Novel food information: Insect resistant MON 94637 soybean

On this page

• Background
• Introduction
• Development of the modified plant
• Characterization of the modified plant
• Product information
• Dietary exposure
• Nutrition
• Chemistry
• Toxicology
• Allergenicity
• Conclusion

Background

Health Canada has notified Bayer CropScience Inc., that it has no objection to the food use of insect resistance soybean line MON 94637 (hereafter referred as MON 94637). MON 94637 will not be cultivated nor commercialized in North America. However, grains could be imported into Canada for food usage. The company also indicated that MON 94637 is intended to be conventionally bred with approved soybean varieties to yield products exhibiting additional characteristics, such as herbicides tolerance and improved insect protection.

The Department conducted a comprehensive safety 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 characteristics. The following provides a summary of the notification from Bayer CropScience Inc. and the evaluation by Health Canada. This document contains no confidential business information.

Introduction

Bayer CropScience Inc. has developed a genetically modified (GM) soybean line (Glycine max L. Merr.),MON 94637, that exhibits resistance toward certain lepidopteran pests found in South American countries. The insect resistance trait was achieved through expression of two novel chimeric insecticidal proteins (i.e., Cry1A.2 and Cry1B.2 proteins). Cry1A.2 and Cry1B.2 proteins follow the same general mode of action as other Bacillus thuringiensis insecticidal proteins currently in commercial use for insect crop protection.

The safety assessment performed by the Food and Nutrition 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 insect resistant soybean MON 94637 was developed, how the composition and nutritional safety of this variety compared to its unmodified comparator, and what the potential is for this variety to present a toxic or allergenic concern. Bayer CropScience Inc. has provided data to support that this variety is safe for use as food in Canada.

The Food and Nutrition Directorate has a legislated responsibility for the pre-market assessment of novel foods and novel food ingredients, as detailed in Division 28 of Part B of the Food and Drug Regulations (Novel Foods). Insect resistant soybean MON 94637 is considered to be 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

Development of the Modified Plant

The petitioner provided information to support the safety and long history of use of the host organism, soybean (Glycine max L. Merr.).This organism does not pose a health or safety concern.

MON 94637 was developed by Agrobacterium-mediated transformation with a transformation plasmid PV-GMIR527237 containing two T-DNAs (i.e., T-DNA I and T-DNA II), where each is delineated by right and left border regions.

T-DNA I contains the cry1A.2 and cry1B.2 expression cassettes while T-DNA II contains two selection markers (i.e., the aadA and splA genes). The aadA gene encodes an aminoglycoside modifying enzyme conferring spectinomycin and streptomycin resistance. The splA gene encodes a sucrose phosphorylase, which interferes with the sucrose metabolism during embryo development by converting sucrose into fructose and glucose–1–phosphate resulting in a recognizable wrinkled seed phenotype.

Transformants were selected based on spectinomycin resistance, wrinkled seed phenotype, and the use of sequencing to confirm the absence of plasmid backbone in the host genome and that a single copy of the T-DNA I insert integrated in non-coding, non-repetitive region and was unlinked to the T-DNA II insert.

Subsequent self-pollination steps and conventional breeding practices were used to remove the T-DNA II insert from the MON 94637 genome.

The novel chimeric cry1A.2 and cry1B.2 genes are codon-optimized for expression in soybean and are composed of multiple domains of existing Cry1 proteins derived from B. thuringiensis. Each domain was either synthetically synthesized or PCR-amplified from an existing vector. As a result, no other genes from B. thuringiensis are carried over into the MON 94637 genome. B. thuringiensis has a long history of safe use producing microbially-derived products with insecticidal activity, i.e., Bt applications^{Footnote 1}. The NIH's Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules lists B. thuringiensis as a safe source of extrachromosomal elements obtained from gram-positive organisms^{Footnote 2}. Further, B. thuringiensis is not listed as a risk group 2 or higher pathogen in the PHAC's ePATHogen database.

