Novel food information : Herbicide tolerant (HT4) soybean – MON 94313

Background

Health Canada has notified Bayer CropScience Inc. (Bayer) that it has no objection to the food use of herbicide-tolerant (HT4) soybean – MON 94313 (MON 94313). 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 characteristics.

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

Introduction

Bayer has developed a novel soybean (Glycine max L. Merr.) variety, MON 94313 that exhibits tolerance to glufosinate (2-amino-4-(hydroxymethylphosphinyl) butanoic acid), dicamba (3,6-dichloro-2-methoxybenzoic acid), 2,4-D (2,4-dichlorophenoxyacetate), and mesotrione (2-[4-(methylsulfonyl)-2-nitrobenzoyl]-1,3-cyclohexanedione) herbicides.

MON 94313 was developed through the introduction of four gene expression constructs for the expression of a phosphinothricin N-acetyltransferase (PAT) protein, a dicamba mono-oxygenase (DMO) protein, a modified R-2,4-dichlorophenoxypropionate dioxygenase (FT_T.1) protein, and a triketone dioxygenase (TDO) protein. Expression of the PAT and DMO proteins confers tolerance to glufosinate and dicamba herbicides, respectively. Expression of the FT_T.1 protein confers tolerance to 2,4-D herbicides, while expression of the TDO protein confers tolerance to mesotrione herbicides.

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 94313 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 has provided data to support that this variety is safe for use 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 Division 28 of Part B of the Food and Drug Regulations (Novel Foods). Foods derived from MON 94313 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

MON 94313 was created by Agrobacterium-mediated transformation of meristem explants (variety A3555) using the PV-GMHT529103 vector. The PV-GMHT529103 vector contains two T-DNA sequences: T-DNA I and T-DNA II. T-DNA I encodes the pat, dmo, ft_t.1, and tdo gene expression cassettes (i.e., the herbicide tolerance characteristics), while T-DNA II encodes splA and aadA gene expression cassettes (i.e., selectable markers). When splA is expressed during embryo development, it results in wrinkled seeds as a result of interference with sucrose metabolism. As a result, seeds containing the T-DNA II can be selected based on the visual phenotype. Also, the expression of aadA provides antibiotic resistance to select for plants containing the T-DNA II.

After co-culturing A3555 soybean seeds with Agrobacterium AB30 carrying PV-GMHT529103 binary plasmid vector, explants were placed on selection media containing spectinomycin to select transgenic events, and carbenicillin, cefotaxime and timentin to inhibit Agrobacterium growth.

Plants that survived on selection media were denoted as R0. R0 plants were screened and selected based on the presence of a single T-DNA I copy that was unlinked with scorable/selectable markers, absence of vector backbone, and the absence of insertions in repetitive or genic regions.

R0 plants that were selected for advancement were self-pollinated to generate R1 seed and enable the segregation of the unlinked T-DNA I and T-DNA II insertions. Removal of T-DNA II in R1 plants was determined by absence of wrinkled phenotype and a PCR analysis for aadA. R1 plants that were homozygous for T-DNA I were selected for further development, and a lead event was identified based on superior agronomic, phenotypic and molecular characteristics.

Characterization of the modified plant

The number of insertions, intactness of insertions, and the presence/absence of unintentional insertions in the MON 94313 genome were determined through the bioinformatic mapping and subsequent analysis of short reads generated using Next-Generation Sequencing (NGS) technologies. In addition, directed sequencing (locus specific PCR and DNA sequence analyses) was used to obtain the sequence of the DNA insert and the adjacent flanking DNA.

The bioinformatic strategy to determine the number of insertion loci and the number of copies of the integrated plasmid DNA was designed to ensure that all transgenic segments were identified. Genomic DNA from MON 94313 and the conventional controls were used to generate short (~150 bp) randomly distributed sequence fragments (sequencing reads) in sufficient numbers to yield comprehensive coverage of the soybean genomes. The number of DNA inserts was determined by identifying and analyzing the sequences at apparent junction points. The junctions yield a characteristic signature that can be identified through read mappings which flank or span the junction point. Each insertion will produce two unique junctions characteristic of the genomic locus, one at the 5′ end of the insert and one at the 3′ end of the insert. Insertion sites can be recognized through the identification of a signature which includes both overlapping junction sequences and reads mapping adjacent to the junction points where their mate pair maps to flanking genomic DNA. By evaluating the number of junction signatures and the sequences of the unique junctions contained within these signatures, the number of plasmid sequence insertions into the genome and the T-DNA copy number can be determined. For a single copy T-DNA insert, two junction sequence signatures are expected, each originating from the ends of the insert, with junctions sequences composed of portions of the T-DNA sequence and the flanking plant genomic DNA sequence and flanked by unpaired alignments mapping to the T-DNA.

