Novel Food Information: High Oleic Soybean

On this page

• Background
  1. Introduction
  2. Development of the Modified Plant
  3. Characterization of the Modified Plant
  4. Product Information
  5. Dietary Exposure
  6. Nutrition
  7. Chemistry/Toxicology
• Conclusion

Background:

Health Canada has notified Calyxt Inc. that it has no objection to the food use of high oleic soybean. The Department conducted a comprehensive assessment of this 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 Calyxt, Inc. and the evaluation by Heath Canada and contains no confidential business information.

1. Introduction

Calyxt, Inc. developed this soybean line to produce oil with approximately 80 % oleic acid content and 20 % less saturated fatty acids compared to commodity soybean oil. High oleic soybean is an alternative to partially hydrogenated soybean oil, providing a more shelf stable oil than conventional soybean oil, without the trans fatty acids of partially hydrogenated oils.

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 high oleic soybean was developed; how the composition and nutritional quality of high oleic soybean compared to non-modified varieties; and the potential for high oleic soybean to be toxic or cause allergic reactions. Calyxt, Inc. provided data that demonstrates that high oleic soybean is as safe and of the same nutritional quality as traditional soybean 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). Food use of high oleic soybean 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
  • (i) 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 high oleic soybean and data that characterize the gene edits that result in the soybean oil profile of approximately 80% oleic acid content through the knock out of the FAD2-1A and FAD2-1B genes. High oleic soybean was produced through TALEN-mediated gene editing to deactivate the target genes resulting in no functional FAD2-1A and FAD2-1B proteins present in the plant.

A. rhizogenes-mediated transformation of half-seed soybean plants was used to deliver the TALEN expression vector construct into the soybean genome. The TALEN expression vector is composed of the coding sequences for the desired TAL effector repeat array targeting the FAD2-1A and FAD2-1B genes, a promoter at the 5' end, and a terminator at the 3' end. A cassette expressing a selectable marker was also part of the T-DNA and used to identify successfully transformed soybean plants. These T0 soybean plants were regenerated from resistant tissue and grown to maturity, seeds harvested, and re-sown to result in T1 plants used for the molecular characterization.

The TALEN-mediated gene edits in high oleic soybean targeted the FAD2-1A and FAD2-1B genes specifically using a proprietary TALEN Hit software which aligned DNA sequences for the FAD2 gene family (contains 7 total genes involved in fatty acid biosynthesis). A conserved region of the FAD2-1A and FAD2-1B genes was targeted to minimize potential off-target binding at any of the other 5 FAD2 genes.

3. Characterization of the Modified Plant

PCR and Sanger sequencing were used to assess the T1 plants for mutations in the FAD2-1A and FAD2-1B genes in order to characterize the TALEN-mediated gene edits. The analysis showed a T1 plant that was homozygous for a 63 base pair deletion in the FAD2-1A gene and a 23 base pair deletion in FAD2-1B gene. The 63 base pair deletion in FAD2-1A is in-frame with the coding region and results in a 21 amino acid deletion which could produce a FAD2-1A protein of 366 amino acids compared to the full-length 387 amino acid protein. The 21 amino acid deletion is found in the highly conserved catalytic site of the protein and likely renders the protein non-functional. The 23 base pair deletion in FAD2-1B is out of coding frame and could result in the production of a 147 amino acid truncated protein compared to the full-length 387 amino acid protein.

The T1 plant that contained the desired mutations was self-pollinated, grown to maturity, seeds harvested, and re-sown to produce T2 plants. A multiplex PCR analysis was conducted on the T2 plants to identify those which lack the TALEN T-DNA. A total of twelve null segregants, lacking the TALEN T-DNA, were identified. These 12 were used to breed subsequent generations as they possess the desired mutations and lack the T-DNA.

Whole genome sequencing of the twelve null-segregant plants was conducted to confirm the absence of TALEN T-DNA in the soybean genome. Genomic DNA libraries were prepared and sequenced by Illumina high-throughput DNA sequencing. This analysis demonstrated no evidence of integrated TALEN T-DNA plasmid fragments in any of the plants tested. This result was in agreement with the PCR results from the progeny of the original self-fertilized mutant plant.

A search of the entire soybean genome was conducted to look for potential off-target sites using not only the combination of the left and right TALEN monomers, but also potential combination of two-left and two-right TALEN monomer binding sites. Seven potential TALEN off-target sites were identified, of which 5 were FAD2-2 genes. All seven contained seven or more mismatches, which makes it highly unlikely the TALENs targeted these genes based on established TALEN design criteria. Whole genome sequencing was also conducted to demonstrate no unintended modifications in the other closely related FAD2 genes. The analysis demonstrated no evidence of mutations occurring in any of the other FAD2 genes, which supports that there are no off-target effects.

The stability of the genetic change was demonstrated using whole genome sequence analysis, which showed the FAD2-1A and FAD2-1B genes had been edited in their original chromosomal location. Molecular analysis of plants from 2 generations showed the expected genotype ratios based on Mendelian inheritance principles. Also, the compositional analysis of the T7 generation demonstrated the high oleic trait was maintained stably after seven generations. The results confirm the inheritance and stability of the genetic changes across multiple generations of Calyxt high oleic soybean.

4. Product Information

The FAD2 proteins are a minor component of the total protein found in soybean seeds because they are membrane-bound and not seed storage proteins. The non-functional FAD2-1A and FAD2-1B proteins are not likely to be present at higher levels than the wild-type protein because the same endogenous promoter controls their expression.

