Novel food information: Insect-resistant soybean – COR23134

Background

Health Canada has notified Pioneer Hi-Bred Canada Company (Pioneer) that it has no objection to the food use of insect resistant soybean – COR23134. Health Canada 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.

The safety of COR23134 soybean for food use was assessed as part of the Health Canada-Food Standards Australia New Zealand (FSANZ) shared assessment process. For this product, Health Canada was the primary assessor and conducted the safety assessment of this soybean line. FSANZ acted as the secondary assessor, peer-reviewing Health Canada's initial assessment report for COR23134 soybean and providing feedback regarding the report.

The following provides a summary of the original notification from Pioneer and the evaluation by Health Canada and contains no confidential business information.

Introduction

A novel food submission was received from Pioneer Hi-Bred Canada Company (hereafter Pioneer) on March 5, 2024, regarding the acceptability for food use of genetically modified (GM) soybean (Glycine max) event COR23134 which exhibits resistance to lepidopteran insect pests and tolerance to acetolactate synthase (ALS)-inhibiting herbicides. To achieve these traits, 4 gene expression cassettes were introduced to the soybean genome by Agrobacterium-mediated transformation.

Soybean event COR23134 is resistant to lepidopteran insect pests. Soybean event COR23134 expresses 3 novel insecticidal proteins: Cry1B.34.1 and Cry1B.61.1, both derived from Bacillus thuringiensis, and IPD083Cb, derived from giant maidenhair fern (Adiantum trapeziforme var. braziliense). The Cry1B.34.1 protein is a truncated form of the full-length Cry1B.34 protein expressed in corn event DP910521 which was also developed by Pioneer and was previously assessed by Health Canada.

Soybean event COR23134 is resistant to ALS-inhibiting herbicides. This trait is the result of the expression of a modified soybean ALS protein (GM-HRA_1). The amino acid sequence of the mature GM-HRA_1 protein is identical to the mature GM-HRA protein which is expressed in soybean events DP305423 and DP356043 which were also developed by Pioneer and previously assessed by Health Canada.

Soybean event COR23134 was assessed under the Health Canada and Food Standards Australia New Zealand (FSANZ)'s Safety Assessment Sharing Initiative. For this assessment, Health Canada was the primary assessor and the safety assessment was conducted according to Health Canada's Guidelines for the Safety Assessment of Novel Foods. FSANZ evaluators peer reviewed the assessment conducted by Health Canada. The assessment considered: how soybean event COR23134 was developed, how the composition and nutritional safety of this variety compared to its conventional comparator, and the potential for this variety to present a toxic or allergenic concern. Pioneer 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 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). Foods derived from soybean event COR23134 are considered novel foods 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

the plant, animal or microorganism exhibits characteristics that were not previously observed in that plant, animal or microorganism."

Development of the modified plant

Soybean event COR23134 was created through Agrobacterium-mediated transformation of immature cotyledons from soybean variety 93Y21 with plasmid PHP90315. The T-DNA region of the plasmid was integrated into the soybean genome and contained 4 gene expression cassettes which confer to soybean event COR23134 resistance to lepidopteran insect pests and tolerance to ALS-inhibiting herbicides.

The T-DNA insert in soybean event COR23134 expresses 3 novel proteins which confer insect resistance: Cry1B.34.1 and Cry1B.61.1 which were derived from Bacillus thuringiensis and the IPD083Cb protein which was derived from giant maidenhair fern (Adiantum trapeziforme var. braziliense). These proteins target lepidopteran pests of soybean such as velvetbean caterpillar (Anticarsia gemmatalis), soybean looper (Chrysodeixis includens), and fall armyworm (Spodoptera frugiperda). The Cry1B.34.1 and Cry1B.61.1 proteins are encoded by the cry1B.34.1 and cry1B.61.1 genes, respectively, and function in a manner similar to other previously assessed Cry proteins through binding to specific receptors in the insect digestive system, causing disruption of the midgut epithelial cells and leading to insect death. The Cry1B.34.1 protein is a truncated form of the full-length Cry1B.34 protein expressed in corn event DP910521 which was also developed by Pioneer and was previously assessed by Health Canada. The IPD083Cb protein is encoded by the ipd083Cb gene and functions through disrupting midgut epithelial cells of lepidopteran species, leading to insect death.

