Novel Food Information - Stearidonic Acid (SDA) Producing Soybean MON 87769

Health Canada has notified Monsanto Canada Inc. that it has no objection to the food use of Stearidonic Acid (SDA) Producing Soybean MON 87769. The Department conducted a comprehensive assessment of these varieties 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 Monsanto Canada Inc. and the evaluation by Heath Canada and contains no confidential business information.

1. Introduction

Monsanto has developed Stearidonic Acid (SDA) Producing Soybean MON 87769 using recombinant DNA techniques to introduce two coding sequences: Pj.D6D, derived from Primula juliae (Juliana primrose), which encodes a delta (Δ)6 desaturase and Nc.Fad3, derived from Neurospora crassa (fungus), which encodes an w3 desaturase. The PjΔ6D protein expressed in MON 87769 creates a double bond at the 6th position from the carboxyl end of a fatty acid, while the NcΔ15D protein creates a double bond between the third and fourth carbon from the methyl end of a fatty acid. Tissue-specific expression of these two enzymes creates a shift in the fatty acid metabolic pathways, yielding significant levels of SDA in the seeds of MON 87769.

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 MON 87769 was developed; how the composition and nutritional quality of MON 87769 compared to non-modified varieties; and the potential for MON 87769 soybeans to be toxic or cause allergic reactions. Monsanto Canada Inc. has provided data that demonstrates that MON 87769 soybeans are 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 28). Food use of Stearidonic Acid (SDA) Producing Soybean MON 87769 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

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 Stearidonic Acid (SDA) Producing Soybean MON 87769 and the molecular biology data that characterize the genetic change, which results in elevated levels of SDA in the seeds of MON 87769. This phenotype was achieved by transformation of the conventional soybean variety A3525 with a transgenic expression cassette containing the novel Pj.D6D and Nc.Fad3 genes and their associated regulatory elements.

Stearidonic Acid (SDA) Producing Soybean MON 87769 was genetically modified using Agrobacterium-mediated transformation of commercial soybean variety A3525 with the 2T-DNA transformation vector PV-GMPQ1972. The transformation vector PV-GMPQ1972 carried two separate transfer DNA (T-DNA I and T-DNA II) sequences, each comprised of an expression cassette, one containing the Pj.D6D and Nc.Fad3 coding sequences (T-DNA I) and one containing the cp4 epsps coding sequence (T-DNA II).

The Pj.D6D and Nc.Fad3 expression cassette contains the following genetic elements: the promoter and leader sequences of the Sphas1 gene from Glycine max encoding beta-conglycinin storage protein (alpha'-bcsp), the Pj.D6D coding sequence from P. juliae encoding the Δ6 desaturase, the terminator and 3' non-translated sequences of the tml gene from the A. tumefaciens octopine-type Ti plasmid, the promoter and leader sequences of the Sphas2 gene from G. max encoding the alpha subunit from beta-conglycinin, the codon-optimized Nc.Fad3 coding sequence from N. crassa encoding the w3 desaturase, the terminator and 3' non-translated sequences of the RbcS2 gene from Pisum sativum encoding the Rubisco small subunit, which functions to direct polyadenylation of the mRNA. Both coding sequences are regulated under seed-specific promoters.

The cp4 epsps expression cassette contained the following genetic elements: the promoter of the 35S RNA from figwort mosaic virus (FMV), the 5¢ non-translated leader sequence from the A. thaliana ShkG gene encoding EPSPS, the targeting sequence encoding the chloroplast transit peptide from the ShkG gene of A. thaliana encoding EPSPS, the codon-optimized cp4 epsps coding sequence of the aroA gene from Agrobacterium sp. strain CP4 encoding the CP4 EPSPS protein, and the 3¢ non-translated sequence from RbcS2 gene of Pisum sativum encoding the Rubisco small subunit, which functions to direct polyadenylation of the mRNA. The T-DNA II transfer DNA containing the cp4 epsps expression cassette was inserted into the plant genome as a selective marker of transformation. The phenotype conferred by the expression of the CP4 EPSPS protein (i.e., tolerance to glyphosate-based herbicides) allowed for the selection of successful transformations. Using traditional methods of breeding, the T-DNA II transfer DNA was subsequently removed from the soybean genome, producing the MON 87769 genetic event containing only the Pj.D6D and Nc.Fad3 expression cassette (T-DNA I).

