Decision Document for Male Sterile Herbicide Tolerant Brassica napus Event MS11
Health Canada has notified Bayer CropScience Inc. that it has no objection to the food use of male sterile herbicide tolerant Brassica napus event MS11 (henceforth referred to as event MS11). The Department conducted a comprehensive assessment of this canola event 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 notification from Bayer CropScience Inc. and the evaluation by Heath Canada and contains no confidential business information.
Bayer CropScience Inc. has developed a genetically modified B. napus (canola) event which exhibits a male sterility phenotype as well as tolerance to the herbicide glufosinate ammonium.
Current Bayer CropScience Inc. canola hybrid varieties are based on B. napus events MS8 and RF3. Event MS11 will replace event MS8 and will only be used for the production of B. napus MS11 hybrid seed. According to the petitioner, event MS11 will not be commercialized as a standalone product.
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 event was developed; how the composition and nutritional quality of this event compared to non-modified canola varieties; and the potential for this canola event to be toxic or cause allergic reactions. Bayer CropScience Inc. has provided data that demonstrate that event MS11 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). Event MS11 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 event MS11 and the molecular biology data that characterize the genetic change, which results in a male sterility phenotype as well as tolerance to the herbicide glufosinate-ammonium.
The male sterility phenotype exhibited by event MS11 is achieved through expression of the barnase gene derived from Bacillus amyloliquefaciens which encodes a ribonuclease. Expression of the barnase gene is controlled under an anther-specific promoter (Pt29), which restricts expression to the tapetum cells during anther development of the plant. Expression of the Barnase protein in the tapetum cells of event MS11 results in a lack of viable pollen (i.e. male sterility). A second gene (barstar) also derived from B. amyloliquefaciens, was introduced into the event MS11 genome for the purpose of increasing transformation. While the barstar gene encodes an inhibitor of the Barnase protein, presence of the barstar gene in event MS11 with expression controlled by a Pnos promoter does not significantly affect Barnase activity (and hence the male sterility phenotype is still exhibited)
The tolerance to glufosinate ammonium exhibited by event MS11 is achieved through expression of the bar gene derived from Streptomyces hygroscopicus which encodes a phosphinothricin acetyltransferase (PAT). The PAT protein catalyzes the acetylation of phosphinothricin (glufosinate) rendering the herbicide non-toxic to the plant. Expression of the bargene is controlled under a plant promoter that is active in all green tissues of the plant (PssuAt).
All three genes were introduced into the host genome as a single gene construct (T-DNA cassette) on the transformation plasmid pTCO113. The T-DNA cassette contains the following genetic elements: the 3¢ untranslated region of the TL-DNA gene 7 of the octopine Ti plasmid (3¢g7); the coding sequence of the phosphinothricin acetyltransferase gene derived from S. hygroscopicus (bar); the promoter region of the ribulose-1,5-biphosphate carboxylase small subunit gene derived from Arabidopsis thaliana (PssuAt); the 3¢ untranslated region of the nopaline synthase gene from the T-DNA of the plasmid pTiT37 (3¢nos); the 3¢ untranslated region of the barnase gene derived from B. amyloliquefaciens (3¢barnase); the coding sequence of the barnase gene derived from B. amyloliquefaciens(barnase); the promoter of the anther-specific gene TA29 derived from Nicotiana tabacum (tobacco) (Pta29); the promoter region of the nopaline synthase gene derived from Agrobacterium tumefaciens (Pnos); the coding sequence of the barstar gene derived from B. amyloliquefaciens (barstar); and the 3¢ untranslated region of the TL-DNA gene 7 of the octopine Ti plasmid (3¢g7).
B. amyloliquefaciens is a ubiquitous organism in nature and found throughout the world as common soil bacteria. S. hygroscopicus is a common saprophytic bacterial species that is found worldwide, predominantly in soil. B. amyloliquefaciens is a permitted source organism for many food additives enzymes in Canada. S. hygroscopicus is not considered pathogenic and is commonly used as a source for the PAT protein.
Event MS11 was developed via Agrobacterium-mediated transformation using the transformation plasmid pTCO113.
3. Characterization of the Modified Plant
To determine the integrity and copy number of the pTCO113T-DNA insert within event MS11 genomic DNA isolated from event MS11 plants was digested with different restriction enzymes and was subjected to Southern blot analysis using the different features of the T-DNA region and the complete T-DNA region as probes. The results of the analysis demonstrated the presence of a single, intact copy of the T-DNA insert in the event MS11 genome.