The petitioner provided information to support the safety and historical use of each donor organism and the recipient organism (i.e., the elite in-bred A3555 soybean line). None of these organisms poses a health or safety concern.

Characterisation of the Modified Plant

Next Generation Sequencing (NGS) of five generations (R3 to R7) of MON 94637was used to determine the insert copy number of T-DNA I and characterize the junction sequences. Additionally, sequencing of specific PCR amplicons from the inserted DNA was used to determine the T-DNA I insert intactness as well as integration site and flanking genomic sequences. The molecular characterization confirmed that the T-DNA I insert integrated as expected in the MON 94637 genome at a unique location in chromosome 19 without disrupting endogenous genetic elements. NGS of the same five generations of MON 94637 also confirmed the absence of plasmid backbone sequence and of T-DNA II.

With regards to the NGS quality metrics, the company reported a minimal coverage depth of 75 X for each sequenced plant, which has been reported to be adequate for providing comprehensive coverage and detection of all inserted DNA as well as potential unintended inserted fragments. The native le1 gene^{Footnote 3}, which is present in a single copy locus in soybean, was used by the petitioner to estimate the depth coverage in both the conventional (A3555) control and MON 94637. The company also reported complete genome coverage. The coverage breadth was determined by identifying all single native copy genes throughout the soybean reference genome before randomly selecting two of those per chromosome. Then, the company fully mapped these two random single copy soybean genes from each soybean chromosomes in both conventional control and MON 94637 (R3 generation) providing a proper indication of the coverage breadth.

The integrated T-DNA I insert exhibits Right and Left Border termini deletions and a 14-bp deletion is observed in the 5' flanking region of the T-DNA I insertion site. These deletions often occur and are attributed to double-stranded break repair mechanisms in the plant during the Agrobacterium-mediated transformation process. The petitioner's open-reading frames (ORF) analysis demonstrates that these deletions do not result in any putative ORFs with similarity to known toxins or allergens.

Bioinformatics analyses of putative ORFs (defined as any sequence from stop-to-stop codons) in all six reading frames were performed on both the sequenced T-DNA I insertion site and junction sequences for similarity to known and putative allergens and toxins. Putative sequenced proteins were searched against the AD_2022 allergen database, the Uniprot toxin protein database (TOX_2022), and the GenBank protein database (PRT_2022). No biologically relevant alignments were found for the T-DNA I insert and junction sequences within the MON 94637 genome.

Genetic stability of the T-DNA I insert in the MON 94637 genome was demonstrated by assessing individual MON 94637 plants from five generations (R3 to R7) by means of NGS. The results indicate that the T-DNA I insert is intact and stable over five generations of MON 94637. PCR analysis on three generations (F2, F3 and F4) also demonstrated that T-DNA I segregated in accordance with the Mendelian principles of inheritance for a single genetic locus.

Product Information

MON 94637 differs from its traditional counterparts by the expression of Cry1A.2 and Cry1B.2 insecticidal proteins. The insecticidal protein levels were determined in grains at maturity (R8 growth stage) by means of validated enzyme-linked immunosorbent assay (ELISA) through a multi-site field trial using a randomized complete block (RCB) design. Five field trial sites located in the U.S. were used with four blocks at each site, resulting in a total of 20 samples per tissue tested for each novel protein.

At maturity, mean (±SE) Cry1A.2 expression levels in grain were 21 (±2.0) µg/g fresh weight and 24 (±2.2) µg/g dry weight. At maturity, mean (±SE) Cry1B.2 expression levels in grain were 11 (±0.69) µg/g fresh weight and 12 (±0.75) µg/g dry weight.

The chimeric, full-length, protoxin Cry1A.2 protein encoded by the cry1A.2 gene is 1189 amino acids in length and has a molecular weight of approximatively 135 kDa. This protein is composed of four domains identical to their B. thuringiensis (Bt) wildtype sequence: domain I originates from the Bt Cry1Ah protein, domains II and IV originate from the Bt Cry1Ac protein, and domain III originates from the Bt Cry1Ca protein.