Based on the comprehensive NGS and junction identification, MON 94313 contains one copy of the T-DNA I inserted into a single locus. Furthermore the structure and organization of the T-DNA I insert was characterized using an overlapping PCR strategy. Two amplicons spanning the whole insert, as well as the flanking genomic DNA, were generated. sequencing of the amplicons resulted in a consensus sequence, revealing the organization of the inserted T-DNA I. The MON 94313 T-DNA I insert is 10196 base pairs long, aligning with the PV-GMHT529103 sequence starting at base 215, within the Right Border, and ending at base 10196 in the Left Border. The alignment was shown and demonstrates 100% sequence identity.

A bioinformatics analysis was performed looking for open reading frames (ORFs) both within the T-DNA I insert and those that could be generated across the insert-genome junctions. These searches looked for sequences that encode proteins, which share sequence similarity to known toxins, allergens, or other biologically active proteins across an 8 amino acid sliding window. No ORFs were identified for MON 94313.

Whole genome sequencing followed by bioinformatic mapping and analysis was performed on five generations of MON 94313 to determine genetic stability of the T-DNA I insert. The same two unique junction sequences were detected in all five generations. This demonstrates the stability of the T-DNA I insert over multiple generations of MON 94313.

To evaluate the mode of inheritance, zygosity of the T-DNA I insert in three segregating generations of MON 94313 was evaluated using Real-Time PCR. No statistical differences were observed between observed and expected segregation ratios for each of the segregating generations. As a result, it can be confidently concluded that the inserted DNA locus segregates according to Mendelian rules of inheritance.

Based on the available data provided, the Bureau of Microbial Hazards (BMH) has no safety concerns regarding MON 94313 from a molecular perspective.

Product information

MON 94313 differs from its conventional counterpart by the expression of a phosphinothricin N-acetyltransferase (PAT) protein, a dicamba mono-oxygenase (DMO) protein, a modified R-2,4-dichlorophenoxypropionate dioxygenase (FT_T.1) protein, and a triketone dioxygenase (TDO) protein.

The PAT protein, encoded by a pat gene from Streptomyces viridochromogenes, confers tolerance to the herbicidal active ingredient glufosinate-ammonium at current labelled rates by acetylating phosphinothricin to an inactive form. The PAT protein present in MON 94313 is identical to the corresponding protein found in a number of approved events across several different crops that are currently commercialized and have a history of safe use.

The DMO protein, encoded by a dmo gene from Stenotrophomonas maltophilia strain DI-6, confers tolerance to dicamba herbicides by conversion of dicamba into non-herbicidal 3,6-dichlorosalicylic acid (DCSA) and formaldehyde. The DMO protein present in MON 94313 is identical to the corresponding protein found in a number of approved events across several different crops that are currently commercialized and have a history of safe use.

The FT_T.1 protein, encoded by a ft_t.1 gene, a modified version of the R-2,4 dichlorophenoxypropionate dioxygenase (RdpA) gene from Sphingobium herbicidovorans. This protein confers tolerance to 2,4-D herbicide in soybean by catalysing the degradation of 2,4-D. The FT_T.1 protein present in MON 94313 is based off of the sequence of the FT_T protein found in MON 87429 maize that was previously assessed by Health Canada. (2020). The FT_T.1 differs from the FT_T protein by 3 amino acids (less than 1.5 % of the total amino acid residues). According to the petitioner, these modifications do not impact substrate specificity of FT_T.1 relative to FT_T (both proteins are still active on the same herbicide substrate molecules), although FT_T.1 exhibits higher enzymatic activity for 2,4-D herbicide (Larue et al., 2019^{Footnote 1}).

The TDO protein is encoded by a TDO gene from Oryza sativa (rice). Expression of the TDO protein confers tolerance to mesotrione (an inhibitor of 4-hydroxyphenylpyruvate dioxygenase [HPPD]) through a sequential double oxidation of the mesotrione molecule. This allows plants expressing the TDO protein to grow properly in the presence of the herbicide. The TDO protein has not previously been assessed by Health Canada.