3. Characterization of the Modified Plant

Southern blot analysis and DNA sequencing of Stearidonic Acid (SDA) Producing Soybean MON 87769 demonstrated the presence of a single copy of the Pj.D6D and Nc.Fad3 expression cassette in the soybean genome at a single locus. Southern blot analysis confirmed the absence of any plasmid backbone DNA in Stearidonic Acid (SDA) Producing Soybean MON 87769.

The stability of the inserted Pj.D6D and Nc.Fad3 expression cassette was evaluated from the progeny of four different generations. The results of Southern blot analysis and segregation data demonstrated the stability of Stearidonic Acid (SDA) Producing Soybean MON 87769 at the genomic level.

4. Product Information

Stearidonic Acid (SDA) Producing Soybean MON 87769 differs from its traditional counterpart by the addition of the Pj.D6D and Nc.Fad3 genes and their associated regulatory elements. The insertion of these gene results in the tissue-targeted expression of the novel PjΔ6D and NcΔ15D proteins in MON 87769. Tissue-specific expression of these two enzymes creates a shift in the fatty acid metabolic pathways, yielding significant levels of SDA in the seeds of MON 87769.

The coding sequence, Pj.D6D, was isolated from Primula juliae and encodes a single polypeptide, designated PjΔ6D. The PjΔ6D protein is a Δ6 desaturase, which creates a double bond at the 6th position from the carboxyl end of a fatty acid. Primula belong to a large genus of plants commonly known as Primrose. These plants contain significant levels of SDA in their leaves. Several Primula species are used for medicinal practices in Bulgaria, Romania, the Czech Republic, Germany, and other European countries. Primrose plants are also used as a food source in soups and as a tea substitute. Contact dermatitis has been reported involving certain varieties of Primula; however, the allergic response is triggered by a non-protein allergen called primin.

The coding sequence, Nc.Fad3, was isolated from Neurospora crassa and encodes a single polypeptide, designated NcΔ15D. The NcΔ15D protein is a w3 desaturase, which creates a double bond between the third and fourth carbon from the methyl end of a fatty acid. N. crassa is a type of bread mould of the phylum Ascomycota and a widely used model organism in genetics and biological research. The organism is ubiquitous in the environment and is used to manufacture food in a variety of different world areas including Indonesia, Brazil, and southern France. N. crassa is considered a non-pathogenic and non-allergenic organism.

The petitioner has provided data to demonstrate the level of expression of the PjΔ6D and NcΔ15D proteins in MON 87769. This study used plant samples from five field trial locations conducted in the United States: Iowa (2 sites), Illinois, Michigan, and Ohio. All locations are relevant soybean-growing regions and represent a range of environmental conditions typically encountered in the production of soybean. At each field site, three replicated plots of MON 87769 and a conventional soybean control (A3525) were planted using a randomized complete block field design. Tissues of MON 87769 that were collected include: Over-season leaf (OSL), forage, root, mature and immature seed. The quantities of PjΔ6D and NcΔ15D proteins were determined by western immunoblot analysis coupled with densitometric analysis. Protein quantities for the tissues were calculated on a microgram (mg) per gram (g) dry weight (dwt) basis. The expression of the PjΔ6D and NcΔ15D proteins is controlled by a 7Sa' and 7Sa seed-specific promoter, respectively. Both proteins were detected in tissues of immature seed, mature seed, and at low levels in forage, as this tissue tends to contain small amounts of immature seed. The mean PjΔ6D protein levels across all sites for immature seed, mature seed, and forage were 100, 1.8, and 16 µg/g dwt, respectively. The mean NcΔ15D protein levels across all sites for immature seed, mature seed, and forage were 200, 10, and 14 µg/g dwt, respectively. Neither protein was detected in the conventional control soybean A3525.

