Novel Food Information - Cibus Canola Event 5715 (Imidazolinone and Sulfonylurea Herbicide Tolerant)

Health Canada has notified Cibus US LLC, a legal affiliate of Cibus Canada Inc., that it has no objection to the food use of Cibus canola event 5715. The Department conducted a comprehensive assessment of this canola variety according to its Guidelines for the Safety Assessment of Novel Foods. These Guidelines are based upon internationally accepted principles for establishing the safety of foods with novel traits.

Background

The following provides a summary of the notification from Cibus US LLC and the evaluation by Heath Canada and contains no confidential business information.

1. Introduction

Cibus US LLC has developed a canola event with an increased tolerance to imidazolinone and sulfonylurea herbicides compared to conventional canola varieties. This canola event possesses a specific single nucleotide mutation in the BnAHAS1C and BnAHAS3A genes, respectively. Both the BnAHAS1C and BnAHAS3A genes encode a protein subunit (i.e., a monomer) of an acetohydroxyacid synthase (AHAS) enzyme that functions as a homodimer made of two subunits from either gene, or as a heterodimer made of one subunit from each gene, respectively. The AHAS enzyme is involved in the biosynthesis of branched chain amino acids including leucine, isoleucine, and valine. Imidazolinone and sulfonylurea herbicides bind to the active sites of the AHAS enzyme, preventing synthesis of these crucial amino acids. The single nucleotide mutation in each subunit of the AHAS enzyme results in conformational changes in the configuration of the enzyme, reducing the binding affinity of imidazolinone and sulfonylurea herbicides, thus conferring herbicide tolerance to the canola plant.

Similar mutations have been previously assessed by Health Canada in the canola lines marketed as Smart® canola (imidazolinone tolerant canola) produced by Pioneer Hi-Bred International Inc. (Health Canada, 1995).

The safety assessment performed by Food Directorate evaluators was conducted according to Health Canada's Guidelines for the Safety Assessment of Novel Foods. These Guidelines are based on harmonization efforts with other regulatory authorities and reflect international guidance documents in this area (e.g., Codex Alimentarius). The assessment considered: how Cibus canola event 5715 was developed; how the composition and nutritional quality of this canola event compared to conventional varieties; and the potential for this event to be toxic or cause allergic reactions. Cibus US LLC has provided data that demonstrates that Cibus canola event 5715 is as safe and of the same nutritional quality as conventional canola varieties used as food in Canada.

The Food Directorate has a legislated responsibility for pre-market assessment of novel foods and novel food ingredients as detailed in the Food and Drug Regulations (Division 28). Cibus canola event 5715 is considered a novel food under the following part of the definition of a novel food:

"c) a food that is derived from a plant, animal or microorganism that has been genetically modified such that

1. the plant, animal or microorganism exhibits characteristics that were not previously observed in that plant, animal or microorganism."

2. Development of the Modified Plant

The petitioner has provided information describing the methods used to develop Cibus canola event 5715, in addition to the molecular biology data that characterize the genetic change, which results in an increased tolerance to imidazolinone and sulfonylurea herbicides.

Cibus canola event 5715 was developed as a cross between Cibus canola line BnALS-57 (donor of the single nucleotide mutation in the BnAHAS1C gene) and the commercially available CLEARFIELD canola variety SP Cougar CL (donor of the single nucleotide mutation in the BnAHAS3A gene). Additional crosses and backcrosses were accomplished to strengthen the desired canola event (i.e., Cibus canola event 5715). With the herbicide tolerant trait fixed in the F4 generation, DNA sequencing was used to confirm homozygosity of the single nucleotide mutation in both the BnAHAS1C and BnAHAS3A genes. Furthermore, plants from an advanced breeding generation of Cibus canola event 5715 demonstrated consistent imidazolinone and sulfonylurea herbicide tolerance, phenotypically confirming the stable inheritance of the trait.

