Novel Food Information: High oleic acid soybean line SVX-4003
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- Background
- Introduction
- Development of the Modified Plant
- Characterization of the Modified Plant
- Product Information
- Dietary Exposure
- Nutrition
- Chemistry/Toxicology
- Conclusion
Background:
Health Canada has notified Sevita Genetics that it has no objection to the food use of the high oleic acid soybean variety SVX-4003 derived from the soybean line KK21-B12. The Department conducted a comprehensive assessment of this soybean line 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 Sevita Genetics and the evaluation by Heath Canada and contains no confidential business information.
1. Introduction
The KK21-B12 soybean line has been subject to classical mutagenesis in order to induce loss of function mutations in two genes, GmFAD2-1a and GmFAD2-1b, which encode fatty acid desaturase-2 enzymes. These enzymes are responsible for the conversion of oleic acid to linoleic acid in soybean seeds. Loss of function of the GmFAD2-1 genes results in the accumulation of high levels of oleic acid in KK21-B12. The soybean variety to be commercialized, designated SVX-4003, is derived from the variety KK21-B12.
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 reflects international guidance documents in this area (e.g., Codex Alimentarius). The assessment considered: how SVX-4003 was developed; how the composition and nutritional safety of SVX-4003 compares to non-modified varieties; and the potential for SVX-4003 to present a toxic or allergenic concern. Sevita Genetics has provided data to support the safety of this soybean variety for use as food in Canada.
The Food Program has a legislated responsibility for the pre-market assessment of novel foods and novel food ingredients as detailed in the Food and Drug Regulations (Division 28). Food use of soybean event SVX-4003 is considered novel 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
- (iii) one or more characteristics of the plant, animal or microorganism no longer fall within the anticipated range for that plant, animal or microorganism."
2. Development of the Modified Plant
The GmFAD2-1b mutation in the B12 line was generated through methanesulfonic acid methyl ester (EMS) treatment and results in an above average oleic acid content in the plant. An identified C to A nucleotide change in GmFAD2-1b causes a T189P amino acid change that results in a strong reduction of GmFAD2-1b protein activity. The GmFAD2-1a mutation in the KK21 line was generated through X-ray irradiation and results in a 20 % increase of oleic acid content in the plant. The GmFAD2-1a mutation is a single nucleotide deletion located 232 bases from the start codon that results in a frameshift and truncation of the protein. KK21 and B12 were crossed and advanced to the F2 stage where the population was screened using molecular markers for both mutant genes (Anai et al., 2008; Canadian Patent No. CA2765560; Hoshino et al., 2010). The resulting KK21-B12 line contained both GmFAD2-1a and GmFAD2-1b loss of function mutations. This new variety was observed to have 80 % oleic fatty acid as a percent of total fatty acids (Hoshino et al., 2010; Canadian Patent No. CA2765560, 2017). The soybean variety to be commercialized, designated SVX-4003, is derived from the donor parent KK21-B12 and carries the GmFAD2-1a and GmFAD2-1b loss of function mutations resulting in the accumulation of high levels of oleic acid in its seed. In developing SVX-4003, molecular markers were used by the developer to select plants that were homozygous for the mutations of interest that resulted in the high-oleic trait.
3. Characterization of the Modified Plant
The presence of the trait-conferring mutations in the GmFAD2-1a and GmFAD2-1b genes was confirmed by sequencing of leaf tissue from eight soybean lines, three non trait-carrying lines (parents) and five trait-carrying lines (KK21-B12, SVX-4003, and three other proposed commercial lines). For both genes, the causative mutations were present and homozygous in the five trait-carrying lines and not present in the three non trait-carrying lines. Sequencing of the genome regions upstream and downstream from the mutations of interest (GmFAD2-1a and GmFAD1b) revealed no additional nucleotide differences in SVX-4003 when compared to non trait-carrying lines.
RNA expression data was provided to estimate expression of the novel proteins in high oleic soybean line SVX-4003. Using a reverse transcription quantitative polymerase chain reaction (RT-qPCR) method, data was provided showing that there was no statistically significant difference in the expression of GmFAD2-1a and GmFAD2-1b RNA in line SVX-4003 as compared to a non trait-carrying parental line.