The potential presence/absence of vector backbone sequences in event MS11 was assessed by means of Southern blot analysis using four overlapping vector backbone probes covering the vector backbone sequence of the pTCO113 vector and PCR analysis. The results of the Southern blot analysis demonstrated the absence of vector backbone sequences in event MS11 genomic DNA samples. The absence of a barstar sequence in the vector backbone (not the barstarsequence present in the T-DNA insert) was verified by PCR analysis. The results of the analysis demonstrated the absence of this barstarsequence in the vector backbone (as opposed to the barstar sequence in the T-DNA insert which is present in event MS11).
Stability of the T-DNA insert in the event MS11 genome was demonstrated by assessing individual event MS11 plants from five generations (T2, T3, F1, BC1, and BC2) by means of Southern blot analysis. Genomic DNA from individual event MS11 plants was digested with an EcoRV restriction enzyme. For all individual plants confirmed as positive for the presence of the event MS11 T-DNA insert in all five generations, both expected restriction fragments were obtained. These results demonstrate the genetic stability of the event MS11 T-DNA insert over multiple generations.
Genetic inheritance of the T-DNA insert in event MS11 was demonstrated by assessing individual event MS11 plants from five generations for the absence or presence of the event MS11 T-DNA insert by PCR analysis. Additionally, the presence or absence of the barnase, barstar, and bargenes in event MS11 was conducted with a gene-specific analysis. Chi-square analysis was performed for the five event MS11 generations mentioned above to confirm the segregation of the event MS11 T-DNA insert. Segregation ratios determined for the five event MS11 generations confirmed that the event MS11 T-DNA insert is inherited in a predictive manner and as expected for a single T-DNA insert. These data are consistent with Mendelian principles and support the conclusion that event MS11 consists of a single T-DNA insert integrated at a single chromosomal locus within the B. napus nuclear genome. Additionally, the presence of the barnase, barstar, and bar genes was confirmed for all five event MS11 generations.
A bioinformatics analysis was performed on the event MS11 insertion locus sequence to identify the position of the insertion locus in the genome and to determine whether endogenous B. napus genes were interrupted upon the insertion of the T-DNA sequences. The bioinformatics analysis demonstrated that the event MS11 insertion locus originates from B. napus chromosome A03. The results indicate that it is unlikely that the insertion of the T-DNA sequences in the event MS11 insertion locus interrupts endogenous B. napus genes.
Bioinformatics analysis was performed on the event MS11 T-DNA insert sequence to identify potential open reading frames (ORFs). An ORF was defined as the region between two translation stop codons (TAA, TAG, or TGA) with a minimum size coding for 3 amino acids. All ORFs crossing a junction or overlapping the inserted T-DNA were reported. In the event MS11 T-DNA insert sequence, the GetORF search program identified 554 ORFs (corresponding to 526 unique sequences). After elimination of duplicates, translated amino acid sequences of at least 30 amino acids in length represented 107 unique sequences.
Translated amino acid sequences from all identified ORFs with a minimum size of 30 amino acids were used as query sequences for a homology search with known sequences available in the allergen database (FARRP; www.allergenonline.org). The overall homology search used the FASTA program (version 35.04, January 15, 2009). Only matches of ≥35% identity over at least 80 amino acids were considered potentially relevant. For all ORFs shorter than 80 amino acids, the percentage identity was calculated over a hypothetical 80 amino acid window with gaps treated as mismatches. In addition, an 8-mer homology search was carried out to identify any short sequences of 8 amino acids or longer that share 100% identity to an allergenic protein. No 100% identities were found between the 8 or longer linearly contiguous amino acid blocks that compose the query sequences and known allergens.
Each complete query sequence was also compared with all the sequences available in the NCBI non-redundant protein database using the FASTA program. The biological relevance of the matches in terms of toxicity potential was assessed by examining the alignments (e.g., identity, length of alignment, presence of gaps, E-value = 0.1), as well as the published information on toxicity of the matching proteins.
In the event MS11 T-DNA insert sequence, GetORF identified 554 ORFs (corresponding to 526 unique sequences). After elimination of duplicates, translated amino acid sequences of at least 30 amino acids in length represented 107 unique sequences.
None of the matches obtained from the NCBI non-redundant database were toxicologically relevant (i.e., indicative of a potential identity with a toxin). These results indicate that the identified ORF sequences of >30 amino acids in length show no biologically relevant sequences identities with known allergens or known toxins. There are no in silico findings indicating that the identified potential ORFs of the event MS11 T-DNA insert sequence are allergenic or toxigenic.