As donor of the single nucleotide mutation in the BnAHAS1C gene, Cibus canola line BnALS-57 was selected as a mutant variant of the wild-type parental line BN2. Mutants of BN2 were generated using the Rapid Trait Development System (RTDS™); an oligonucleotide-directed mutagenesis method. A critical step of the RTDS protocol is the application of tissue culture techniques to generate plant cells more receptive to mutagenesis. BN2 protoplasts were subjected to the RTDS then cultured on media containing imazethapyr (an imidazolinone herbicide). Actively growing calli were isolated from a background of brownish cells. The herbicide tolerant calli were genotyped and the presence of the single nucleotide mutation in the BnAHAS1C gene was confirmed. The petitioner hypothesized that the single nucleotide mutation was the result of a spontaneous somaclonal variation that occurred during the tissue culture process, and not due to the specific oligonucleotide used in the RTDS protocol. The selected mutant was subsequently regenerated into the canola line that would be designated as BnALS-57. Following regeneration, both the BnAHAS1C and BnAHAS3A genes were sequenced in their entirety, confirming that the only mutation (compared to these genes in the wild-type BN2) was the single nucleotide mutation in the BnAHAS1C gene. No changes were observed in the promoter regions or locations that would affect endogenous gene expression. This was confirmed by sequencing a 2500-base pair (bp) region upstream of the BnAHAS1C gene which was determined to be identical with GenBank accession Z11524.

3. Characterization of the Modified Plant

As previously mentioned, Cibus canola event 5715 was developed as a cross between Cibus canola line BnALS-57 and CLEARFIELD canola variety SP Cougar CL. As the single nucleotide mutation is a single nucleotide change that results in a single amino acid substitution DNA sequencing of both the BnAHAS1C and BnAHAS3A genes in Cibus canola event 5715 was used to confirm homozygosity of the single nucleotide mutation in both genes.

The stability of the single nucleotide mutation in the BnAHAS1C and BnAHAS3A genes was demonstrated by observing consistent imidazolinone and sulfonylurea herbicide tolerance from an advanced breeding generation of Cibus canola event 5715.

4. Product Information

Cibus canola event 5715 differs from its conventional counterpart by the presence of the single nucleotide mutation in both the BnAHAS1C and BnAHAS3A genes. Both genes encode a protein subunit of an AHAS enzyme which functions as a dimer. The AHAS enzyme is involved in the biosynthesis of branched chain amino acids including leucine, isoleucine, and valine. The native AHAS enzyme is sensitive to imidazolinone and sulfonylurea herbicides which can inhibit enzymatic activity. The single nucleotide mutation in both the BnAHAS1C and BnAHAS3A genes results in a single amino acid substitution in each protein subunit which induces conformational changes in the configuration of the enzyme, conferring tolerance to imidazolinone and sulfonylurea herbicides.

5. Dietary Exposure

It is expected that Cibus canola event 5715 will be used in applications similar to conventional canola varieties. The petitioner does not anticipate a significant change in the food use of canola oil with the introduction of Cibus canola event 5715.

6. Nutrition

Confined yield trials were conducted in multiple sites in Canada in 2011 to test the Cibus canola event 5715 variety and hybrids containing the same single nucleotide mutation, compared to several commercially grown (non-mutant) canola hybrids. The canola line BY5525 was selected as the comparator line for nutritional assessment since it is an approved canola line planted commercially on many acres in Canada and as such would be representative of the canola quality currently expected for commercial varieties. This variety was also selected for its similarity to Cibus canola event 5715 as it also possesses the single nucleotide mutation in the BnAHAS3A gene. Compositional data was analyzed from 6 Canadian locations in the canola growing regions of Manitoba and Saskatchewan.