Expression of the PAT, DMO, FT_T.1, and TDO proteins in MON 94313 soybean are controlled by promoter, intron, and terminator elements in each expression cassette. In all four cases, the expression of proteins is expected to be constitutive to provide tolerance to in-crop herbicide applications. While variability in protein expression can and does exist in different tissues of MON 94313 as seen from protein expression data provided, this variability is not intentionally designed into any of the expression cassettes.

PAT, DMO, FT_T.1, and TDO protein levels in tissues were determined by enzyme-linked immunosorbent assay (ELISA). PAT, DMO, and FT_T.1 were measured in a multiplex ELISA format, while TDO was evaluated using a standard ELISA.

For human food use, only soybean grain is expected to be used. PAT protein was observed at 3.5 mg/g FW (+ 0.13). DMO protein was observed at 37 mg/g FW (+ 1.1). FT_T.1 protein was observed at 5.7 mg/g FW (+ 0.15). TDO protein was observed at 4.6 mg/g FW (+ 0.33).

Dietary exposure

It is expected that MON 94313 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 94313.

Nutrition

Compositional data for genetically modified soybean MON 94313 and genetically similar conventional control A3555 were obtained from grain samples harvested from five field trials conducted in the US during the 2020 growing season. Each field trial was planted with modified and conventional soybean in a randomized complete block design with four replicates. Samples were analyzed using acceptable methods for proximates, fibres, amino acids, fatty acids, minerals, anti-nutrients, and iso-flavones.

When a statistically significant difference was identified between the modified soybean and its conventional control (P <0.05), the nutritional relevance of the difference was determined through comparison to the control range value (maximum value minus the minimum value) of the conventional control and the natural variability defined by the range of values observed in the literature and reported in the Agriculture and Food Systems Institute's Crop Composition Database for soybean.

There were no statistically significant differences for 48 of the 55 components analyzed. Seven components in grain (cystine, tryptophan, palmitic acid, linolenic acid, carbohydrates by calculation, vitamin K1, and glycitein) showed a statistically significant difference between MON 94313 and the conventional control. For these seven components, the mean difference between MON 94313 and the conventional control was less than the control range value of the conventional control, suggesting that the genetic modification does not impact levels of these components more than the natural variation observed within the conventional control grown at multiple locations.

The Bureau of Nutritional Sciences (BNS) has not identified any nutritional concerns related to the proposed use of MON 94313.

Chemistry

Chemical contaminant residue data have not been provided, nor have any unique contaminant considerations been identified with respect to MON 94313. 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 or 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, including soybean, 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.

Toxicology

The Pre-Market Toxicology Assessment Section (PTAS) evaluated the toxicological safety of the genetically modified soybean MON 94313, which expresses DMO, PAT, TDO, and FT_T.1 proteins.

The DMO and PAT proteins expressed by MON 94313 soybean share high amino acid sequence similarity with previously assessed DMO and PAT proteins present in MON 88701 cotton, MON 87419 corn and MON 87429 corn, which were previously approved by Health Canada^{Footnote 2}. Toxicological studies consisting of acute oral toxicity and in vitro heat stability and digestibility studies were provided as part of the MON 88701 submission. The BMH confirmed that the DMO and PAT proteins in MON 94313 soybean are equivalent from a safety perspective to the previously assessed DMO and PAT proteins.

The DMO protein did not show any evidence of toxicity in mice following a single oral dose of 283 mg/kg body weight (bw). Heat stability and digestion studies indicated that DMO protein loses functional activity when heated to temperatures above 55°C and is completely digested within 0.5 minutes in simulated gastric fluid (SGF) and within 5 minutes in simulated intestinal fluid (SIF). According to a published study by Wang et al. (2016)^{Footnote 3}, DMO proteins with similar sequence, structure, and function as the notified DMO protein are well tolerated in mice without any associated toxicity at acute oral doses of up to 1000 mg/kg bw. The heat inactivation of DMO protein at temperatures above 55°C and its rapid and complete digestion in SGF and SIF was also substantiated in this study.