The chimeric, full-length, protoxin Cry1B.2 full-length protein encoded by the cry1B.2 gene is 1187 amino acids in length and has a molecular weight of approximatively 135 kDa. This protein is composed of four domains identical to their B. thuringiensis (Bt) wildtype sequence: domains I and II originate from the Bt Cry1Be protein, domain III originates from the Bt Cry1Ka2 protein, and domain IV originates from the Bt Cry1Ab protein.

To characterize both Cry1A.2 and Cry1B.2 proteins, the petitioner performed SDS-PAGE, western blot, protein glycosylation, N-terminal sequence/mass fingerprint analysis using LC-MS/MS, an insect bioassay, and a storage stability assay. The provided protein characterization data confirmed the identity and functionality of both MON 94637-produced Cry1A.2 and Cry1B.2 proteins and demonstrated that they are not glycosylated. The provided data also revealed that MON 94637 expresses a version of the notified Cry1B.2 protein that is missing the N-terminal methionine residue likely due to the activity of native methionine aminopeptidase or other aminopeptidases. Cleavage of the N-terminal methionine residue is common in many organisms, does not affect the protein function, and does not pose a safety concerns.

The Cry1A.2 and Cry1B.2 proteins used to perform toxicological studies were sourced from B. thuringiensis expression systems as opposed to being sourced from MON 94637. Equivalence between MON 94637-produced proteins and their microbial counterparts was established using several analysis including: SDS-PAGE, western blot, protein glycosylation, N-terminal sequence/mass fingerprint analysis using LC-MS/MS, and an insect bioassay. The results of these analyses concludes that the microbially-produced proteins are equivalent to their full-length plant-produced counterparts in MON 94637 and therefore can be used as test articles for protein safety studies.

Based on the information provided, there are no concerns regarding the food use of MON 94637 soybean from a molecular perspective.

Dietary Exposure

It is expected that MON 94637 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 94637. Food and Nutrition Directorate evaluators agree with this statement.

Nutrition

Compositional data for genetically modified soybean MON 94637 and genetically similar conventional control A3555 were obtained from grain samples harvested from five field trials conducted in the US during the 2021 growing season. Each field trial was planted with test and control soybean in a randomized complete block design with four replicates.

Grain samples were analyzed, using acceptable methods, for proximates and fibres, amino acids, fatty acids, vitamins, minerals and anti-nutrients as suggested by the Organization for Economic Co-operation and Development consensus document on compositional considerations for new varieties of soybean.

The petitioner conducted statistical analyses comparing the composition of the modified soybean with its conventional control. Where a statistically significant difference (P-value < 0.05) was identified, the nutritional relevance of the difference was determined through comparison to the natural variability defined by the range of values reported in the scientific literature and the Agriculture and Food Systems Institute Crop Composition Database.

Three components (palmitoleic acid, heptadecanoic acid and behenic acid) showed a statistically significant (p<0.05) difference between MON 94637 and the conventional control. In all cases, the mean composition of MON 94637 was within the range of values reported for conventional soybean.

The petitioner has demonstrated that MON 94637 soybean has similar nutritional composition to its control. Based on the information provided, no nutritional concerns were found with the sale of foods derived from MON 94637 soybean in Canada.

Chemistry

Chemical contaminant residue data have not been provided, nor have any unique contaminant considerations been identified with respect to MON 94637 soybean. As well, there are no maximum levels for contaminants specific to this food set out in Health Canada's List of Contaminants and Other Adulterating Substances in Foods and the List of Maximum Levels for Chemical Contaminants in Foods.

As with any food or food ingredient sold in Canada, it is the responsibility of the food manufacturer to ensure that its use does not result in a violation of Section 4(1)(a) and (d) of the Food and Drugs Act, which states that no person shall sell an article of food that has in or on it any poisonous or harmful substance or is adulterated. If an elevated concentration of any chemical contaminant is found in any type of food, the Bureau of Chemical Safety (BCS) may conduct a human health risk assessment to determine if there is a potential safety concern and whether risk management measures are required.