The analytes measured and evaluated in the extracted oil of the Cibus canola event 5715 and BY5525 control varieties were: fatty acids (palmitic, palmitoleic, stearic, oleic, linoleic, linolenic, arachidic, eicosenoic, eicosadienoic, behenic, erucic, lignoceric, and nervonic fatty acids). The analytes measured and evaluated in the defatted meal of the Cibus canola event 5715 and BY5525 control varieties were: protein, oil, ash, crude fibre, acid detergent fibre (ADF), neutral detergent fibre (NDF), glucosinolates (aliphatic, methylthiobutenyl [Msgl], indol, and total glucosinolates), calcium, phosphorus, and amino acids (alanine, arginine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, and valine). The analytes measured and evaluated in the whole seed of Cibus canola event 5715 and BY5525 control varieties were: fatty acids (palmitic, palmitoleic, stearic, oleic, linoleic, linolenic, arachidic, eicosenoic, eicosadienoic, behenic, erucic, lignoceric, and nervonic fatty acids), phytic acid, and trypsin inhibitors.

Results indicated that several of the analytes (13 of 57) in Cibus canola event 5715 showed a statistically significant difference compared to the BY5525 control variety. However, these differences were considered acceptable because the level of these analytes in Cibus canola event 5715 were within the ranges published in the Organization for Economic Co-operation and Development (OECD) consensus document for canola (2011); or in the case of glucosinolates, the levels were significantly lower in Cibus canola event 5715, which would likely have a positive impact on nutritional safety.

The BY5525 variety was the prominent comparator line used in the nutritional assessments conducted for Cibus canola event 5715. Additionally, comparison to the progenitor parent line BN2 was included based on the analysis of seed samples harvested from one field trial location in Langdon, ND. The Langdon location is on the same soil association as represented in southern Manitoba and shares similar weather patterns. As such, it is expected to provide results that are predicative of performance on the Canadian prairie of Manitoba. The line included in this analysis contained the BN2 genotype (i.e., the non-mutant parental line of Cibus canola line BnALS-57) as the female component of the hybrid. Three replicate plots for both Cibus canola event 5715 and BN2 were harvested and analyzed individually for nutritional composition.

The analytes measured and evaluated in the extracted oil of the Cibus canola event 5715 and BN2 control varieties were: fatty acids (palmitic, palmitoleic, stearic, oleic, linoleic, linolenic, arachidic, eicosenoic, eicosadienoic, behenic, erucic, lignoceric, and nervonic fatty acids). The analytes measured and evaluated in the defatted meal of the Cibus canola event 5715 and BN2 control varieties were: amino acids (alanine, arginine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, valine, and tryptophan). The analytes measured and evaluated in the whole seed of the Cibus canola event 5715 and BN2 control varieties were: protein, oil, ash, crude fibre, ADF, NDF, glucosinolates (aliphatic, methylthiobutenyl [Msgl], indol, and total glucosinolates), calcium, and phosphorus.

Results indicated that several analytes (19 of 43) in the Cibus canola event 5715 showed a statistically significant difference compared to the BN2 control variety. However, these differences were considered acceptable because the level of these analytes in Cibus canola event 5715 were within the ranges published in at least one of the OECD consensus documents for canola (2011, 2001); or the magnitude of the difference was too small to impact dietary intakes and pose a nutritional safety concern; or in the case of glucosinolates, the values were significantly lower in Cibus canola event 5715, which would likely have a positive impact on nutritional safety.

Based on the information provided, the petitioner has demonstrated that Cibus canola event 5715 has a substantially equivalent composition to the progenitor parent line BN2 and the commercial canola line BY5525.

7. Chemistry/Toxicology

The single nucleotide mutation in the BnAHAS3A gene has previously been incorporated in other canola lines approved by Health Canada such as Smart® canola and Clearfield® canola. Health Canada approved these canola lines for food use based on the lack of similarity to known protein toxins, evidence that the mutant protein is heat labile, is degraded in simulated gastric fluid (SGF) and the negligible anticipated exposure from the consumption of canola oil. Since the novel trait in Cibus canola event 5715 is the single nucleotide mutation in the BnAHAS1C gene, which is 99.7% identical to the BnAHAS3A gene, it is unlikely that the potential for toxicity would differ significantly between the AHAS proteins expressed by these two genes.