The PAT protein demonstrated no signs of toxicity in mice following a single oral dose of 1086 mg/kg bw. In a heat stability assay, the functionality of PAT protein was reduced to 9% of control when heated to 95°C. PAT protein was completely digested in SGF within 0.5 minutes and in SIF within 5 minutes. These results are corroborated in a publication by Hérouet et al. (2005)^{Footnote 4} in which PAT protein equivalent to the notified PAT protein was not associated with any adverse effects following a single intravenous dose of 10 mg/kg bw to mice, was inactivated when heated to temperatures above 55°C and was digested in SGF within 0.5 minutes and in SIF within 5 minutes. PAT protein equivalent to the notified PAT protein is expressed in a number of genetically modified foods previously approved by Health Canada and has been shown to have low order of toxicity (LD50 5000 mg/kg bw)^{Footnote 5}.

For the TDO and FT_T.1 proteins, the petitioner submitted a new set of toxicological studies using surrogate proteins produced in bacterial expression systems. The BMH confirmed that the microbially produced TDO and FT_T.1 proteins were equivalent to the plant produced TDO and FT_T.1 proteins in MON 94313.

In acute oral toxicity studies in mice with either TDO or FT_T.1 protein, there were no signs of toxicity when animals were dosed up to 2000 mg/kg bw.

Both TDO and FT_T.1 proteins were heat inactivated at temperatures above 55°C and were digested in SGF within 0.5 minutes and in SIF within 5 minutes. In the digestion assay with FT_T.1, lower molecular weight peptide fragments were observed through to 20 minutes when treated in SGF alone, but no such fragments were observed following sequential digestion in which the FT_T.1 protein was treated in SGF for 2 minutes followed by SIF for 0.5 minutes. No persistent peptide fragments were observed with TDO in SGF or SIF. These results suggest that TDO and FT_T.1 are rapidly and completely digested in SGF and/or SIF.

The petitioner compared the sequences of the four novel proteins in MON 94313 soybean against known toxins using a toxin database (TOX_2021^{Footnote 6}) and the GenBank protein database (PRT_2021^{Footnote 7}). The searches did not identify any significant amino acid homologies to protein sequences of known toxins.

The potential dietary exposure to each of the novel protein, in the worst case scenario in the most sensitive population, infants of 0 to 12 months of age fed soy based formula (95th percentile), was determined to be 130 µg/kg bw per day for DMO, 12 µg/kg bw per day for PAT, 16 µg/kg bw per day for TDO, and 20 µg/kg bw per day for FT_T.1. The margins of exposures (MOEs) based on the highest doses tested in the in vivo acute toxicity studies, wherein no adverse effects were observed with any of the novel proteins, are at least three orders of magnitude 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 information, the PTAS did not identify any toxicological food safety concerns with the use of the MON 94313 soybean, as proposed.

Allergenicity

The PTAS assessed the potential allergenicity of the four novel proteins and the potential impact of gene modification on the expression of endogenous allergens in MON 94313 soybean.

The petitioner conducted an amino acid sequence search of an allergen database (described as AD_2021^{Footnote 8}) obtained from the Comprehensive Protein Allergen Resource (COMPARE) of the Health and Environmental Sciences Institute (HESI) to identify potential sequence homology between the four novel proteins in MON 94313 soybean and known allergens. Searches included a full FASTA alignment, a sliding window of 80 amino acid stretches with greater than 35% identity, and a search for 8 contiguous amino acids to assess for the presence of epitopes^{Footnote 9}. For each of the novel proteins, the searches did not identify any matches against known allergens.

As noted in the 'Toxicological Assessment' 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 petitioner conducted a quantitative comparison of endogenous soybean allergens to assess if genetic modifications could affect the levels of these allergens in MON 94313 soybean compared to a conventional soybean variety. Protein extracts from the seeds of MON 94313 soybean and conventional soybean were quantified using a LC MS/MS^{Footnote 10} method to measure the levels of specific clinically relevant soybean allergens^{Footnote 11}. No significant differences in the quantity of these allergens were identified between the two types of soybeans suggesting that there is no expectation of increased dietary exposure to endogenous soybean allergens from consumption of soybean products derived from MON 94313 soybean.

The PTAS does not consider the MON 94313 soybean to pose any additional allergenic health concerns in comparison to conventional soybean.

Conclusion

Health Canada's review of the information presented in support of the use of MON 94313 does not raise concerns related to food safety.

