Novel food information: Insect Protected Soy – MON 87751

Health Canada has notified Monsanto Canada Inc. that it has no objection to the food use of Insect Protected Soy – MON 87751. The Department conducted a comprehensive assessment of this soybean 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.

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 Insect Protected Soy – MON 87751 using recombinant DNA techniques to introduce two coding sequences: cry1A.105 and cry2Ab2, which encode the Cry1A.105 and Cry2Ab2 proteins, respectively. Both proteins are insecticidal ð-endotoxins that bind to specific cadherin receptors on the midgut epithelium of targeted lepidopteran insects. Both the cry1A.105 and cry2Ab2 coding sequences are derived from the common soil bacterium Bacillus thuringiensis, subspecies kurstaki.

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 87751 was developed; how the composition and nutritional quality of MON 87751 compared to non-modified varieties; and the potential for MON 87751 soybeans to be toxic or cause allergic reactions. Monsanto Canada Inc. has provided data that demonstrates that MON 87751 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 Insect Protected Soy – MON 87751 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 Insect Protected Soy – MON 87751 and the molecular biology data that characterize the genetic change, which results in a resistance to specific lepidopteran pests. This phenotype was achieved by transformation of the conventional soybean variety A3555 with a transgenic expression cassette containing the novel cry1A.105 and cry2Ab2 genes and their associated regulatory elements.

Insect Protected Soy – MON 87751 was genetically modified using Agrobacterium-mediated transformation of commercial soybean variety A3555 with the the plasmid PV-GMIR13196. The plasmid PV-GMIR13196 carried two separate transfer DNA (T-DNA I and T-DNA II) sequences, each comprised of an expression cassette, one containing the cry1A.105 and cry2Ab2 coding sequences (T-DNA I) and one containing the aadA and splA coding sequences (T-DNA II). The aadA sequence encodes an aminoglycoside-modifying enzyme that confers spectinomycin and streptomycin resistance and allows for selection of successfully transformed tissue. The splA sequence encodes the sucrose phosphorylase protein. When the protein is expressed during embryo development, it interferes with sucrose metabolism, leading to a shrunken appearance. Seeds without splA appear normal, thus allowing a visual demonstration that T-DNA II is absent from the plant genome.

The cry1A.105 and cry2Ab2 expression cassette contains the following genetic elements: the promoter, leader and intron sequences from the act2 gene of Arabidopsis thaliana that directs transcription in plant cells; the CTP2 targeting sequence of the ShkG gene from A. thaliana encoding the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) transit peptide region that directs transport of the protein to the chloroplast; the cry2Ab2 coding sequence encoding the Cry2Ab2 protein that provides insect resistance; the 3′ untranslated region (UTR) sequence from the Oryza sativa (rice) Mt gene encoding a metallothionein-like protein that directs polyadenylation of the mRNA; the promoter, leader, and targeting sequences from the A. thaliana rbcS gene family encoding small subunit ats1A that directs transcription in plant cells and transport of the protein to the chloroplast; the cry1A.105 coding sequence encoding the Cry1A.105 protein that provides insect resistance; and the 3′ UTR sequence from the Medicago truncatula (alfalfa) PT1 gene encoding a phosphate transporter that directs polyadenylation of the mRNA. The cry1A.105 coding sequence encodes a single synthetic, chimeric protein, Cry1A.105, comprised of various domains from the Cry1Ab, Cry1Ac, and Cry1F proteins of B. thuringiensis, subspecies kurstaki. The cry2Ab2 coding sequence encodes a single protein, Cry2Ab2.

The aadA and splA expression cassette contained the following genetic elements: the enhancer sequence from the 35S RNA of Figwort Mosaic Virus (FMV) that enhances transcription in most plant cells; the promoter, leader, and intron sequences of the EF-1 gene from A. thaliana encoding elongation factor EF-1α that directs transcription in plant cells; the targeting sequence of the ShkG gene from A. thaliana encoding the EPSPS transit peptide region that directs transport of the protein to the chloroplast; the bacterial aadA coding sequence for an aminoglycoside-modifying enzyme, 3"(9)-O-nucleotidyltransferase from the transposon Tn7, that confers spectinomycin and streptomycin resistance; the 3′ UTR sequence from Pisum sativum (pea) rbcS gene family encoding the small subunit of ribulose bisphosphate carboxylase protein that directs polyadenylation of the mRNA; the 5′ UTR leader, promoter, and enhancer sequence from Vicia faba (broad bean) encoding a seed protein that directs transcription in plant cells; the splA coding sequence encoding the sucrose phosphorylase protein that catalyzes the conversion of sucrose to fructose and glucose-1-phosphate; and the 3′ UTR sequence of the nopaline synthase (nos) gene from Agrobacterium tumefaciens pTi encoding NOS that directs polyadenylation of the mRNA. Using traditional methods of breeding, the T-DNA II transfer DNA was subsequently removed from the soybean genome, producing the MON 87751 genetic event containing only the cry1A.105 and cry2Ab2 expression cassette (T-DNA I).

