Novel Food Information: Dicamba Tolerant Canola - MON 94100
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Health Canada has notified Bayer CropScience Inc. that it has no objection to the food use of Dicamba Tolerant Canola - MON 94100. 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.
The following provides a summary of the original notification from Bayer CropScience Inc. and the evaluation by Health Canada and contains no confidential business information.
Bayer CropScience Inc. has developed a genetically modified Brassica napus L. (canola) variety which exhibits tolerance to the herbicide dicamba (3,6-dichloro-2-methoxybenzoic acid).
MON 94100 was developed through the introduction of a dmo gene encoding a dicamba mon-oxygenase (DMO). This gene and its corresponding trait have been previously evaluated by Health Canada in several genetically modified (GM) crop lines including Herbicide Tolerant HT4 Maize - MON 87429 (Health Canada, 2020), Herbicide Tolerant Maize - MON 87419 (Health Canada, 2016), Dicamba and Glufosinate Tolerant Cotton - MON 88701 (Health Canada, 2014), and Dicamba Tolerant Soybean - MON 87708 (Health Canada, 2012).
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 this canola variety was developed; how the composition and nutritional quality of this variety compared to non-modified canola varieties; and the potential for this canola variety to be toxic or cause allergic reactions. Bayer CropScience Inc. has provided data that demonstrate that MON 94100 is as safe and of the same nutritional quality as traditional 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 B.28). MON 94100 is considered a novel food under the following part of the definition of novel foods:
"c) a food that is derived from a plant, animal or microorganism that has been genetically modified such that:
- 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 MON 94100 and the molecular biology data that characterize the genetic change, which results in tolerance to dicamba.
MON 94100 was developed through Agrobacterium-mediated transformation of spring canola variety 65307 with a transformation vector PV-BNHT508701.
The PV-BNHT508701 vector contains a construct (T-DNA) of the dmo gene, encoding a dicamba mon-oxygenase (DMO). The coding sequence for the DMO protein was isolated from the bacterium Stenotrophomonas maltophilia strain DI-6. The organism was originally named Pseudomonas maltophilia, but was subsequently re-named to Xanthomonas maltophilia, before it was given its own genus (Palleroni and Bradbury, 1993).
The petitioner provided information to support the safety and long history of use of the donor organism (S. maltophilia) and the recipient organism (Brassica napus L.). None of these organisms pose a health safety concern.
3. Characterization of the Modified Plant
The number of integration sites of the T-DNA insert in MON 94100 was characterized by using a combination of whole genome sequencing (WGS), Junction Sequence Analysis (JSA), and directed sequencing (locus-specific PCR, DNA sequencing and analyses). The results of the analyses demonstrated the presence of a single transgenic locus which consists of a single copy of the T-DNA insert.
Bioinformatics analyses were performed to assess potential toxicity and allergenicity of any putative peptides encoded by the translation of the six reading frames within the T-DNA insert and the flanking genomic DNA (gDNA) sequences. Open reading frames (ORFs) were defined by searching the encompassed nucleotide sequence for any initiation and stop codons producing a peptide of eight amino acids or greater in length. A total of 9 putative ORFs were identified and used as query sequences in homology searches to known toxins and allergens.
Each query sequence was compared with all protein sequences present in the NCBI's allergen (AD_2019), toxin (TOX_2019), and protein (PRT_2019) databases. The biological relevance of each match was assessed. No biologically relevant matches were found with known allergens, toxins, or other biologically active proteins that could affect human health.
Stability of the T-DNA insert in the MON 94100 genome was demonstrated by assessing individual MON 94100 plants from five generations (R3, R3F1, R4, R5, R6) by means of WGS and JSA. The results indicate that the T-DNA insert is intact and stable over all five generations of MON 94100.
Segregation of the T-DNA insert was assessed in three back-crossed generations of MON 94100 (BC1F1, BC2F1, BC3F1). Segregation ratios confirmed that the dmo gene contained within the T-DNA insert is inherited in a manner as expected for a single insertion. The results are consistent with Mendelian principles of inheritance and support the conclusion that the MON 94100 genome contains a single T-DNA insert integrated into a single chromosomal locus within the canola genome.
4. Product Information
MON 94100 differs from its traditional counterparts by the addition of a single dmo gene expression construct which expresses a DMO protein conferring tolerance to the herbicide dicamba.
Protein expression levels of the DMO protein in tissues of MON 94100 were determined by Enzyme-Linked Immunosorbent Assay (ELISA) in field-grown canola matrices from MON 94100 plants treated and not treated with trait-specific herbicide grown at three field trials in the USA and two sites in Canada in 2018.
Root tissue at BBCH 19-59 growth stage demonstrated the highest mean DMO protein expression levels. Mean (±SE) DMO expression levels in treated root at BBCH 19-59 of MON 94100 was 5.0 ± 0.37 mg/g dry weight (DW).
Seed tissue at maturity (BBCH 99) growth stage demonstrated the lowest mean DMO protein expression levels. Mean (±SE) DMO expression levels in treated seed at maturity (BBCH 99) of MON 94100 was 0.64 ± 0.068 mg/g DW.
Similar expression levels of the DMO protein were observed in the untreated samples of MON 94100.