Stability of the trait-conferring mutations was demonstrated by assessing the inheritance in the SVX-4003 line by means of Sanger sequencing. Results showed that the trait-conferring mutations are stable and inherited in a homozygous configuration, as expected. Sequencing and SNP data was used by the petitioner to show that both GmFAD2-1a and GmFAD2-1b alleles segregate according to the expected Mendelian inheritance ratio (1:2:1) in all three F2 populations that were tested. Analysis of the oleic acid content of seeds over several generations also showed that the fatty acid phenotype was stable over this period, with average oleic acid content greater than 75 %.
4. Product Information
The KK21-B12 soybean line, and the SVX-4003 variety derived from it, differ from their traditional counterparts by the loss of function mutations in two genes GmFAD2-1a and GmFAD2-1b. The GmFAD2-1 genes encode for fatty acid desaturase-2 enzymes that are responsible for the conversion of oleic acid to linoleic acid in soybean seeds. The loss of function of these enzymes results in the accumulation of high levels of oleic acid in KK21-B12, and varieties derived from it (e.g. SVX-4003).
5. Dietary Exposure
Sevita Genetics stated that the primary intended food uses for SVX-4003 include tofu, soymilk, sprouts and meat alternatives, but can also be used as miso, tempeh, natto and other soy-food products.
It is expected that high oleic soybean will be used in applications similar to conventional soybean varieties. The petitioner does not anticipate a significant change in the food use of soybean with the introduction of this high oleic soybean.
6. Nutrition
Compositional data for the high oleic soybean event and its closely related conventional counterpart were obtained from several field trials. Seed samples were harvested and analyzed, using acceptable methods, for proximates and fibres, mineral, amino acids, fatty acids, and anti-nutrients and a statistical analysis comparing the modified cultivar with its conventional counterpart was provided. If a statistically significant difference (P-value <0.05) was indicated, the nutritional relevance of the difference was determined through comparison to the expected range for conventional soybean as defined by values reported in the Agriculture & Food Systems Institute Crop Composition Database, the Organisation for Economic Co-operation and Development consensus document regarding new varieties of soybean (2012), and the peer reviewed scientific literature.
Sevita stated that statistically significant differences in fatty acid composition in the soybean seed were observed between the modified cultivar and its conventional counterpart such that the modified cultivar was outside the expected range for conventional soybean. These included an elevated level of oleic acid (14.43 % vs 3.43 % of fatty acid relative to total soybean DW) and a lower level of linoleic acid (1.52 % vs 10.56 %). These differences were not considered to pose a nutritional safety concern primarily because (a) the Institute of Medicine has not set a Tolerable Upper Intake Level for oleic acid, (b) it is considered very unlikely that the lower linoleic acid content of SVX-4003 oil would compromise the essential fatty acid status of Canadians consuming a mixed diet, and (c) labelling of the derived oil will allow consumers requiring a specific fatty acid composition to differentiate it from conventional soybean oil. It was also noted that the levels of these fatty acids in the modified cultivar were similar to other high oleic soybeans for which the Food Directorate has expressed no objection following a pre-market safety assessment.
Heptadecanoic acid was significantly higher in the modified cultivar compared to control (0.0314 vs 0.0089 % fatty acids) and the amount in the modified cultivar was outside the expected range found in the literature. However, soybean is not a significant source of heptadecanoic acid and the amount present is still very low. In addition, the Institute of Medicine (IOM) did not identify a Tolerable Upper Intake Level for heptadecanoic acid or monounsaturated fatty acids (MUFAs; IOM, 2006) indicating that excess consumption is not a nutritional safety concern per se, even if levels were greatly elevated in the notified line compared to control.
Several statistically significant differences in fatty acids and other components were found, however, the values for the modified cultivar were within the expected range for conventional soybean (modified cultivar vs. conventional counterpart, units): crude protein (36.88 vs 35.71 % DW), crude fat (18.1 vs 17.54 % DW), carbohydrate (31.32 vs 33.37, % DW), ash (4.5 vs 4.32, % DW), calcium (0.173 vs 0.198 % DW), potassium (1.71 vs 1.57 % DW), zinc (42.79 vs 40.44 ppm), alanine (1.60 vs 1.52 % DW), arginine (2.81 vs 2.65 % DW), aspartic acid (4.5 vs 4.19 % DW), glutamic acid (7.49 vs 6.97 % DW), glycine (1.65 vs 1.56 % DW), histidine (1.04 vs 0.98, % DW), isoleucine (1.84 vs 1.75 % DW), leucine (2.84 vs 2.75 % DW), lysine (2.59 vs 2.41 % DW), proline (2.04 vs 1.88 % DW), valine (1.90 vs 1.79 % DW), palmitic acid (1.40 vs 1.94 %fa), palmitoleic acid (0.0181 vs 0.0139 %fa), stearic acid (0.571 vs 0.638 %fa), linlolenic acid (1.22 vs 1.45 %fa), arachidic acid (0.0640 vs 0.0563 %fa), eicosenoic acid (0.0591 vs 0.0280 %fa), and behenic acid (0.0751 vs 0.0457 %fa). These differences were not considered to pose a nutritional safety concern as, in all cases, the composition of the modified cultivar was within the expected range for conventional soybean.
