Novel food information: Insect resistant and herbicide tolerant DAS-Ø1131-3 Maize

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Background

Health Canada has notified Pioneer Hi-Bred Canada Company that it has no objection to the food use of insect resistance and herbicide tolerant maize line DAS-Ø1131-3 (hereafter referred as DAS1131-3). The Department conducted a comprehensive safety assessment of this maize 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 traits.

The following provides a summary of the notification from Pioneer Hi-Bred Canada Company and the evaluation by Health Canada. This document contains no confidential business information.

Introduction

Pioneer Hi-Bred Canada Company has developed a genetically modified (GM) maize line (Zea mays L.), DAS1131-3, that exhibits resistance to Lepidopteran maize pests and tolerance to glufosinate-ammonium herbicide. The insect resistance trait was achieved through expression a novel chimeric cry1DA2 gene protein. The tolerance to glyphosate herbicide trait was achieved through expression of a chimeric dgt-28 epsps gene.

The chimeric Cry1Da2 protein, encoded by the cry1Da2 gene, is composed of cry1Da2 and cry1Ab genes, which are derived from Bacillus thuringiensis strain PS81I and B. thuringiensis subsp. Kurstaki, respectively. Cry1Da2 protein binds to receptors in the brush border membrane of susceptible lepidopteran pests and causes cell death through the formation of non-specific, ion conducting pores in the apical membrane of the midgut epithelial cells.

The chimeric DGT-28 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) protein, encoded by the dgt-28 epsps gene, is composed of the Streptomyces sviceus epsps gene fused to a chloroplast transit peptide (TraP8) derived from Brassica napus and Brassica rapa via a 2-amino acid linker. The TraP8 peptide ensures transport of the DGT-28 EPSPS protein into the chloroplast by the cellular expression machinery where it catalyzes the same enzymatic reaction as other natural and modified EPSP synthases that have been shown to confer tolerance to glyphosate.

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 western corn rootworm resistant and herbicide tolerant DAS1131-3 maize 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. Pioneer Hi-Bred Canada Company 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). Western corn rootworm resistant and glufosinate-tolerant DAS1131-3 maize 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

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

Development of the modified plant

Maize event DAS1131-3 was developed using Agrobacterium-mediated transformation through means of transformation of immature maize embryos of public inbred maize line B104 with plasmid PHP88492, which contains two expression cassettes, two engineering landing pads, and three zinc-finger nuclease target sites present in a single T-DNA. One cassette resulted in the expression of the novel chimeric Cry1DA2 protein, while the second cassette resulted in the expression of the chimeric DGT-28 EPSPS protein, which confer resistance to certain susceptible lepidopteran pests and tolerance to glyphosate herbicide, respectively. The introduced engineering landing pads and zinc-finger nuclease target sites do not result in the expression of any substances and do not impart a characteristic.

Following embryo and Agrobacterium co‐cultivation and a recovery phase, the embryos were transferred to a medium containing both carbenicillin antibiotic and glyphosate herbicide to kill Agrobacterium cells and for selection purposes, respectively. Then, transformed callus were transferred to a germination medium and incubated to initiate shoot and root development (T0). PCR analysis was performed on healthy regenerated transformants in order to confirm presence of the PHP88492 T-DNA insert.

In plasmid PHP88492, both the cry1Da2 and dgt-28 epsps genes are under the transcriptional control of the endogenous ubiZM1 promotor, ubiZM1 5' UTR and ubiZM1 terminator. In addition, the endogenous ubiZM1 intron serves as a post-transcriptional control of both cry1Da2 and dgt-28 epsps expression.

The petitioner provided information to support the safety and historical use of each donor organism and the recipient organism (i.e., elite-inbred line B104). Neither B. thuringiensis, B. thuringiensis subsp. Kurstaki, nor S. sviceus poses a health safety concern. B. napus and B. rapa contain mustard-related allergens. However, the TraP8 peptide coding sequence is the only genetic element derived from those species, preventing mustard allergen coding sequences from being introduced into maize event DAS1131-3.

The PHP88492 T-DNA also contains a landing pad, which consist in two engineering landing pad (ELP) regions and three zinc finger nuclease (ZFN) target sites flanked by both expression cassettes.