4. Product Information
Event MS11 differs from its traditional counterparts by the addition of three genes: barnase, barstar, and bar, which encode a ribonuclease, an inhibitor to the ribonuclease, and a phosphinothricin acetyltransferase (PAT), respectively. Expression of the Barnase (ribonuclease) protein in the tapetum cells of event MS11 results in a lack of viable pollen (i.e. male sterility). While the barstar gene encodes an inhibitor of the Barnase protein, the presence of the barstar gene in event MS11 under the control of the Pnos promoter does not significantly affect Barnase activity. Expression of the PAT protein allows the canola plant to grow in the presence of the herbicide glufosinate ammonium.
Protein expression levels of Barnase, Barstar, and PAT were analysed in event MS11 plants both treated with trait-specific herbicide at a nominal rate and untreated. The parental canola variety of event MS11 was also grown as the negative control and was not treated with the herbicide.
Protein expression levels of Barnase, Barstar, and PAT were determined on a fresh weight (FW) and dry weight (DW) basis by sandwich Enzyme-Linked ImmunoSorbent Assay (ELISA).
The level of Barnase expression was below the lower limit of quantification (LLOQ) in all untreated and treated event MS11 tissue samples.
The level of Barstar expression in untreated and treated event MS11 tissue samples ranged from below the LLOQ (observed in whole plant samples at 3-5 leaf stage, stem elongation, and first flowering growth stage as well as raceme and grain samples) to 1.04 mg/g DW (observed in root at stem elongation growth stage). Mean (±SD) Barstar expression levels in untreated and treated root samples varied within the range of 0.39 ± 0.10 mg/g DW and 0.50 ± 0.24 mg/g DW. Only two samples of treated event MS11 raceme and three samples of treated event MS11 whole plant at first flowering growth stage were above the LLOQ. Mean Barstar expression of the two raceme samples and three whole plant at first flowering samples of treated event MS11 was 0.68 ± 0.31 mg/g DW and 0.21 ± 0.08 mg/g DW, respectively.
The level of PAT expression in untreated and treated event MS11 tissue samples ranged from below the LLOQ to 74.44 mg/g DW. Root (BBCH 30-39 and BBCH 57-65 growth stages) and grain samples all exhibited lower mean PAT DW expression levels relative to mean DW values for other tissue samples of event MS11. Mean PAT DW expression levels were highest in whole plant samples of untreated and treated event MS11 at BBCH 30-39 and BBCH 57-65 growth stages, respectively.
The level of Barnase, Barstar, and PAT expression in all tissue samples were similar between event MS11 treated with trait-specific herbicide and untreated event MS11. In other words, treatment with a trait-specific herbicide does not appear to affect novel protein expression in event MS11.
Furthermore, expression levels of both the Barnase and Barstar proteins are low in all tissue samples of event MS11 while the expression level of PAT protein in mature grain tissue is also low. Thus, the exposure to all three novel proteins via the consumption of grain-derived food/feed is minimal.
5. Dietary Exposure
It is expected that progeny of event MS11 hybrids will be used in applications similar to conventional canola varieties. The petitioner does not anticipate a significant change in the food use of canola with the introduction of this transformed event.
Compositional data for the transgenic event MS11 (treated and not-treated with glufosinate ammonium) and its non-transgenic conventional counterpart (variety N90-740) were collected from nine field trials in the B. napus growing regions of Canada and the USA in 2014. In addition to the control, six commercial non-genetically modified (GM) B. napus varieties were also grown to provide reference ranges for the composition assessment. On each site, the field trial consisted of six entries replicated four times (24 plots total) in a randomized complete block design.
Seed samples were analyzed for proximates (ash, carbohydrates, moisture, protein, acid detergent fiber, neutral detergent fiber, and fat), amino acid profile, fatty acid profile, vitamins, minerals, and anti-nutrients. The compositional analyses were conducted using standard methods published by AOAC or other international organizations.
The data were analyzed for potential statistical differences using SAS version 9.3. Differences for each analyte were estimated and presented with 95% confidence intervals, along with the p-values (t-test). Statistical significance was evaluated at p<0.05 level.
No significant differences were observed between event MS11 not treated with trait-specific herbicides and the non-GM conventional counterpart for any of the proximates, amino acids, fatty acids, minerals, vitamins, and anti-nutrients, except gluconapin, and insoluble tannins. However, the means for gluconapin and insoluble tannins were within the range of the reference varieties and the tolerance intervals.