The T-DNA insert in soybean event COR23134 contains a gm-hra_1 gene which encodes a soybean modified acetolactate synthase (GM-HRA_1) protein. Expression of the GM-HRA_1 protein confers tolerance to ALS-inhibiting herbicides and allows for selection of successful transformants with chlorsulfuron. The GM-HRA_1 protein expressed in soybean event COR23134 has only 2 amino acid substitutions compared to the native soybean ALS protein. The amino acid sequence of the mature GM-HRA_1 protein is identical to the mature GM-HRA protein which is expressed in soybean events DP305423 and DP356043 that were also developed by Pioneer and were previously assessed by Health Canada. The gm-hra_1 sequence lacks the following features which are present in the gm-hra sequence: 15 additional nucleotides in the 5' untranslated region and 5 additional amino acids at the N-terminus of GM-HRA within the chloroplast transit peptide (CTP). Following cleavage of the CTP, the mature protein sequences of GM-HRA_1 and GM-HRA are identical.

The petitioner provided information to support the safety and historical use of the source organisms: B. thuringiensis, A. trapeziforme var. braziliense, and G. max. Based on the information provided, it was demonstrated that none of the source organisms are pathogenic to mammals.

Characterization of the modified plant

The molecular characterization of soybean event COR23134 was conducted through a combination of Southern-by-Sequencing (SbS) and Sanger sequencing. SbS is a molecular characterization method that combines probe-based capture techniques with next-generation sequencing (NGS) and was used to determine the insert copy number, organisation of the inserted DNA, and to confirm the absence of any unintended plasmid sequences. Genomic DNA from plants from soybean event COR23134 as well as the non-genetically modified (GM) control soybean line 93Y21 was analysed by SbS. Plasmid DNA from PHP90315 was spiked into genomic DNA from the non-GM soybean control line to be used as the positive control. The sequencing reads obtained by SbS were compared to the intended T-DNA insertion sequence, the PHP90315 plasmid sequence, and to a soybean reference genome to identify unique junctions attributable to inserted DNA. Sanger sequencing was performed on genomic DNA from soybean event COR23134 as well as the non-GM control soybean line 93Y21.

The results from the SbS analysis and Sanger sequencing indicated that a single copy of the intended insertion, with the intended organization, was integrated into the genome of soybean event COR23134 with the exception of three small deletions. A 21-bp deletion was identified at position 21,740 in the pv-ubi2 promoter of the ipd083Cb cassette compared to the intended T-DNA insertion sequence. As this deletion is in the gene promoter, it may affect the gene expression levels but it is considered unlikely to affect the food safety of soybean event COR23134. There were additionally small deletions of 26 bp from the right border and 185 bp from the left border of the T-DNA insertion in soybean event COR23134. Border region deletions of a T-DNA insertion are common during Agrobacterium-mediated plant transformation due to DNA repair mechanisms. Given that the left and right borders are not part of the expression cassettes, it is considered unlikely to affect the function of the inserted genetic elements or the food safety of soybean event COR23134.

Alignment of the SbS reads from soybean event COR23134 and the non-GM control soybean line 93Y21 to the PHP90315 sequence confirmed that there was no integration of the plasmid backbone sequence into soybean event COR23134, including any antibiotic resistance genes.

To determine the location of the T-DNA insertion in the soybean event COR23134 genome, the identified soybean sequences flanking the insertion site were subjected to homology search against a reference soybean genome. These searches located the single T-DNA insert on chromosome 20. The soybean event COR23134 T-DNA insertion did not disrupt any endogenous genes or any other known annotated feature in the soybean genome.

Bioinformatics analyses were performed to assess the potential toxicity, allergenicity, or biological activity of putative peptides that may result from the T-DNA insertion. All potential open reading frames (ORFs) of ≥ 8 amino acids in length spanning the 5′ and 3′ insert-flank junctions of soybean event COR23134, or contained within the insert itself, were translated in silico from stop codon to stop codon in all six reading frames. A total of 1,695 theoretical ORFs ≥ 8 amino acids were identified and queried against toxin and allergen databases.

To assess potential toxicity, similarity searches against a Pioneer internal database of protein toxin sequences (updated 2023) and against NCBI non-redundant (nr) protein sequences was performed using the Basic Local Alignment Search Tool (BLAST; January 2023; https://blast.ncbi.nlm.nih.gov/Blast.cgi).

From the 1,695 theoretical ORFs, no alignments were returned between the putative peptides and any protein sequence in the internal toxin database. Additionally none of the alignments returned by the NCBI search are proteins known to be toxic to humans or mammals.

To assess potential allergenicity, similarity searches against the Comprehensive Protein Allergen Resource (COMPARE) (2023; https://comparedatabase.org) were performed. A full length sequence search using a FASTA (Fast Alignment Search Tool – All) alignment was performed comparing each predicted ORF sequence to the database entries. A 80-mer sliding window FASTA search was performed comparing all contiguous 80 amino acids within the ORFs to the database entries. Matches were identified if there was greater than 35% homology. A 8-mer exact match FASTA search was performed comparing contiguous 8 amino acids within the ORF to the database entries. Matches were identified if there was 100% homology.