The company explains that although ELP regions and ZFN target sites were originally designed to be components of a site-directed integration system, they were not involved in the development of DAS1131-3 and are simply present, but inactive. The company further explained that the presence of these sequences alone does not cause any recombination since a specific recombinase enzyme, which is not naturally present in plants, needs to be expressed in order to function. No genetic modifications were described that may allow the plant to express such recombinase enzyme. Therefore, BMH has no concern pertaining to the presence ELP regions and ZFN target sites in final maize line DAS1131-3.

Following the transformation of public inbred line B104 and development of T0 generation, Pioneer Hi-Bred Canada Company undertook a breeding program using Pioneer in-bred lines PH1V5T and PH4257 to develop generations suitable for analysing the characteristics of DAS1131-3 maize and incorporating the trait into elite lines for commercialisation.

Characterization of the modified plant

Genomic DNA from leaf tissues of DAS1131-3 maize was analyzed by Southern-by-Sequencing (SbS) and Sanger sequencing to determine the insertion copy number, the organization within the plant genome, and to confirm the absence of plasmid backbone sequences.

Results of the SbS analysis and Sanger sequencing revealed that PHP88492 T-DNA integrated in DAS1131-3 maize genome at a unique location in chromosome 1 with no evidence of endogenous gene being disrupted at both 5' and 3' genomic border sequences. The insert lacks the first 27 bp at the 5' end and the last 390 bp deletion at the 3' end, and a single A-to-G point mutation was confirmed in the first copy of the ubiZM1 promoter. These discordances with the expected PHP88492 T-DNA insert sequence have no safety implications since right and left border termini deletions often occur in Agrobacterium-mediated transformation. All remaining sequences are intact and identical to that of the PHP88492 T-DNA. No plasmid-derived off-target insertions, nor antibiotic resistance genes, nor plasmid backbone sequences were incorporated into DAS1131-3 maize genome.

Plasmid PHP88492 employed in the development of DAS1131-3 maize contained the spectinomycin resistance genes in their backbone. The petitioner showed the absence of antibiotic resistance genes in the DAS1131-3 maize genome through the absence of plasmid backbone sequences by SbS analysis.

A Bioinformatics assessment of open-reading frame (ORF) analysis was performed on both sequenced insertion site and junction sequences for similarity to known and putative allergens and toxins following established international criteria. All translated stop codon-bracketed reading framesFootnote 1 exhibiting a length equal or higher than eight amino acids in DAS1131-3 maize were considered. Putative translated stop codon-bracketed reading frames were searched against the Comprehensive Protein Allergen Resource (COMPARE) 2021 databaseFootnote 2 and the Pioneer toxin databaseFootnote 3. None of the putative translated stop codon-bracketed reading frames at the DAS1131-3 maize insertion site and border junctions returned alignments from the search against both databases.

Stability of each expression cassettes in DAS1131-3 maize genome was demonstrated by assessing individual DAS1131-3 maize from five generations (T1, T2, T3, T4, and T6) by means of Southern blot analysis. Hybridization of all probes resulted in a single band of the expected size in all five generations of DAS1131-3 maize samples analysed. Hybridization patterns exhibited event-specific bands unique to the DAS1131-3 maize insertion and therefore demonstrated the genetic stability of this event across all tested generations.

Genomic DNA from individual plants of seven DAS1131-3 maize generations were tested to verify the hypothesis that the maize event DAS1131-3 T-DNA insert was inherited in a manner that is predictable according to Mendelian principles for a single genetic locus using qPCR. The probes employed were specific to the event. Also, a phenotypic analysis used a visual herbicide injury evaluation to confirm the presence or absence of injuries corresponding to an herbicide-susceptible phenotype and herbicide-tolerant phenotype, respectively. The qPCR results were compared to the phenotype results in order to verify co-segregation of genotype with phenotype.

Segregation ratios determined for the seven generations of the maize event DAS1131-3 demonstrated that dgt-28 epsps, cry1Da2 genes, and the maize event DAS1131-3 as a whole segregated together and in accordance with the Mendelian rule of inheritance for a single genetic locus. In addition, these results support that the DNA insertion co-segregated with the phenotype trait and was stable through traditional breeding.

Product information

Event DAS1131-3 differs from its traditional counterparts by the expression of DGT-28 EPSPS and Cry1Da2 proteins. ELISA assays were used to measure expression levels of introduced proteins in plant tissues (leaf, root, pollen, stalk, and grain samples) through a field trial. The multi-site field trial was a randomized complete block design conducted at six sites in commercial maize-growing regions located in the U.S.A. and Canada using four blocks at each site, resulting in a total of 24 samples per tissue tested for each given notified protein. In addition to the analysis of different plant tissues, the company assessed the expression of notified proteins in different maize growth stages, which are described by Abendroth et al. (2011)Footnote 4. None of the notified proteins were expected to be expressed in a tissue-specific manner since none of the promotors that drive the transcriptional regulation of notified proteins are tissue-specific.