3. Characterization of the Modified Plant

Southern blot analysis and DNA sequencing of Insect Protected Soy – MON 87751 demonstrated the presence of a single copy of the cry1A.105 and cry2Ab2 expression cassette in the soybean genome at a single locus. Southern blot analysis confirmed the absence of any plasmid backbone DNA in Insect Protected Soy – MON 87751.

The stability of the inserted cry1A.105 and cry2Ab2 expression cassette was evaluated from the progeny of five different generations. The results of Southern blot analysis and segregation data demonstrated the stability of Insect Protected Soy – MON 87751 at the genomic level.

4. Product Information

Insect Protected Soy – MON 87751 differs from its traditional counterpart by the addition of the cry1A.105 and cry2Ab2 genes and their associated regulatory elements. The insertion of these gene results in the tissue-targeted expression of the novel Cry1A.105 and Cry2Ab2 proteins in MON 87751. Tissue-specific expression of these two insecticidal proteins confers resistance to specific lepidopteran insects.

The coding sequence, cry1A.105, encodes a single synthetic, chimeric protein, Cry1A.105, comprised of various domains from the Cry1Ab, Cry1Ac, and Cry1F proteins of B. thuringiensis, subspecies kurstaki. The cry2Ab2 coding sequence encodes a single protein, Cry2Ab2, also derived from B. thuringiensis, subspecies kurstaki.

B. thuringiensis is a Gram-positive, soil-dwelling bacterium that has not been reported to be a source of known allergens. Sprays of sporulated B. thuringiensis have been used commercially since 1958 to produce microbial-derived products with insecticidal activity. The extremely low toxicity of B. thuringiensis-based insecticide products has been demonstrated in numerous safety studies, and there are no confirmed cases of allergic reactions to Cry proteins in applicators of microbial-derived B. thuringiensis-based products during 50 years of use.

The petitioner has provided data to demonstrate the level of expression of the Cry1A.105 and Cry2Ab2 proteins in MON 87751. This study used plant samples from five field trial locations conducted in the United States during the 2012 growing season: Arkansas, Iowa, Kansas, North Carolina, and Pennsylvania. 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, four replicated plots of MON 87751 were planted using a randomized complete block field design. Tissues of MON 87751 that were collected include: over season leaf (OSL) 1-4, root, seed, forage, and pollen/anther tissue. Flowers for the extraction of pollen/anther tissue of MON 87751 were collected at one U.S. growing location (Illinois). This site was planted in a non-randomized manner. The quantities of Cry1A.105 and Cry2Ab2 proteins were determined by enzyme-linked immunosorbent assay (ELISA). The across-site means, standard deviations (SDs), and ranges for Cry1A.105 and Cry2Ab2 protein levels in soybean OSL 1-4, root, forage, and seed tissues were reported on a µg/g dry weight (dwt) basis. The Cry1A.105 and Cry2Ab2 protein levels in soybean pollen/anther tissue were reported on a µg/g fresh weight (fwt) basis.

Cry1A.105 was reported at the following levels in the following tissues: OSL1 (580±250 µg/g dwt, range 260-1100 µg/g dwt); OSL2 (590±270 µg/g dwt, range 68-1100 µg/g dwt); OSL3 (400±220 µg/g dwt, range 50-780 µg/g dwt); OSL4 (790±280 µg/g dwt, range 430-1600 µg/g dwt); root (<LOD, LOD = 0.563 µg/0.322 fwt); forage (230±91 µg/g dwt, range 110-440 µg/g dwt); and seed (2.4±0.50 µg/g dwt, range 1.7-3.2 µg/g dwt). The mean level of Cry1A.105 protein in pollen/anther tissue was observed to be 11 µg/g fwt however the SD and range could not be determined due to an insufficient amount of tissue. The mean Cry1A.105 protein level in MON 87751 across all sites was highest in OSL4 at 790 µg/g dwt and lowest in root which was below the limit of detection.

Cry2Ab2 was observed at the following levels in the following tissues: OSL1 (24±5.9 µg/g dwt, range 17-37 µg/g dwt); OSL2 (26±3.1 µg/g dwt, range 20-33 µg/g dwt); OSL3 (32±5.2 µ/g dwt, range 25-43 µg/g dwt); OSL4 (24±2.7 µg/g dwt, range 18-29 µg/g dwt); root (15±2.7 µg/g dwt, range 11-22 µg/g dwt); forage (14±2.2 µg/g dwt, range 11-18 µg/g dwt); and seed (4.0±0.77 µg/g dwt, range 2.6-5.1 µg/g dwt). The mean level of Cry2Ab2 protein in pollen/anther tissue was observed to be 7.7 µg/g fwt however the SD and range could not be determined due to an insufficient amount of tissue. The mean Cry2Ab2 protein level in MON 87751 across all sites was highest in OSL3 at 32 µg/g dwt and lowest in seed at 4.0 µg/g dwt.