From the 1,695 theoretical ORFs, one putative peptide (COR23134_784) contained within the GM-HRA_1 coding sequence displayed > 35% identity with five known allergens over a sliding window of 80 amino acids. However, COR23134_784 lies entirely within the GM-HRA coding sequence and is out-of-frame with respect to it, therefore it is not expected to be translated. This alignment is considered low significance and unlikely to pose a food safety risk. There were no other putative peptides identified with similarity to any known human allergens.

The genetic stability of the T-DNA insert in soybean event COR23134 was assessed by Southern blot analysis. Leaf-derived genomic DNA from five generations of soybean event COR23134 (T1, T2, T3, T4 and T5) was used. Genomic DNA from the non-GM soybean control line 93Y21 served as a negative control, and the non-GM soybean control line DNA spiked with plasmid PHP90315 served as a positive control. The results showed equivalent bands of the expected sizes across all five generations tested. These results demonstrate that the inserted T-DNA is maintained stably in soybean event COR23134.

The pattern of inheritance of the T-DNA insert in soybean event COR23134 was demonstrated through segregation analysis of three segregating generations (T1, T2, and T3). Plants from the segregating generations were evaluated by qualitative real-time polymerase chain reaction assays. A Chi-square test was used to compare the observed segregation ratios of the soybean event COR23134 T-DNA insert to the expected ratios (1 homozygous T-DNA insert : 2 hemizygous : 1 homozygous wildtype). The results of the Chi-square analysis of the segregating progeny of soybean event COR23134 indicated no statistically significant difference between the observed and expected segregation ratios of the T-DNA insert. These results support the conclusion that the T-DNA insert resides at a single locus within the soybean event COR23134 and is inherited according to Mendelian principles of inheritance.

The phenotypic stability of the herbicide tolerance trait in soybean event COR23134 was assessed based on plant survival after exposure to diclosulam herbicide. Three homozygous generations (T4, T5, and T6) of soybean event COR23134 were tested. Based on the results, the intergenerational stability of the herbicide tolerance phenotype was confirmed.

Product information

Expression of the Cry1B.34.1, Cry1B.61.1, IPD083Cb, and GM-HRA_1 proteins in soybean event COR23134 was analyzed in various tissues including leaf (V5, R1, and R3 growth stages), root (R3 growth stage), flowers (R1-R2 growth stage), forage (R3 growth stage), and seed (R8 growth stage). Protein concentrations were quantified for each sample type using a validated enzyme-linked immunosorbent assay (ELISA) in nanograms per milligram dry weight (ng/mg DW).

Cry1B.34.1 was detected in soybean event COR23134, with the highest expression at the V5 stage in leaf tissue ranging from 190 to 960 ng/mg DW. Leaf is the target tissue for lepidopteran pest consumption. Cry1B.34.1 was detected in the seed ranging from 140 to 210 ng/mg DW. Root had the lowest level of Cry1B.34.1 expression ranging from 15 to 170 ng/mg DW.

Cry1B.61.1 was detected in soybean event COR23134, with the highest expression at the R1 stage in leaf tissue ranging from 250 to 1300 ng/mg DW. Cry1B.61.1 was detected in the seed ranging from 11 to 22 ng/mg DW. Root had the lowest level of Cry1B.61.1 expression ranging from <0.14 to 0.93 ng/mg DW.

IPD083Cb was detected in soybean event COR23134, with the highest expression at the R3 stage in leaf tissue ranging from 59 to 130 ng/mg DW. Seed had the lowest level of IPD083Cb expression ranging from 13 to 18 ng/mg DW.

GM-HRA_1 was detected in soybean event COR23134, with the highest expression at the V5 stage in leaf tissue ranging from <2.2 to 17 ng/mg DW. Seed had the lowest level of GM-HRA_1 expression ranging from <0.54 to 1.7 ng/mg DW.

The Cry1B.61.1 and IPD083Cb proteins are expressed at low levels in soybean event COR23134 so surrogate proteins were produced from alternate sources for use in toxicological safety studies. The surrogate Cry1B.61.1 protein was expressed in Escherichia coli and included a C-terminal histidine tag. The surrogate IPD083Cb protein was expressed in a tobacco-based protein expression system and included a C-terminal histidine tag.