Expression levels of the DMO protein in MON 94100 were too low to allow for purification of sufficient quantities for use in safety assessment studies. Previously, the safety of the DMO protein was demonstrated in MON 87708 soybean. Equivalence of the MON 94100- and MON 87708-produced DMO protein was established using a panel of analytical tests and assays including analysis of a Coomassie-strained SDS-PAGE; western blot analysis; glycostaining analysis; Mass Spectroscopy; N-terminal sequence analysis; and a functional activity assay. Based on this panel of tests and analyses, the MON 94100-produced and MON 87708-produced DMO proteins are considered equivalent. As such, it is appropriate for studies demonstrating the safety of MON 87708-produced DMO protein to be used to demonstrate the safety of the MON 94100-produced DMO protein
5. Dietary Exposure
It is expected that MON 94100 will be used in applications similar to conventional canola varieties. No significant change in the food use of canola is expected with the introduction of MON 94100.
To evaluate if there were any unanticipated consequences of the genetic modifications to MON 94100, the nutritional and anti-nutritional components of the MON 94100 were analysed and compared to a non-GM conventional control. This was done as part of a field trial conducted in 2018 at four different sites in the USA (Bonneville County, Idaho; Jerome County, Idaho; Brookings County, South Dakota; and Grant County, Washington) and one site in Canada (Portage la Prairie Rural Municipality, Manitoba). The field trial was a randomized complete block design with four blocks at each site and each block containing MON 94100 and the control.
The compositional analytes measured in MON 94100 and non-GM control canola seed were: carbohydrates (by calculation), protein, total fat, acid detergent fibre, neutral detergent fibre, ash, minerals (calcium and phosphorus), vitamins (vitamin E and vitamin K1), fatty acids (21 fatty acids measured), amino acids (18 amino acids measured), total glucosinolates, total alkyl glucosinolates, total indolyl glucosinolates, sinapine, phytic acid, and tannins.
Of the analytes measured, sinapine showed a statistically significant treatment effect in the MON 94100 compared to the control (higher in MON 94100 by 2.7 %). Sinapine is an anti-nutrient that can impart a fishy odour and flavour in the tissues and eggs of some animals fed canola meal (OECD, 2011). The difference between the MON 94100 canola and the control is acceptable as the analyte level remained within the International Life Sciences Institute Crop Composition Database range (renamed the Agriculture & Food Systems Institute Crop Composition Database as of May 1, 2020). The remaining analytes were not statistically significantly different in MON 94100 compared to control.
Based on the information provided on the composition of MON 94100 and control, there are no safety concerns with the use of MON 94100 as a food in Canada from a nutritional perspective.
The safety of the DMO protein has previously been reviewed by Health Canada in products approved for food use including MON 87708 soybean, MON 88701 cotton, MON 87419 corn, and MON 87429 corn. Based on the data provided by the petitioner, evaluators confirmed the equivalency of the DMO protein expressed in MON 94100 canola to a previously assessed DMO protein in MON 87708 soybean.
Updated bioinformatics analyses demonstrated that the amino acid sequence of the DMO protein does not share significant homology with known protein toxins identified in the National Center for Biotechnology Information database (search date: January 2019). This suggests the protein is unlikely to function as a toxin when consumed.
Previously submitted experimental data suggests that the DMO protein is readily digestible in simulated gastric and simulated intestinal fluid, thus it is expected to be completely digested in the gastrointestinal tract. The protein's functionality is heat sensitive at temperatures above 55 °C and it is likely inactivated and degraded during processing of canola oil. An acute oral toxicity study in mice failed to demonstrate any signs of clinical toxicity, at the highest dose tested (i.e., 140 mg/kg body weight).
Dietary exposure to canola is primarily refined, bleached, and deodorized (RBD) oil that is highly processed and expected to contain very low amounts of protein (<0.2 ppm). Results from field expression studies provided by the petitioner showed that the DMO protein represents a minimal amount (0.00022 %) of total seed protein. On this basis, the dietary exposure to the DMO protein from MON 94100 is considered negligible.
The petitioner conducted a sequence homology search to identify potential cross-reactivity between the DMO protein and known allergens, gliadin, and glutenins in the Comprehensive Protein Allergen Resource (COMPARE, 2019) database. Searches included a FASTA alignment to assess overall structural similarity; a sliding window search for sequences of 80 amino acids with a linear identity greater than 35 %; and a search for eight contiguous amino acids to assess for the presence of potential immunologically significant epitopes. No matches were identified for the DMO protein. As such, the DMO protein is not expected to be cross-reactive with known allergens upon digestion.
Many food allergens show relative stability during digestion. The results from in vitro digestion assays using simulated gastric and intestinal fluids suggest that the DMO protein is extensively digested under conditions similar to that of the human gastrointestinal system. This further supports that the DMO protein is unlikely to trigger an allergic reaction.
Given that most allergens are proteins and human dietary exposure to canola is primarily RBD oil that is not expected to contain appreciable amounts of protein, RBD oil produced from MON 94100 would not be expected to pose an allergic risk.
Based on the information provided, evaluators did not identify any safety concerns regarding the food use of MON 94100 from a toxicological or allergenic perspective.
Health Canada's review of the information presented in support of the food use of MON 94100 does not raise concerns related to food safety. Health Canada is of the opinion that food derived from this canola variety is as safe and nutritious as food derived from current commercial canola varieties.
Health Canada's opinion deals only with the food use of MON 94100. Issues related to its environmental release and use as animal feed have been addressed separately through existing regulatory processes in the CFIA.
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.
(Également disponible en français)
For further information, please contact:
Novel Foods Section
Health Products and Food Branch
Health Canada, PL2204A1
251 Frederick Banting Driveway
Ottawa, Ontario K1A 0K9
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