5. Dietary Exposure

The petitioner states that MON 87769 can serve as an alternate source of an omega-3 fatty acid to help meet the need for long-chain omega-3 fatty acids in food. The oil from MON 87769 contains approximately 20-30% stearidonic acid (% of total fatty acids) and SDA-enriched soybean oil can be used for the production of margarine, mayonnaise, shortenings, salad dressings, ready-to-eat foods, and other food categories. The petitioner states that MON 87769 is expected to be a low-acreage product planned initially for production in North America. The oil will be used in food applications where omega-3 products are currently being used. The co-product, soybean meal, has been shown to be compositionally comparable to other commodity soybean meal and it will be used in a manner similar to conventional soybean meal in livestock feed.

Monsanto is not currently requesting approval for MON 87769 SDA-enriched oil as livestock feed and has indicated that they will notify the Food Directorate and the Canadian Food Inspection Agency (CFIA) as required by the Food and Drug Regulations and the Feed Regulations, respectively, if they decide to pursue this soybean oil as livestock feed. The CFIA, however, formally requested an opinion from the Food Directorate regarding whether the levels of SDA, gamma linolenic acid (GLA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) that could be present in meat, eggs, and milk (from direct addition of 310 mg SDA to the eligible foods and from addition of 3-5% MON 87769 SDA-enriched oil to livestock feeds) would be of concern to human health.

Using the aforementioned estimated exposure/intakes, the Food Directorate calculated that the projected intakes of EPA and DHA resulting from the consumption of eligible foods, meat, and alternative choices from MON 87769 soybean-fed livestock could exceed the current recommendations of the Food Directorate of 1 g/day but they are unlikely to exceed 3 g/day. The Food Directorate is currently reviewing this upper limit value to determine if an increase is scientifically justified. The expected SDA and GLA intakes are expected to be below 7 g/day and 2 g/day, respectively.

6. Nutrition

The nutritional and anti-nutritional components of Stearidonic Acid (SDA) Producing Soybean MON 87769 were measured and compared with the conventional control variety A3525 and commercially available soybean varieties.

For soybean and forage analysis, MON 87769, A3525, and reference soybean varieties were grown at five locations in the United States: Iowa (2 sites), Illinois, Michigan, and Ohio during the 2006 growing season.

For defatted, toasted soybean meal; refined, bleached, and deodorized soybean (RBD) oil; soy protein isolate, and crude lecithin fractions analysis, MON 87769, A3525, and reference soybean varieties were grown at two locations in the United States (both in Illinois) during the 2006 growing season.

The key nutritional and compositional analytes measured in the MON 87769 and control variety A3525 seed were: protein, fat, carbohydrates, fibre, ash, moisture, amino acids, fatty acids, vitamin E, and anti-nutritional factors (lectin, phytic acid, raffinose, stachyose, trypsin inhibitors, isoflavones (i.e., daidzein, genistein, and glycitein). For the refined oil, the analytes included fatty acids and vitamin E. Only amino acids were measured in the protein isolate. Lecithin was assessed in the crude fractions.

Statistically significant (p<0.05) differences were observed in several analytes in both seed and oil derived from MON 87769 compared to the control variety A3525.

Differences in the seed included carbohydrates (lower in MON 87769), protein (higher), all amino acids (higher) with the exception of tryptophan (not statistically different), palmitic acid (higher), oleic acid (lower), linoleic acid (LA) (lower), alpha-linolenic acid (ALA) (higher), arachidic acid (higher), behenic acid (lower), SDA (not detected in control), GLA (not detected in control), trans-ALA (not detected in control), trans-SDA (not detected in control), and the isoflavones (lower).