Statistically significant differences (p<0.05) were observed between event MS11 treated with trait-specific herbicides and the non-GM conventional counterpart for most of the analytes (30 in total). However, the means for these analytes were within the range of values reported for the reference varieties and their tolerance intervals and were not considered nutritionally important. It is likely that the observed differences in the herbicide-treated plants were due to cross-pollination allowed to generate seeds.
The petitioner has demonstrated that the composition of event MS11 is similar to its control and to conventional canola varieties.
Safe use of canola oil derived from event MS11 (containing the transgenes: barnase, barstar, and bar) is supported by the results of submitted toxicity and stability studies, which were conducted in accordance with Good Laboratory Practices (GLP) Standards. The studies include: bioinformatics, acute toxicity in mice (OECD 420), and heat stability.
The novel proteins (Barnase, Barstar, and PAT) used for testing were microbially produced, due to low expression levels in the plants. The equivalence between the plant-produced proteins and the microbially-produced surrogate proteins was demonstrated by comparing: molecular weight, immuno-reactivity, glycosylation status, N-terminal sequence, and biological activity.
A bioinformatics analysis of the amino acid sequences of the three novel proteins did not find any biologically relevant matches with any known toxic proteins. The National Center for Biotechnology Information (NCBI) non-redundant database and in-house Bayer toxin database were searched in March, 2016.
The acute oral gavage mouse toxicity study used 6 animals per sex per dose for Barnase and Barstar, and 10 animals per sex per dose for PAT. No adverse effects were noted, from 14 days of observation or from necropsy. The No Observable Adverse Effect Level (NOAEL) is the limit dose of 2000 mg/kg body weight (bw).
Four different assays demonstrate that the three novel proteins are sensitive to degradation from heat exposure. Incubation for 30 minutes at temperatures of 55 °C and greater produces: additional bands on SDS-PAGE and western blots, reduction in the amount of protein measured with ELISA, and decreased enzyme activity. All novel proteins show complete loss of functional activity with an incubation at 95 °C for 30 minutes. These proteins are expected to be denatured from heat treatment during oil extraction and refinement.
No dietary exposure to the novel proteins is expected, because none of the novel proteins were detected by ELISA in the final oil food product. A dietary exposure was estimated for the PAT protein, with the assumption that no loss or degradation occurs, using the WHO GEMS/Food databases for refined rape seed oil (includes canola oil). PAT is the only novel protein detectable at measurable levels in the plant. Using a fresh weight average PAT protein content in event MS11 seeds of 0.44 mg/g, acute consumption is estimated to be 0.12 mg/kg bw/day for women (14-50 years) and 0.39 mg/kg bw/day for toddlers (8-20 months). To calculate the margin of exposure (MoE) for PAT protein, the NOAEL of >2000 mg/kg bw/day (obtained from the acute toxicity study in mice) was used. The highly conservative MoE is quite large; >5.1×106 in toddlers and >1.7×107 in adult women. This MoE is considered sufficient from a safety perspective.
There are no toxicological food safety concerns with the use of canola oil derived from event MS11, based on the available toxicity data.
Proteins the pose an allergenic risk are typically resistant to degradation in the digestive system. The three novel proteins are expected to be denatured from heat treatment during oil extraction and refinement, because they lose all functional activity after heat exposure of 95 °C for 30 minutes. The novel proteins are sensitive to digestion in human simulated gastric fluid at pH 1.2; complete degradation occurred within 2 minutes.
Oil of canola quality, extracted from B. napus seeds, is the only fraction used for human consumption. Proteins are not expected to be present in food-grade oil, due to the heat and chemical treatment during oil extraction and refinement. ELISA analysis did not detect any of the novel proteins (Barnase, Barstar, and PAT) in refined, bleached, deodorized oil.
The bioinformatics analysis of the amino acid sequences of the three novel proteins, derived from S. hygroscopicus and B. amyloliquefaciens, did not detect any sequence homology matches with known allergens. Searches were conducted according to current guidelines, using the database of allergens in AllergenOnline in March, 2017.
Based on the information provided, use of canola oil derived from event MS11 is not expected to pose an additional allergenic concern.
Health Canada's review of the information presented in support of the food use of event MS11 does not raise concerns related to food safety. Health Canada is of the opinion that food derived from this transformed event is as safe and nutritious as food from current commercial canola varieties.
Health Canada's opinion deals only with the food use of event MS11. Issues related to its use as animal feed have been addressed separately through existing regulatory processes in the Canadian Food Inspection Agency (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.
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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