The petitioner states that the AHAS enzyme is not a known toxin and that there is no evidence in the scientific literature suggesting that the presence of the single nucleotide mutation would change this. A bioinformatics analysis was performed using the GenBank protein database (accessed April, 2013) to compare both the wild type and mutant AHAS proteins derived from the BnAHAS1C gene to known toxins. A total of 689,560 sequences were searched. Under the search criteria used, no significant matches were found for the mutant BnAHAS1C protein that were not already obtained when the same database was queried with the wild type BnAHAS1C sequence. In total, 56 matches were found, all of which were with thiamine pyrophosphate-dependent enzymes. These enzymes are related to the AHAS enzyme and play a key role in energy metabolism. In searching the peer-reviewed scientific literature and to the knowledge of the petitioner, there is no evidence that these enzymes are toxic or that they yield products that are directly toxic.

The petitioner submitted studies to determine if the mutant BnAHAS1C is degraded when exposed to SGF and denatured at high temperatures. The results from the SGF study were not considered valid due to difficulties in interpreting the methodology and the results. The heat lability study demonstrated that the single nucleotide mutation did not affect the protein's sensitivity to heat inactivation as the enzyme activity of both the wild type and mutant proteins were significantly reduced when exposed to high temperatures similar to those used in the processing of canola oil. However, the validity of these studies could not be confirmed because the petitioner failed to provide information confirming the equivalency between the microbial-derived proteins used in the studies to the plant-derived proteins. Nonetheless, there are negligible amounts of the protein present in the oil due to the extensive chemical processing, filtering, and high temperatures used in its production.

8. Allergenicity

The petitioner conducted an in silico search for amino acid homology between the single nucleotide mutation AHAS proteins and allergens listed in the Allermatch database. No matches were found using the search criteria of eight contiguous amino acids with a greater than 35% identity. Additionally, no matches were found using the same criteria with the wild type AHAS proteins.

No foreign DNA was used to produce Cibus canola event 5715 and therefore no additional sources of potential allergens are present in this canola event.

Canola is highly processed and undergoes many treatments before it becomes canola oil. These treatments include processes such as heating, solvent extraction, toasting, bleaching (i.e., filtration through clay), and steam distillation (i.e., de-odorization). In general, these processes denature and remove protein, such that the final canola oil is a negligible source of protein and would not be expected to pose an allergenic concern.

Conclusion

Health Canada's review of the information presented in support of the food use of Cibus canola event 5715 does not raise concerns related to food safety. Health Canada is of the opinion that food derived from Cibus canola event 5715 is as safe and nutritious as food from current commercial canola varieties.

Health Canada's opinion deals only with the food use of Cibus canola event 5715. Issues related to its use as animal feed have been addressed separately through existing regulatory processes in the Canadian Food Inspection Agency (CFIA). The CFIA evaluated information provided on the environmental, animal, and human health safety of Cibus canola event 5715 with the intended use in animal feed. From their assessment, the CFIA concluded that there are no concerns from an environmental and feed safety perspective. This perspective is applicable to the food and feed products derived from Cibus canola event 5715 destined for commercial sale.

It is the continuing responsibility of the food manufacturer or importer to ensure that their products are in compliance with all applicable statutory and regulatory requirements. Any new information obtained in relation to these products which have potential health and safety implications should be forwarded to Health Canada for our consideration in order to ensure the continued safety and integrity of all foods available in the Canadian marketplace. The sale of a food which poses a hazard to the health of consumers would contravene the provisions of the Food and Drugs Act.

This Novel Food Information document has been prepared to summarize the opinion regarding the subject product provided by the Food Directorate, Health Products and Food Branch, Health Canada. This opinion is based upon the comprehensive review of information submitted by the petitioner according to the Guidelines for the Safety Assessment of Novel Foods.

For further information, please contact:

Novel Foods Section
Food Directorate
Health Products and Food Branch
Health Canada, PL2204A1
251 Frederick Banting Driveway
Ottawa, Ontario K1A 0K9
novelfoods-alimentsnouveaux@hc-sc.gc.ca