The toxicological safety studies for Cry1B.34.1 and GM-HRA_1 were bridged from previously assessed novel foods. Pioneer provided protein characterization data on the Cry1B.34.1 and GM-HRA_1 proteins expressed in soybean event COR23134. The surrogate Cry1B.34.1 protein was expressed in E. coli and included a C-terminal histidine tag. The surrogate GM-HRA_1 protein was expressed in E. coli and included a N-terminal histidine tag which was removed by thrombin cleavage.

The surrogate Cry1B.34.1, Cry1B.61.1, IPD083Cb, and GM-HRA_1 proteins were shown to be biochemically and functionally equivalent to their respective soybean event COR23134-derived proteins based on molecular weight, immunoreactivity, peptide mapping, N-terminal sequence analysis, and glycosylation analysis. The equivalency of the surrogate and soybean event COR23134-derived Cry1B.34.1, Cry1B.61.1, and IPD083Cb proteins was additionally demonstrated through insect bioassay using velvetbean caterpillar (A. gemmatalis).

Based on the results provided, the surrogate Cry1B.61.1 and IPD083Cb proteins were found to be equivalent to the soybean event COR23134-derived proteins and acceptable for use in safety studies. The protein characterization of Cry1B.34.1 and GM-HRA_1 confirmed the identity of these proteins expressed in soybean event COR23134.

Based on the available data provided, Health Canada has no safety concerns regarding soybean event COR23134 from a molecular perspective.

Dietary exposure

The intended purpose of soybean event COR23134 is to provide a soybean with insect resistance to lepidopteran pests and tolerance to ALS-inhibiting herbicides. It is not expected by the petitioner that the introduction of soybean event COR23134 into the marketplace will result in a significant change in the food use of soybeans.

Nutrition

The petitioner provided compositional data for soybean event COR23134, the non-GM soybean control line 93Y21, and eighteen conventional reference varieties collected from eight field trials in the United States and Canada during the 2022 growing season. In each trial, four replicates of the novel and control cultivars were planted in a randomized complete block design (n=32 per cultivar). Typical commercial agriculture production practices were used for the field trials.

Seed samples were harvested at physiological maturity and analyzed using acceptable methods for proximates and fibres, amino acids, fatty acids, vitamins, minerals, antinutrients and isoflavones as suggested by the Organization for Economic Co-Operation and Development (OECD) Revised Consensus Document on Compositional Considerations for New Varieties of Soybean [Glycine max (L) Merr.]: Key Food and Feed Nutrients, Anti-nutrients, Toxicants and Allergens (2012).

When statistically significant differences between the non-GM soybean control line 93Y21 and soybean event COR23134 were noted (P-value < 0.05), the nutritional relevance of these differences was further examined by comparing the results to the expected range for conventional soybeans based on the OECD consensus document, analysis of the in-study reference varieties, and other publicly available data sources including the Agriculture and Food Systems Institute Crop Composition Database.

Statistically significant differences between the soybean event COR23134 and the non-GM soybean control line 93Y21 were observed for 23 analytes: crude protein, ash, carbohydrates, myristic acid, palmitic acid, heptadecanoic acid, heptadecenoic acid, linolenic acid, arginine, glutamic acid, glycine, isoleucine, leucine, proline, serine, valine, calcium, magnesium, phosphorus, vitamin B5, α-tocopherol, total daidzein equivalent, and total genistein equivalent. However, the soybean event COR23134 values were within the expected range for conventional soybeans in all cases.

Therefore, Health Canada has not identified any nutritional concerns related to the proposed food use of soybean event COR23134.

Chemistry

Chemical contaminant residue data have not been provided, nor have any unique contaminant considerations been identified with respect to soybean event COR23134. 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, Health Canada may conduct a human health risk assessment to determine if there is a potential safety concern and whether risk management measures are required.

Toxicology

The petitioner provided evidence to support the safety of the four introduced proteins (Cry1B.34.1, Cry1B.61.1, IPD083Cb, and GM-HRA_1) expressed in soybean event COR23134.

The Cry1B.34.1 protein is a truncated form of the full-length Cry1B.34 protein expressed in corn event DP910521 which was also developed by Pioneer and was previously assessed by Health Canada. Studies used to support toxicological safety (i.e., in vivo oral acute toxicity study, and in vitro heat stability and digestion assays) evaluated previously for the full-length Cry1B.34 protein were considered suitable for supporting the safety of the truncated Cry1B.34.1 protein on the basis that: 1) the only change in the truncated protein is the removal of the C-terminal crystal forming domain, which functions to stabilize Cry proteins but does not contribute to toxicity; 2) both proteins share an identical toxin core, suggesting similar modes of action and activity; 3) the removal of the crystal forming domain is unlikely to enhance the protein's resistance to digestion or heat degradation; and 4) Cry proteins have a history of safe use in food, a well-characterized mode of action, and a spectrum of activity that does not include mammals.