The average level of DGT-28 EPSPS in plant tissues varied between 3.8 to 68 ηg/g of tissue dry weight. Roots contain the least amount of DGT-28 EPSPS protein (3.8±1.8 ηg/g of tissue dry weight at R4) while leaves contain the highest (68±32 ηg/g of tissue dry weight at R4). Grains contain between 14 to 39 ηg/g of tissue dry weight at R6 growth phase.

The average level of Cry1Da2 in plant tissues varies between 8.1 and 41 ηg/g of tissue dry weight. At maturity, grains contain the least amount of Cry1Da2 protein (8.1 ±2.2 ηg/g of tissue dry weight) while pollens contain the highest (41± 4.1 ηg/g of tissue dry weight).

The notified full-length Cry1Da2 and DGT-28 EPSPS proteins are 603 and 481 amino acids in length and have a molecular weight of approximatively 68 and 51 kDa, respectively. To characterize both proteins, the petitioner provided a SDS-PAGE, Western blot, protein glycosylation analysis, peptide mapping by liquid chromatography mass spectrometry, and N-terminal amino acid sequencing using Edman degradation method. In addition, the Cry1Da2 protein was subjected to a bioactivity study against S. frugiperda larvae.

The provided protein characterization data demonstrated that the maize event DAS1131-3 might express a truncated version of the notified Cry1Da2 protein missing the first 19 amino acids of the maize-derived Cry1Da2. The company explained that N-terminal truncation is likely the result of "proteolysis by trypsin-like protease in planta or during extraction and purification processes", which is common for Cry1 proteins when expressed in or isolated from plant tissues (Gao et al 2006Footnote 5). The truncation did not affect the protein function and does not raise any safety concerns. Further, the N-terminal amino acid analysis revealed that the maize-derived DGT-28 EPSPS protein may contain at its N-terminal end an additional arginine residue, which is a remnant of a 2-amino acid linker, which does not represent a safety issue.

Based on the available data provided, the Bureau of Microbial Hazards has no safety concerns regarding maize event DAS1131-3 from a molecular perspective.

Dietary exposure

It is expected that DAS1131-3 maize will be used in applications similar to conventional maize varieties. The petitioner does not anticipate a significant change in the food use of maize with the introduction of DAS1131-3 maize.

Chemistry

Chemical contaminant residue data have not been provided, nor have any unique contaminant considerations been identified with respect to DAS1131-3 maize. 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.

Nutrition

The petitioner provided compositional data for DAS1131-3 maize, non-GM near-isoline control maize (control), and sixteen non-GM reference varieties collected from eight field trials in the United States and Canada during the 2020 growing season. In each trial, four replicates of each entry were planted in a randomized complete block design. Typical commercial agriculture production practices were used for the field trials.

Grain samples (n=32) were harvested and analysed using acceptable methods for proximates and fibres, amino acids, fatty acids, vitamins, minerals and anti-nutrients. The data provided was for all key nutrients and anti-nutrients as described by the Organization for Economic Co-Operation and Development (OECD)Footnote 6.

When statistically significant differences between the conventional control and DAS1131-3 maize were noted (P-values were < 0.05), the nutritional relevance of these differences were further examined by comparing the results to expected ranges for conventional maize as described in the OECD consensus document (2002), the in-study reference range, and the tolerance interval which was based on proprietary accumulated data from field studies conducted between 2003 and 2019 consisting of a total of 184 non-GM commercial reference maize lines and 185 unique environments representative of commercial maize-growing regions in the United States, Canada, Chile, Brazil, and Argentina.

A statistically significant difference between DAS1131-3 maize and the conventional control maize was observed for manganese (p<0.02) in the across site analysis even when adjusted for False Discovery Rate (FDR). However, the value is within tolerance levels and reference ranges provided by the petitioner and the expected range for conventional maize. There were no differences in proximates, fibre, fatty acid, amino acid, vitamins, mineral, or secondary metabolite content between the DAS1131-3 and the control maize.

Based on the information provided, no nutritional concerns were found with the sale of foods derived from DAS1131-3 in Canada.