Based on the information provided, MON 94637 soybean would not be expected to pose a concern from a chemical contaminant perspective.

Toxicology

The toxic mode of action of Cry proteins towards certain insects would not be expected to occur in humans due to differences between species in the digestive tract (e.g. receptors are not present in humans and the more acidic environment of the mammalian gut leads to degradation of Cry proteins.

Both tested Cry1A.2 and Cry1B.2 proteins were expressed in B. thuringiensis before being purified and used as test articles. However, it has been concluded above (see "Product Information" section) that microbially-produced Cry1A.2 and Cry1B.2 proteins are equivalent to their MON 94637-produced counterparts. Therefore, these assay results can be applied to MON 94637 soybean.

The novel proteins (Cry1A.2 and Cry1B.2) were not toxic in acute oral rodent toxicity tests at 5000 mg/kg body weight (bw). Mice (10/sex/group) were exposed to 0 or 5000 mg/kg Cry1A.2 or Cry1B.2 by oral gavage. The studies followed EPA Health Effects Test Guideline OPPTS 870.1100 and Good Laboratory Practice (GLP). No unscheduled deaths, test substance-related clinical observations, necropsy findings or statistical differences in body weight, body weight gains or food consumption were observed in the Cry1A.2 group or the Cry1B.2 group compared to vehicle controls.

Comparison of the highest estimated exposures to Cry1A.2 (37.8 ug/kg bw per day) or Cry1B.2 (19.8 µg/kg bw per day) to their corresponding acute toxicity values (LD50 >5000 mg/kg bw per day) results in margins of >130 000 or >250 000, respectively. These margins are considered sufficient from a safety perspective.

Based on the available data, from a toxicological perspective, MON 94637 soybean is considered as safe as conventional soybean currently available on the Canadian market

Allergenicity

Soy is a priority food allergen in Canada. No significant differences in endogenous allergens between MON 94637 soybean and conventional soybean would be expected based on the intended genetic modifications.

No statistically significant differences between MON 94637 and conventional soybean were identified in the concentrations of 6 clinically relevant endogenous soy allergens (Gly m4, Gly m5, Gly m6, Gly m Bd 28 K, Gly m Bd 30K^{Footnote 4} and KTI).

The gene donor(B. thuringiensis) occurs ubiquitously in the environment and is not commonly known for human allergenicity.

The novel proteins (Cry1A.2 and Cry1B.2) do not share characteristics (i.e., stability in heat and digestion assays) associated with some known food allergens. Cry1A.2 and Cry1B.2 were inactivated after 15 minutes at 75 degrees Celsius. In simulated gastric (stomach) fluid, these proteins were degraded within 0.5 minutes, with only small peptides (4 kDa) remaining. When the simulated gastric fluid (2 minutes) was followed by simulated intestinal fluid, the peptide fragments were degraded within 0.5 minutes.

The amino acid sequences of Cry1A.2 and Cry1B.2 were compared to known or suspected allergens in the Petitioner's in-house allergen, gliadin and glutenin protein sequence database (AD_2022, 2463 sequences). No amino acid homology matches, consistent with international guidance^{Footnote 5} were identified between these novel proteins and known allergens.

Based on the available data, from an allergenicity perspective, MON 94637 soybean is considered as safe as conventional soybean currently available on the Canadian market.

Conclusion

Health Canada's review of the information presented in support of the use of insect resistant soybean variety MON 94637 does not raise concerns related to food safety.

Health Canada's opinion refers only to the food use of MON 94637 soybean. 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 and Nutrition 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:

Health Canada
Novel Food Section
Food and Nutrition Directorate
Health Products and Food Branch
251 Sir Frederick Banting Driveway
PL2204E
Ottawa, Ontario, K1A 0K9
Email: bmh-bdm@hc-sc.gc.ca