The GM-HRA_1 protein expressed in soybean event COR23134 is identical to the mature form of the GM-HRA protein expressed in soybean events DP305423 and DP356043 which were also developed by Pioneer and were previously assessed by Health Canada. Given that the mature forms of both GM-HRA_1 and GM-HRA proteins are identical, the studies used to support toxicological safety (i.e., in vivo oral acute toxicity study, and in vitro heat stability and digestion assays) evaluated previously for the GM-HRA protein were considered suitable for supporting the safety of the GM-HRA_1 protein.

For the Cry1B.61.1 and IPD083Cb proteins, the petitioner submitted a new set of studies to support toxicological safety (i.e., in vivo oral acute toxicity study, and in vitro heat stability and digestion assays) using surrogate proteins produced in E. coli- or tobacco-based expression systems. As described above, the surrogate Cry1B.61.1 and IPD083Cb proteins were found to be equivalent to their respective plant produced proteins in soybean event COR23134.

In acute toxicity studies in mice, no evidence of toxicity was observed following single oral doses of 5000 mg/kg body weight (bw) of Cry1B.34, 5000 mg/kg bw of Cry1B.61.1, 5000 mg/kg bw of IPD083Cb, or 436 mg/kg bw of GM-HRA proteins. The dose of 5000 mg/kg bw for the full-length Cry1B.34 protein is equivalent to a dose of 2907 mg/kg bw for the truncated Cry1B.34.1 protein following adjustment of the dose using molecular weight ratios for the two proteins.

The activity of Cry1B.34, Cry1B.61.1 and IPD083Cb proteins was fully diminished or significantly reduced when heated to ≥75˚C for approximately 30 minutes. The activity of GM-HRA protein was fully diminished when heated to ≥50˚C for 15 minutes. The results of the heat stability assays suggest that the novel proteins in soybean event COR23134 are heat labile and will likely be inactivated during processing or cooking of soybeans.

The Cry1B.34, Cry1B.61.1, IPD083Cb, and GM-HRA proteins were completely digested within 5 minutes in sequential simulated gastric fluid (SGF), simulated intestinal fluid (SIF), and/or sequential SGF and SIF assays, suggesting that these proteins are susceptible to digestion in the mammalian stomach.

Bioinformatics analysis confirmed the absence of significant similarity of Cry1B.34.1, Cry1B.61.1, IPD083Cb, and GM-HRA_1 proteins to known toxins.

The potential dietary exposure to each of the novel protein was estimated by the petitioner. In the worst case scenario, the estimated intake in the most sensitive population, non-nursing infants (95^th percentile), was determined to be 0.626 mg/kg bw per day for Cry1B.34.1, 0.055 mg/kg bw per day for Cry1B.61.1, 0.052 mg/kg bw per day for IPD083Cb, and 0.003 mg/kg bw per day for GM-HRA_1. The margins of exposures (MOEs) based on the highest doses tested in the acute toxicity studies, wherein no adverse effects were observed with any of the novel proteins, are at least 4600-fold greater than the estimated human exposure levels of each protein under worst case assumptions. In the absence of any evidence of toxicity, these MOEs are considered sufficient from a safety perspective.

Based on the available data provided, Health Canada has no safety concerns regarding soybean event COR23134 from a toxicological perspective.

Allergenicity

Bioinformatics analysis confirmed the absence of significant similarity of Cry1B.34.1, Cry1B.61.1, IPD083Cb, and GM-HRA_1 proteins to known allergens.

As noted in the 'Toxicology' section above, the novel proteins are sensitive to heat denaturation and susceptible to digestion in SGF and SIF in vitro. This suggests that little to no exposure to intact proteins or their resultant peptides is expected and therefore, the novel proteins will not persist in the gastrointestinal tract for any considerable period of time to elicit an allergenic response.

The genetic modifications employed in the development of soybean event COR23134 are unlikely to increase the expression of endogenous soybean allergens. The inserted T-DNA (containing gene cassettes for cry1B.34.1, cry1B.61.1, ipd083Cb, and gm-hra_1) in soybean event COR23134 does not encode for allergens or disrupt genes related to known endogenous allergens. It is therefore not expected to alter the expression of endogenous soybean allergens.

Based on the available data provided, Health Canada has no safety concerns regarding soybean event COR23134 from an allergenicity perspective.

Conclusion

Health Canada's review of the information presented in support of the use of soybean event COR23134 does not raise concerns related to food safety.

Health Canada's opinion refers only to the food use of soybean event COR23134. 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:

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

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2025-09-08

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