Toxicology

For Cry1Da2 protein, no mortality was observed in mice (LD50>5000 mg/kg bw) in an acute oral toxicity study of the Cry1Da2 protein produced in a microbial expression system. The study followed Organisation for Economic Co-operation and Development (OECD) guidelines and was compliant with Good Laboratory Practices (GLP). Evidence were provided to demonstrate equivalency of the microbially produced Cry1Da2 protein and the protein produced in DAS1131-3 maize.

Comparison of the highest estimated human exposure to Cry1Da2 from corn consumption (24 µg/kg bw per day) to the LD50 (> 5000 mg/kg bw) results in a margin of > 200 000. The margin was considered sufficient from a safety perspective.

For DGT-28 EPSPS protein, no mortality was observed in mice (LD50>2000 mg/kg bw) in an acute oral toxicity study of the DGT-28 EPSPS protein produced in a microbial expression system. The study followed OECD guidelines and was compliant with GLP. Evidence were provided to demonstrate equivalency of the microbially produced DGT-28 EPSPS protein and the protein produced in DAS1131-3 maize.

Comparison of the highest estimated human exposure to DGT-28 EPSPS from corn consumption (74 µg/kg bw per day) to the LD50 (> 2000 mg/kg bw) results in a margin of > 20 000. The margin was considered sufficient from a safety perspective.

It was therefore concluded that DAS1131-3 maize would be as safe as conventional maize in terms of toxicity.

Allergenicity

The host organism, maize (Zea mays), has a long history of safe food use in Canada and is not associated with allergenic concerns.

The amino acid sequences of the novel proteins were compared to the sequences of known or suspected allergens in the Comprehensive Protein Allergen Resource (COMPARE) database (Jan., 2021: 2348 sequences).

For Cry1Da2, an alignment was identified with >35% homology over 80 a.a. for an allergen in rainbow trout and cattle. However, the percent homology (38.6%) was only slightly above the international guidance value of 35% and no exact 8 a.a. matches were identified, suggesting the lack of an epitope. Additionally, the E-value was high (>0.75), suggesting that the match was likely random and not indicative of cross-reactivity. In addition, no alignments were identified between this sequence of Cry1Da2 and epitopes of this specific allergen (i.e. collagen alpha 2(I) chain) as identified in the immune epitope database (IEDB).

Cry1Da2 protein is inactivated by heating conditions (30-35 minutes at 75 C), expected to be met in the processing/cooking of some corn products (e.g. baking). Cry1Da2 was completely and quickly broken down after incubation in simulated gastric fluid for 1 minute, followed by simulated intestinal fluid for 0.5 minutes. These data suggest that the Cry1Da2 protein would likely be quickly and completely digested under conditions of the human stomach and upper intestine, therefore having little opportunity to interact with the immune system and result in allergic reactions. No food allergy concerns were identified for this protein.

For DGT28-EPSPS, three alignments were identified with >35% homology over 80 a.a. for allergens from rainbow trout (40%), cattle (40%) and peanut (37%). However, the percent homologies were only slightly above the international guidance value of 35% and no exact 8 a.a. matches were identified suggesting a lack of epitopes. Additionally, the E-values were high (>0.02), suggesting that these matches were likely random and not indicative of cross reactivity. In addition, no alignments were identified between these sequences of DGT-28 EPSPS and epitopes of these specific allergens (i.e. collagen alpha 2(I) chain and Ara h 1) as identified in the immune epitope database (IEDB).

DGT28-EPSPS protein is inactivated by heating conditions (30-35 minutes at 50 C), expected to be reached in the processing/cooking of some corn products (e.g. baking). DGT28-EPSPS was completely and quickly broken down after incubation in simulated gastric fluid for 2 minutes, followed by simulated intestinal fluid for 0.5 minutes. These data suggest that the DGT28-EPSPS protein would likely be quickly and completely digested under conditions of the human stomach and upper intestine, therefore having little opportunity to interact with the immune system and result in allergic reactions. No food allergy concerns were identified for this protein.

It was therefore concluded that DAS1131-3 maize would be as safe as conventional maize in terms of food allergenicity.

Conclusion

Health Canada's review of the information presented in support of the use of insect resistant and herbicide tolerant maize variety DAS1131-3 does not raise concerns related to food safety.

Health Canada's opinion refers only to the food use of DAS1131-3 maize. 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

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