5. Dietary Exposure

It is expected that Insect Protected Soy – MON 87751 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 MON 87751. The dietary exposure estimate for the subpopulation with the greatest consumption on a body weight basis (infants ≤1 year old) was calculated based on the 95th percentile single day intake for eaters-only. A worst case exposure scenario showed that the dietary exposure estimate for proteins Cry1A.105 and Cry2Ab2 were 30.7 µg/kg body weight (bw)/day and 52.7 µg/kg bw/day, respectively. Compared to the NOAELs of the acute oral toxicity studies (see Chemistry/Toxicity below) these figures yielded margins of safety greater than 6.7×10⁵ or 4.1×10⁵, respectively, which are considered sufficient to protect the public.

6. Nutrition

The nutritional and anti-nutritional components of Insect Protected Soy – MON 87751 were measured and compared with the conventional control variety A3555 and commercially available soybean varieties.

For soybean compositional analysis, MON 87751, A3555, and 19 reference soybean varieties were grown at eight locations in the United States: Arkansas, Iowa (2 sites), Illinois, Kansas, North Carolina, Nebraska, and Pennsylvania during the 2012 growing season. These areas were representative of the major growing areas for soybean. All plants were grown under normal agronomic field conditions for their respective geographic regions. Further, within any given individual site, these agronomic treatments were performed uniformly across all plots (test, control, and references).

The key nutritional and compositional analytes measured in the MON 87751 and control variety A3555 seed were: proximate content (protein, fat, ash, moisture, carbohydrate), fiber (acid detergent fiber, neutral detergent fiber), minerals (calcium, phosphorus), 18 amino acids, 22 fatty acids, vitamins [E (-tocopherol) and K (phylloquinone)], anti-nutrients (lectin, phytic acid, trypsin inhibitors), oligosaccharides (raffinose, stachyose), and isoflavones (total daidzein equivalent, total genistein equivalent, total glycitein equivalent). The key nutrients and anti-nutrients were selected according to the Revised Consensus Document on Compositional Considerations for New Varieties of Soybean [Glycine max (L.) merr.]: Key Food and Feed Nutrients, Antinutrients, Toxicants and Allergens (2012).

No statistically significant differences were observed in the combined site analysis between MON 87751 and the conventional control for 42 of 50 components.

The combined-site analysis showed a statistically significant overall treatment effect in 8 of the 50 components. These included: protein (mean increase of 1.15%), proline (mean increase of 1.97%), glycine (mean increase of 1.17%), raffinose (mean decrease of 7.37%), phosphorus (mean increase of 1.89%), vitamin E (mean decrease of 6.83%) for soybeans; and for the forage, total fat (mean decrease of 6.22%) and neutral detergent fiber (mean increase of 7.89%). All combined-site mean values of MON 87751 were within the 99% tolerance intervals established from the conventional commercial reference varieties grown in the same trial. Furthermore, all combined-site mean values and ranges of MON 87751 for all nutrient components, including those that were significantly different, were within the context of natural variability of commercial soybean composition published in the Organization for Economic Co-operation and Development (OECD) document, scientific literature, and/or available in the International Life Sciences Institute (ILSI) Crop Composition Database.

The petitioner has sufficiently demonstrated that Insect Protected Soy – MON 87751 soybeans have similar composition compared to their non-transgenic control and therefore would not pose an increased nutritional risk to consumers.

7. Chemistry/Toxicology

The petitioner provided scientific rationales and bioinformatics data which were considered acceptable for the purpose of the safety assessment. The introduced proteins have been previously assessed by the Food Directorate in support of the transgenic maize variety, MON 89034, and were determined to not pose a toxicological hazard to consumers.

Bioinformatics analysis of the DNA insert and flanking regions was provided by the petitioner. Putative amino acid sequences were compared to available FAARP protein (Genbank release 193) and toxin (TOX_2013) databases using the FASTA alignment tool (v3.4). No statistically significant or toxicologically relevant alignments were detected based on the search criteria and therefore any protein made would not be expected to pose a toxic risk.