5. Dietary Exposure

The oil from Calyxt high oleic soybean is similar to other high oleic soybean oils already consumed in Canada and will be used in a variety of food applications. It is expected that there would be no change in the food use of soybean oils caused by the presence of Calyxt high oleic soybean.

6. Nutrition

The petitioner provided compositional data for the high oleic soybean line and its closely related conventional counterpart obtained from three field trials conducted across three locations in the United States during the 2016 growing season. In each trial, four replicates of each entry were planted in a randomized complete block design. Seed samples were harvested and analyzed, using acceptable methods, for proximates and fibres, amino acids, fatty acids, isoflavones, and anti-nutrients.

The petitioner conducted a statistical analysis comparing the modified cultivar with its conventional counterpart. 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, and the peer-reviewed scientific literature.

As intended, two major changes in fatty acid composition were observed such that differences between the modified cultivar and its conventional counterpart were statistically significant and placed the modified cultivar outside the expected range for conventional soybean, namely an elevated level of oleic acid (77 % vs. 20 % of total fatty acids) and a lower level of linoleic acid (5 % vs. 53 %). These differences were not considered to pose a nutritional safety concern because (a) the levels of these fatty acids are comparable to conventional olive oil, (b) there are no clearly established adverse effects of cis-monounsaturated fatty acids (e.g., oleic acid) per se in humans according to the Institute of Medicine, (c) it is considered very unlikely that the lower linoleic acid content of high oleic soybean oil would compromise essential fatty acid status of Canadians consuming a mixed diet, and (d) 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 genetically modified, high oleic soybeans for which a pre-market safety assessment has been completed.

The following statistically significant differences in fatty acids and other components were found such that values for the modified cultivar were within the expected range for conventional soybean (modified cultivar vs. conventional counterpart, units): palmitic acid (7.8 vs. 12.0, % total FA), stearic acid (3.5 vs. 3.9, % total FA), linolenic acid (4.8 vs. 9.2, % total FA), arachidic acid (0.36 vs. 0.31, % total FA), lignoceric acid (0.17 vs. 0.19, % total FA), histidine (1.1 vs. 1.0, % DW), isoleucine (1.7 vs. 1.6, % DW), threonine (1.4 vs. 1.3, % DW), tyrosine (1.5 vs. 1.4, % DW), valine (1.8 vs. 1.7, % DW). 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

FAD2-1A and FAD2-1B proteins are naturally present in soybean and neither of these proteins are known to be toxic. The petitioner conducted an in silico search of the National Center for Biotechnology Information (NCBI) protein database to compare the amino acid sequences of the truncated FAD2-1A and FAD2-1B proteins to known toxins. The results were analyzed for the keywords "toxin" or "toxic". The search did not identify any similarities to protein sequences of known toxins.

A semi-quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a label-free quantification (LFQ) workflow was conducted to evaluate the presence and relative abundance of full and truncated forms of the FAD2-1A and FAD2-1B proteins in the unedited soybean control and the high oleic soybean. In this analysis, only the full form of FAD2-1B was detected, with its level estimated to be 0.004 % of total protein^{Footnote 1}. Neither of the two truncated proteins were detected suggesting that these proteins in high oleic soybean are present at even lower amounts.

Soybean oils contain negligible quantities of protein due to manufacturing process. On this basis, the potential exposure to the truncated FAD2 proteins from oils is also considered negligible. In considering the non-oil soybean food applications, any dietary exposure to truncated proteins will be lower than exposure to the endogenous native proteins from conventional soybean^{Footnote 2}. It is unlikely that the lowered amounts of exposure to the truncated proteins will pose an increased health risk.

Based on the very low potential for dietary exposure to the truncated FAD2 proteins, the lack of similarity of these proteins to known toxins, and the fact that the native FAD2 proteins have been widely consumed safely for decades, there is no anticipation of any risk of toxicity from the truncated FAD2 proteins in high oleic soybean. Given that there were no other genetic modifications, there is no evidence to suggest that the high oleic soybean poses any increased risk of toxicity.

FAD2-1A and FAD2-1B proteins are naturally present in soybean and neither of these proteins are known to be soy allergens. Health Canada conducted a search of PubMed^{Footnote 3} to identify reports of allergic reactions to FAD2 proteins. There was no evidence in literature that the FAD2 proteins represent a potential allergen.

The petitioner conducted an amino acid sequence homology search using the AllergenOnline database^{Footnote 4} to identify potential cross-reactivity of the truncated FAD2-1A and FAD2-1B proteins against known allergens. Searches included a full FASTA alignment, a sliding window of 80 amino acids stretches with greater than 35 % identity, and a search for 8 contiguous amino acids to assess for presence of epitope^{Footnote 5}. The search did not identify any matches against known allergens.

The petitioner conducted a whole genome sequencing (WGS) analysis to assess if there were any further modifications in the genome that could affect the levels of endogenous soybean allergens. The WGS analysis did not identify any foreign DNA or off-target mutations suggesting that there are no changes to the levels of endogenous soybean allergens.

High oleic soybean does not pose any additional allergenic health concerns in comparison to conventional soybean.

Conclusion:

Health Canada's review of the information presented in support of the food use of high oleic soybean does not raise concerns related to food safety. Health Canada is of the opinion that food derived from high oleic soybean is as safe and nutritious as food from current commercial soybean lines.

Health Canada's opinion deals only with the food use of high oleic soybean.

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
Food Directorate
Health Products and Food Branch
Health Canada, PL2204A1
251 Frederick Banting Driveway
Ottawa, Ontario K1A 0K9
bmh-bdm@hc-sc.gc.ca