Differences in the oil included palmitic acid (higher in MON 87769), stearic acid (higher), lignoceric acid (lower), SDA (not detected in control), GLA (not detected in control), trans-ALA (not detected in control), trans-SDA (not detected in control), and vitamin E (higher).

Differences in the soybean protein isolate included alanine (lower in MON 87769) and leucine (lower).

Differences in the soybean meal included carbohydrates (lower in MON 87769), aspartic acid (higher), glutamic acid (higher), histidine (higher), and tryptophan (higher).

The statistically significant differences between MON 87769 and the conventional control variety A3525 are acceptable for the following reasons: a) the analyte values were within the published ranges for conventional soybean varieties, b) the magnitude of the difference was too small to impact dietary intakes and pose a nutritional safety concern, and c) several of the significant differences in the MON 87769 fatty acid profile could be attributed to the genetic modifications made in this transgenic line.

The omega-6 fatty acids are LA and GLA. The omega-3 fatty acids are ALA and SDA. The omega-6:omega-3 ratio for MON 87769 seed and refined oil (~1:1) are decreased compared to values for canola (~2:1) and refined soybean oils (~7:1). This ratio and the fatty acid profile of MON 87769 seed and refined oil does not pose any nutritional safety concerns.

For MON 87769, the total trans fatty acid levels were approximately 0.83% in the refined oil, which is within the recommended limits of the Health Canada multi-stakeholder Trans Fat Task Force (i.e., trans fat content of less than 2% of total fats for all vegetable oils and soft, spreadable, tub-type margarines).

With regard to the safety of SDA, GLA, EPA, and DHA from the direct addition of 310 mg of SDA to the eligible foods, clinical trials support the nutritional safety of up to 4.2 g of SDA daily (Shawna L Lemke, 2010, William S. Harris, 2008, Michael J James, 2003). The safety of GLA is supported by Health Canada guidance in the Natural Health Products Directorate Borage oil monograph (2009) for a maximum GLA dose of 1350 mg/day, and a systematic review by Cameron M, 2011, reporting no serious adverse effects in people receiving 540 and 2800 mg GLA/day for 6 months to one year. Health Canada guidance (2006) also supports the safety of up to 1 g/day of added EPA and DHA to foods.

With regard to the safety of SDA, GLA, EPA, and DHA from both the direct addition of SDA to the eligible foods and the addition of 3-5% SDA-enriched oil to livestock feeds, SDA intakes could be higher than the dosages used in the clinical trial by Lemke et al. (2010), and reported as safe. However, the lack of adverse effects reported in the clinical trials at dosages ranging from 0.75 g to 4.2 g daily and the undetectable or negligible accumulation of SDA or its intermediate metabolite in erythrocytes or plasma phospholipids suggest that expected exposure levels will not pose any nutritional safety concerns. Following the addition of 3-5% SDA-enriched oil to livestock feeds, the increase in GLA levels per serving is less than 2% (i.e., 0.17% in control fed broiler chickens versus 1.64% in SDA-enriched oil fed broilers). The magnitude of difference is small and suggests that changes in intakes is supported in part by the NHPD monograph (2009) and he review by Cameron M. (2011) which reported that the risk of adverse effects in participants using GLA was not statistically different from those receiving a placebo.