In place of acute oral toxicity studies for Cry1A.105 and Cry2Ab2, an acceptable scientific rationale was provided. Escherichia coli-derived Cry1A.105 and Cry2Ab2 proteins previously assessed as part of MON 89034 corn were considered equivalent to the proteins in this submission. Cry1A.105 and Cry2Ab2 proteins from MON 87751 have >99% and >98% amino acid identity, respectively, when compared to these proteins found in MON 89034. Cry1A.105 expressed in MON 87751 was found to have 4 additional amino acids at its N-terminus, while Cry2Ab2 has 18 fewer amino acids at its N-terminus, compared to MON 89034. However, sequence identity was 100% in the protease-resistant core domains which confer the insecticidal properties. These E. coli-derived proteins were previously assessed in acute oral toxicity studies in CD-1 mice and, at the highest doses tested, demonstrated no observed adverse effect levels (NOAELs) for Cry1A.105 and Cry2Ab2 equal to or greater than 2072 or 2198 mg/kg bw, respectively.

The petitioner also provided an additional acute oral toxicity study in CD-1 mice for E. coli-derived Cry2Ab2. Groups of mice (10/sex/group) were administered Cry2Ab2 at a dose of 2000 mg/kg bw by gavage in a 1mM carbonate/bicarbonate buffer (pH 10.15-10.30). Gavage was performed in 2 doses over a 4 hour period. Control animals received an equivalent dose of bovine serum albumin (BSA), as a control protein. Treatment with Cry2Ab2 was associated with mortality (3/10 males and 4/10 females) and clinical signs of toxicity (5/10 males and 5/10 females) within one day following dosing. In response to the unexpected result the authors of the study conducted an investigation to determine the reason for the anomalous study outcome. A dose formulation analysis indicated a lipopolysaccharide (LPS) concentration of 7.0×10⁵ EU (endotoxin unit)/mL (equivalent to 1.4×10⁶ EU per animal) and Na+ concentrations and ion conductivity 2-fold higher in the treatment dose solution relative to the control dose solution. The study authors hypothesized that toxicity may be associated with elevated LPS concentration and/or solution conductivity.

A follow-up study assessed whether mortality in the main study may be due to deviations in the dosing solutions (i.e. elevated levels of LPS and high conductivity and sodium ion concentration relative to control solution). Groups of female CD-1 mice were administered one of 6 oral dose formulations containing varying solute concentrations at neutral or high pH, with or without high dose LPS (8.0×10⁵ to 1.6×10⁷ EU per animal) and with or without BSA. No Cry2Ab2 protein was administered. Mortality was reported in two animals (2/5 mice) receiving a high pH and conductivity formulation containing BSA following dose administration and one animal (1/10 mice) receiving a high pH and conductivity solution was moribund and was euthanized. No signs of clinical toxicity were observed in any other mice. An additional group was administered approximately 3×10⁵ EU/animal as an intravenous injection. A slight decrease in body weight was observed however there was no mortality.

The study authors concluded that mortality observed in the main study was due to a combination of high LPS, protein, and vehicle formulation. These conditions were not typical of a dosing solution and may have interacted in an unusual manner. A more typical dosing solution, used in the acute oral toxicity assay for MON 89034 and in the second study (see paragraph 40) does not result in clinical toxicity or mortality. However, the opinion of the Scientific Services Division of the Bureau of Chemical Safety (BCS) was that the results of the main study would be considered as consistent with technical error (e.g., contaminated dosing equipment, error in preparation of the dosing solution) rather than a true adverse effect related to the administered protein.

In a second study also in CD-1 mice (10 mice/sex/group), a dose of 2000 mg/kg bw of E. coli-derived Cry2Ab2 protein in a vehicle (2 mM carbonate/bicarbonate, pH 10.15-10.30) with a low LPS concentration and low conductivity was administered in an identical manner as the main study. The E. coli-derived Cry2Ab2 protein was equivalent to that used in the first acute study and to protein derived from MON 87751. Dosing with Cry2Ab2 did not result in clinical signs of toxicity or mortality. A control group showed a similar response. Based on the lack of mortality or adverse effects in this study, and the acute oral toxicity study previously assessed in support of NF-156, the PTAS has concluded that the appropriate NOAEL for Cry2Ab2 is considered to be equal to or greater than 2198 mg/kg bw.

Cry1A.105 and Cry2Ab2 were shown to lack significant amino acid homology with known proteins or toxins, have a low acute oral toxicity, and are rapidly digested in simulated gastric fluid (see paragraph 45). In addition, these proteins are expected to have a very low presence in the diet. Both proteins were present in a previously assessed product, MON 89034 maize, which was considered safe to consume.

Based on the weight of evidence, it was concluded that MON 87751 is as safe for consumption as conventional soybean varieties.

Conclusion:

Health Canada’s review of the information presented in support of the food use of Insect Protected Soy – MON 87751 does not raise concerns related to food safety. Health Canada is of the opinion that food derived from MON 87751 soybeans is as safe and nutritious as food from current commercial soybean varieties.

Health Canada's opinion deals only with the food use of Insect Protected Soy – MON 87751. 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 Insect Protected Soy – MON 87751 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 Insect Protected Soy – MON 87751 destined for commercial sale.