7. Chemistry/Toxicology
Data on toxic trace elements and mycotoxins were not provided; however, based on an understanding of the biochemical and physiological functions of the mutations, it is not expected that SVX-4003 would present an increased risk of contamination for heavy metals or mycotoxins. Furthermore, while toxic trace elements and mycotoxins are occasionally observed at low levels in soy and soy products, these have not been identified as significant contributors to total dietary exposure of these compounds.
It was not possible to directly measure the levels of FAD2-1A and FAD2-1B enzyme (protein) expression in the SVX-4003 soybean. Instead, results of a RT-qPCR gene expression study were presented, which showed no significant differences in messenger RNA expression between the wildtype and mutated GmFAD2-1A or GmFAD2-1B genes. The resultant expression of the modified FAD2 proteins is, therefore, also unlikely to increase compared to the wild-type proteins. Furthermore, the FAD2 proteins constitute only a minor portion of the overall soybean protein fraction, as they are not seed storage (or other abundant) proteins that are present in soybean. Therefore, dietary exposure to the modified FAD2 proteins is anticipated to be low.
FAD2-1A and FAD2-1B proteins are naturally present in soybean, and neither is known to be toxic. Sevita conducted a bioinformatics amino acid sequence homology search of the modified FAD2 proteins produced by SVX-4003 soybean against a database of known toxin sequences. No similarities were identified.
Sevita also conducted a bioinformatics amino acid sequence homology search to confirm that the modified FAD2 proteins produced by SVX-4003 soybean do not share any homologies with known allergens. The search included: full FASTA sequence alignment; 80 amino acid sliding window with an identity threshold of 35%; and 8 contiguous amino acids. The results did not identify any matches against known allergens. Moreover, an enzyme-linked immunosorbent assay (ELISA) showed that the expression of the major endogenous soybean allergen beta‑conglycinin, which shares a genetic linkage with the GmFAD2‑1A and GmFAD2-1B genes, was comparable between SVX-4003 soybean and conventional soybean.
Soybean is a priority allergen in Canada. Soybean products would be considered allergenic if they contain soy proteins. Compositional analysis, including for proximates and fibres, mineral, amino acids, and anti-nutrients, demonstrates that the SVX-4003 cultivar was within the expected range for conventional soybean. There is no expectation that classical mutagenesis techniques used to develop the SVX‑4003 soybean will cause off-target mutations that could result in overexpression of endogenous allergens. Moreover, the similarity between SVX-4003 and conventional soybean in phenotypic and growth characteristics supports the absence of off-target effects from the genetic modification, including increased expression of the novel FAD2 proteins. This suggests that the amounts of endogenous allergens in SVX-4003 products are equivalent to conventional soybean products.
Based on the lack of similarity of the modified FAD2 proteins to known toxins and allergens, as well as the minimal expression of these proteins, in addition to the fact that the native FAD2 proteins have been widely and safely consumed, there is no toxicity or allergenicity concerns from exposure to the modified FAD2 proteins in SVX-4003 soybean.
Based on the information provided, evaluators did not identify any safety concerns regarding the food use of SVX-4003.
Conclusion:
Health Canada's review of the information presented in support of the food use of SVX-4003 does not raise concerns related to food safety. Health Canada is of the opinion that food derived from SVX-4003 soybeans is as safe and nutritious as food from current commercial soybean varieties.
Health Canada's opinion deals only with the food use of soybean SVX-4003. Issues related to its use as animal feed have been addressed separately through existing regulatory processes in the Canadian Food Inspection Agency.
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:
Health Canada
Novel Food Section
Food Directorate
Health Products and Food Branch
251 Sir Frederick Banting Driveway
PL2204E
Ottawa, Ontario, K1A 0K9
bmh-bdm@hc-sc.gc.ca
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