In 2006, the main concern with exceeding 1 g of EPA and DHA combined was evidence of increased bleeding times in some people at high levels of EPA/DHA intake. Since that time, new evidence has supported the safety of higher levels of intake. The FAO/WHO expert consultation (FAO/WHO, 2010) acknowledged that consumption as high as 3 g/day reduced cardiovascular risk factors and was not associated with adverse effects in short and intermediate randomized trials. However, the FAO/WHO recommendation of EPA and DHA is 2 g/day, based on experimental evidence indicating that high supplement intakes may increase lipid peroxidation and reduce cytokine production. Recently, the Norwegian Scientific Committee for Food Safety (VKM, 2011) evaluated the negative and positive health effects of n-3 fatty acids as constituents of food supplements and fortified foods, and reported that an increased bleeding time was observed only after intakes of 6.9 g/day of EPA and DHA combined. The Committee also noted that no negative health complications with regards to bleeding were reported in connection with EPA and DHA supplements. The Committee concluded that it was not possible to identify clear adverse effects from EPA and DHA up to the dosage of 6.9 g/day and that no tolerable upper level intake level could be established. Other potential negative effects from high EPA and DHA supplementation reviewed included reports of increased lipid peroxidation, inflammation and minor increases in LDL-C at doses above 3.5 g/day. The Committee concluded these effects are not established risk factors of disease and that their significance is uncertain and should be further investigated. Overall, it was noted that the intake of EPA and DHA, from both food sources and supplements, could provide positive health effects without any appreciable risk of negative or adverse health effects. As noted above, the Food Directorate is reviewing the current reference safe intake for EPA and DHA combined (i.e., 1 g/day).

The Food Directorate expects that the petitioner would provide detailed information on expected intakes and the safety of SDA, GLA, EPA, and DHA from foods derived from livestock fed SDA-enriched soybean oil should they pursue approval of SDA-enriched soybean oil as livestock feed. Nevertheless, based on the Directorate's very conservative approach to estimating intakes, it is unlikely that levels of SDA, GLA, EPA, and DHA in such foods would present a safety concern. The Food Directorate has no safety concerns regarding the levels of these fatty acids that would occur with the incidental type of exposure through occasional comingling of SDA-enriched soybean commodities with commercial non-SDA-enriched soybean commodities.

7. Chemistry/Toxicology

The MON 87769-produced PjΔ6D and NcΔ15D proteins represent a very small portion of the total protein in mature soybean seed (approximately 0.00043% and 0.00239%, respectively). In addition, desaturase proteins similar in both structure and function to PjΔ6D and NcΔ15D are commonly consumed in the human diet with no reports of adverse health effects associated with exposure to these proteins.

Heat treatment tests designed to simulate the fate of protein after food processing indicated that the activity of PjΔ6D and NcΔ15D present in the final food product after heated to approximately 190 °C for 15 minutes is reduced by at least 84.1% and 80.5%, respectively. The susceptibility of these proteins to denature from heat exposure during food processing greatly reduces the potential exposure to these proteins in food commodities consumed by humans.

Digestive fate experiments conducted with PjΔ6D and NcΔ15D demonstrated that the full-length proteins are rapidly digested in simulated gastric fluid (SGF) and the transiently stable protein fragments in the SGF assay were quickly degraded during the short exposure to simulated intestinal fluid (SIF). This indicates that it is highly unlikely that the PjΔ6D and NcΔ15D proteins or their fragments will reach absorptive cells of the intestinal mucosa, greatly limiting the systemic exposure and reducing the potential for toxic and allergic reactions to intact and biologically active proteins.

The assessment of the potential toxicity of the PjΔ6D and NcΔ15D proteins did not identify any structurally relevant similarities between the PjΔ6D and NcΔ15D proteins and any known toxic proteins. Oral toxicity studies conducted with mice treated with single gavage doses of 4.66 mg/kg bw of PjΔ6D, or of 37.7 mg/kg bw of NcΔ15D did not result in any adverse effects.

The margin of exposure (MOE) between the doses of PjΔ6D and NcΔ15D proteins tested in the acute oral toxicity studies conducted in mice (i.e., 4.7 and 37.7 mg/kg bw, respectively) in which no adverse effects were observed and the estimated human consumption of these proteins range from 700 to 40,000. These MOEs are considered adequate to account for any uncertainties on exposure and effects.