Health Canada's opinion refers only to the food use of MON 94313. 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 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 Directorate
Health Products and Food Branch
Health Canada, PL2204A1
251 Frederick Banting Driveway
Ottawa, Ontario K1A 0K9
bmh-bdm@hc-sc.gc.ca

Footnotes

Footnote 1

Larue, C.T., Goley, M., Shi, L., Evdokimov, A.G., Sparks, O.C., Ellis, C., Wollacott, A.M., Rydel, T.J., Halls, C.E., Van Scoyoc, B., Fu, X., Nageotte, J.R., Madio, A., Zheng, M., Sturman, E.J., Garveya, G.S., and Varagonaa, M.J. 2019. Development of enzymes for robust aryloxyphenoxypropionate and synthetic auxin herbicide tolerance traits in maize and soybean crops, Pest Management Science, 75: 2086–2094.

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Footnote 2

Technical summary of dicamba and glufosinate-tolerant cotton MON 88701. Decision date: June 23, 2014. Available at: https://www.canada.ca/en/health-canada/services/food-nutrition/genetically-modified-foods-other-novel-foods/approved-products.html.
Technical summary of herbicide tolerant maize MON 87419. Decision date: February 8, 2016. Available at: https://www.canada.ca/en/health-canada/services/food-nutrition/genetically-modified-foods-other-novel-foods/approved-products/herbicide-tolerant-maize-mon-87419.html.
Technical summary of herbicide tolerant maize MON 87429. Decision date: August 21, 2020. Available at: https://www.canada.ca/en/health-canada/services/food-nutrition/genetically-modified-foods-other-novel-foods/approved-products/herbicide-tolerant-ht4-maize.html.

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Footnote 3

Wang, C., Glenn, K.C., Kessenich, C., Bell, E., Burzio, L.A., Koch, M.S., et al. 2016. Safety assessment of dicamba mono-oxygenases that confer dicamba tolerance to various crops, Regulatory Toxicology and Pharmacology, 81: 171-82.

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Footnote 4

Hérouet, C., Esdaile, D.J., Mallyon, B.A., Debruyne, E., Schulz, A., Currier, T., Hendrickx, K., van der Klis, R-J., and Rouan, D. 2005. Safety evaluation of the phosphinothricin acetyltransferase proteins encoded by the pat and bar sequences that confer tolerance to glufosinate-ammonium herbicide in transgenic plants, Regulatory Toxicology and Pharmacology, 41: 134-149.

Return to footnote 4 referrer

Footnote 5

Technical summary of Cry1F Insect-resistant/Glufosinate-tolerant Maize Line 1507. Decision date: October 11, 2002. Available at: https://www.canada.ca/en/health-canada/services/food-nutrition/genetically-modified-foods-other-novel-foods/approved-products/novel-food-information-food-biotechnology-cry1f-insect-resistant-glufosinate-tolerant-maize-line-1507.html.
Technical summary of Bacillus thuringiensis (B.t) Cry34/35/Ab1 insect resistant, glufosinate-tolerant transformation corn event DAS-59122-7. Decision date: November 18, 2005. Available at: https://www.canada.ca/en/health-canada/services/food-nutrition/genetically-modified-foods-other-novel-foods/approved-products/bacillus-thuringiensis-cry34-35-insect-resistant-glufosinate-tolerant-transformation-corn-event-59122-7.html.

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Footnote 6

TOX_2021 is a toxin specific database that was derived from the Swiss-Prot database and filtered using keyword searches to remove likely non-toxin proteins. The keyword searches were conducted on January 5, 2021. The final TOX_2021 database contains 7,870 sequences.

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Footnote 7

PRT_2021 is the GenBank protein database that was retrieved from NCBI on January 5, 2021 and contains 139,450,651 sequences.

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Footnote 8

The AD_2021 database was retrieved from the COMprehensive Protein Allergen REsource (COMPARE) database of the Health and Environmental Sciences Institute (HESI) on February 1, 2021 and contains 2,348 sequences.

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Footnote 9

An epitope is the part of an allergenic molecule that binds to an antibody.

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Footnote 10

Liquid Chromatography with tandem mass spectrometry.

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Footnote 11

The levels of the following six allergens in MON 94313 soybean and conventional soybean were quantified: Gly m 4 (SAM22), Gly m 5 (β-conglycinin), Gly m 6 (glycinin), Gly m Bd 28 K (unknown Asn-linked glycoprotein), Gly m Bd 30 K (P34), and KTI (Kunitz trypsin inhibitor).

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Date modified:: 2024-01-08

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