whole seeds and forage derived from the canola event NS-B5ØØ27-4 as livestock feed at this time since no data was provided to support the use of these ingredients as livestock feed. As such, whole seed and parts of omega-3 canola crop (for example, forage) shall not be grazed by livestock or fed as chop feed or forage for livestock.

6. New Information Requirements

If at any time, Nuseed Canada 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 NS-B5ØØ27-4 or lines derived from it, Nuseed Canada Inc. is required to immediately provide such information to the CFIA. On the basis of such new information, the CFIA will re-evaluate the potential impact of canola event NS-B5ØØ27-4 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 NS-B5ØØ27-4.

7. Regulatory Decision

Based on the review of the data and information submitted by Nuseed Canada 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 NS-B5ØØ27-4 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 Nuseed Canada Inc. and input from other relevant scientific sources, the Animal Feed Program of the Animal Health Directorate, CFIA, has authorized the use of canola event NS-B5ØØ27-4 oil as a source of omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs) for fish feeds only. Furthermore, only pre-pressed solvent extracted defatted canola meal has been authorized for use as livestock feed.

Unconfined release into the environment and use as livestock feed of canola event NS-B5ØØ27-4 is therefore authorized by the Plant Biosafety Office of the Plant Health and Biosecurity Directorate and the Animal Feed Program of the Animal Health Directorate, respectively, as of July 28, 2020. Any canola lines derived from canola event NS-B5ØØ27-4 may also be released into the environment and used as livestock feed with the specified conditions stated below, provided that:

• no inter-specific crosses are performed
• the intended uses are similar, and meet the conditions of the authorizations
• it is known based on characterization that these plants do not display any additional novel traits, and
• the novel genes are expressed at a level similar to that of the authorized line

With respect to its unconfined release into the environment, an appropriate herbicide tolerance management plan should be implemented. If canola event NS-B5ØØ27-4 is cultivated in Canada as an individual event or in combination with other canola events in stacked/pyramided products, Nuseed Canada Inc. must submit a herbicide tolerance management plan to the CFIA.

Regarding the feed authorization, canola event NS-B5ØØ27-4 was approved as follows:

• As a result of the modified and introduced fatty acids within canola event NS-B5ØØ27-4 and a different usage than conventional canola oil (that is, as a source of omega-3 LC-PUFAs), the oil derived from canola event NS-B5ØØ27-4 seed was determined to meet the current definition of omega-3 LC-PUFA canola oil as listed in class 8, Part I, Schedule IV of the Feeds Regulations.
• The information presented to the AFP indicated that the whole seed from canola event NS-B5ØØ27-4 will be subjected to prepress solvent extraction resulting in a meal that contains a small fraction of oil (approximately 1%). As per the definition 5.3.3 in Schedule IV, Part I, where applicable, the subsequent addition of spent bleaching clay and vegetable oil refinery lipids will result in the increase in the final oil fraction of the meal. When processed in this way, the meal derived from seeds of the canola event NS-B5ØØ27-4 can be used in the same way as conventional canola meal in livestock feeds.
• The safety and efficacy of the levels of omega-3 LC-PUFAs in foods of animal origin such as milk, eggs and meat by feeding long chain omega-3 canola oil or whole seeds from canola event NS-B5ØØ27-4 to livestock species other than fish were not assessed at this time. As such, Nuseed Canada Inc. is required to implement an identity preservation system to ensure that the oil derived from omega-3 canola seeds are used in fish feeds only and not fed to other livestock species.
• No approval is granted for use of whole seeds and forage derived from the canola event NS-B5ØØ27-4 as livestock feed at this time. As such, parts of omega-3 canola crop (for example, forage) shall not be grazed by livestock or fed as chop feed for livestock.

Canola event NS-B5ØØ27-4 is subject to the same phytosanitary import requirements as unmodified canola varieties. Canola event NS-B5ØØ27-4 is required to meet the requirements of other jurisdictions, including, but not limited to, the Food & 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 NS-B5ØØ27-4.