Toxicological studies conducted with SDA-enriched soybean oil produced from MON 87769 did not indicate any adverse health effects in a 28-day rodent study or a 90-day rodent study combined with a one-generation reproduction study at levels up to 600 and 1000 mg SDA/kg bw/day, respectively, or in a 16-week clinical trial in which participants consumed up to 53 mg of SDA/kg bw/day.

The levels of endogenous allergens in Stearidonic Acid (SDA) Producing Soybean MON 87769 are comparable to those in soybean varieties that are currently available on the market.

Conclusion

Health Canada's review of the information presented in support of the food use of Stearidonic Acid (SDA) Producing Soybean MON 87769 does not raise concerns related to food safety. Health Canada is of the opinion that food derived from MON 87769 soybeans is as safe and nutritious as food from current commercial soybean varieties.

SDA is converted in the body to EPA; however, the reported rate of conversion varies from 6:1 to 3.1:1. The Food Directorate previously assessed the reference safe intake of EPA and DHA combined to be 1 g/day.

The Food Directorate's guidance is that food as offered for sale should contain no more than 100 mg added EPA and DHA, combined, from all sources (not including overage for products declaring up to 100 mg on the label) per reference amount (Schedule M of the Food and Drug Regulations) and per serving of stated size to: all foods except for flours, pasta, rice, butter, suet, lard, fresh fruits and vegetables, nuts, seeds, legumes, sugars and non-nutritive sweeteners, sugar syrups, maple syrup, honey and other sweetening agents, alcoholic beverages, seasonings, leaf tea, coffee beans, prepackaged water and ice, herbs, spices, and leavening agents (i.e., baking powder, yeast, etc.).

Based on the evaluation of the submitted information and the considerations above, the Food Directorate has no objection to the addition of SDA-enriched soybean oil up to a maximum of 310 mg SDA per serving of food to the above list of foods, equivalent to approximately 1033 mg of 30% SDA-enriched soybean oil or 1550 mg of 20% SDA-enriched soybean oil per serving of food. The Directorate's recommended maximum addition of SDA is equivalent to 100 mg EPA and DHA combined based on a 3.1:1 SDA:EPA conversion ratio.

SDA-enriched soybean oil is not permitted to be added to a food for which a standard exists in the Food and Drug Regulations unless provision exists or is made in the standard for the source oil as an ingredient. Any interest in making changes to a standard will be addressed separately and would require a regulatory amendment.

Health Canada's opinion deals only with the food use of Stearidonic Acid (SDA) Producing Soybean MON 87769. 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 Stearidonic Acid (SDA) Producing Soybean MON 87769 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 Stearidonic Acid (SDA) Producing Soybean MON 87769 destined for commercial sale.

References

Cameron M, G. J. (2011). Herbal therapy for treating rheumatoid arthritis (Review). The Cochrane Library, 1-93.

FAO/WHO. (2010). Fats and fatty acids in human nutrition. Report of an expert consultation. 10-14 November 2008, Geneva. FAO Food and Nutrition Paper, 169.

Michael J James, V. M. (2003). Metabolism of steridonic acid human subjects: comparison with the metabolism of other n-3 fatty acids. American Journal of Clinical Nutrition, 1140-1145.

Shawna L Lemke, J. L. (2010). Dietary intake of stearidonic acid-enriched soybean oil increases the omega-3 index: randomized, double-blind clinical study of efficacy and safety. The American Journal of Clinical Nutrition, 776-775.

VKM. (2011). Opinion of the Steering Committee of the Norwegian Scientific Committee for Food Safety: Evaluation of the negative and positive effects of n-3 fatty acids as constituents of food supplements and fortified foods. Doc. No.: 08-707-final, 88.

William S. Harris, S. L. (2008). Stearidonic Acid-Enriched Soybean Oil Increased the Omega-3 Index, an Emerging Cardiovascular Risk Marker. Lipids, 805-811.

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
novelfoods-alimentsnouveaux@hc-sc.gc.ca