Novel Food Information: Insect-resistant and herbicide-tolerant maize - DP-51291-2

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 Pioneer Hi-Bred Canada Company (Pioneer) that it has no objection to the food use of insect resistant and herbicide tolerant maize – DP-51291-2 (DP-51291-2).

The safety of DP-51291-2 for food use was assessed as part of the second pilot of the Health Canada and Food Standards Australia New Zealand (FSANZ)'s Safety Assessment Sharing Initiative. For this pilot, FSANZ was the primary assessor and conducted the safety assessment of this maize line. Health Canada acted as the secondary assessor, peer-reviewing FSANZ's initial assessment report for DP-51291-2, providing feedback regarding the report, and using the finalized report to inform the Department's own regulatory decision regarding this product.

FSANZ conducted a comprehensive assessment of this maize variety according to FSANZ's Application Handbook, specifically its guidelines for applications for new foods produced using gene technology. These guidelines are based on internationally established scientific principles and guidelines developed through work in international fora (e.g., Codex Alimentarius) and align with Health Canada's Guidelines for the Safety Assessment of Novel Foods (updated 2022).

The following provides a summary of the notification from Pioneer and the evaluation conducted by FSANZ and peer-reviewed by Health Canada. This document contains no confidential business information.

Introduction

Pioneer has developed a novel maize (Zea mays L.) variety, DP-51291-2 that which exhibits resistance to corn root worm pests, and tolerance to glufosinate herbicides.

DP-51291-2 was developed through the introduction of three gene expression cassettes for the expression of a IPD072Aa protein, a maize-optimized version of a phosphinothricin N-acetyltransferase (mo-PAT) protein, and a phosphomannose isomerase (PMI) protein. Expression of the IPD072Aa protein confers resistance to corn rootworm larvae, while expression of the mo-PAT protein confers tolerance to glufosinate (2-amino-4-(hydroxymethylphosphinyl) butanoic acid) herbicides. Expression of the PMI protein served as a selectable marker to enable selection of plants containing the desired constructs during the event development process (Negrotto et al., 2000^{Footnote 1}).

The safety assessment peer-reviewed by Food and Nutrition Directorate evaluators was conducted according to FSANZ's Application Handbook, specifically its guidelines for applications for new foods produced using gene technology. These guidelines are based on internationally established scientific principles and guidelines developed through work in international fora (e.g., Codex Alimentarius) and align with Health Canada's Guidelines for the Safety Assessment of Novel Foods (updated 2022). The assessment considered: how DP-51291-2 was developed, how the composition and nutritional safety of this variety compared to its conventional comparator, and what the potential is 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 DP-51291-2 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

Development of the Modified Plant

Three sequential transformation steps were used to construct DP-51291-2: an initial Agrobacterium-mediated transformation to insert a "landing pad" sequence into the maize genome; a second microparticle bombardment-mediated transformation to prepare the landing pad sequence for insertion of the intended expression cassettes, and a third Agrobacterium-mediated transformation to integrate the intended expression cassettes and generate DP-51291-2.

In the first transformation step, the T-DNA region from the plasmid PHP50742 was integrated into the genome to create an intermediate line containing a landing pad sequence, which included target sites for the Cre recombinase (i.e., two loxP sites) flanking the ip3-h5 gene cassette, and target sites for the FLP recombinase (i.e., FRT1 and FRT87) flanking the pmi and mo-pat gene cassettes.

In the second transformation step, a total of 5 plasmids were used to prepare the inserted landing pad site for the integration of the desired expression cassettes:

• The plasmid PHP46438 contains the nptII and AmCyan1 gene cassettes flanked by FRT1 and FRT87 sites. During this transformation step, the pmi and mo-pat cassettes present in the initial landing pad sequence were replaced by the nptII and AmCyan1 cassettes from PHP46438 by Flp-FRT recombination.
• The plasmid PHP5096 contains the maize-optimized flippase recombinase (mo-Flp) gene cassette and allowed for transient expression of the FLP recombinase during transformation but was not integrated into the maize genome.
• The plasmid PHP16072 contains the maize-optimized Cre recombinase gene (mo-cre) and allowed for transient expression of Cre recombinase during transformation but was not integrated into the maize genome. As a result of Cre recombinase expression, the ip3-h5 gene cassette present in the initial landing pad sequence was removed by Cre-lox recombination.
• The plasmid PHP21139 contains the zm-wus2 gene cassette and allowed for transient expression of the maize-derived WUS2 protein, used to promote improved regeneration of maize plants during transformation. No elements from PHP21139 were integrated into the maize genome.

In the third transformation step, the T-DNA region from the plasmid PHP74638 containing the ipd072Aa, mo-pat, and pmi gene cassettes, flanked by FRT1 and FRT87 recombination sites, was incorporated into the DP-51291-2 genome by Flp-FRT recombination, replacing the nptII and AmCyan1 cassettes that were incorporated in the second transformation step. The zm-wus2, zm-odp2, mo-Flp and DsRed2 genes on the plasmid PHP74638 were not integrated into the maize genome but were transiently expressed. WUS and ODP2 proteins allowed for improved regeneration, FLP recombinase allowed for the integration of the intended DNA cassettes and DsRed2 allowed screening for any unintended integration of DNA sequences in plant cells.

Characterization of the Modified Plant

The molecular characterization of DP-51291-2 was conducted through a combination of SbS and Sanger sequencing. SbS is an in-house method that combines probe-based capture techniques with NGS (Zastrow-Hayes et al., 2015^{Footnote 2}) and was used to determine the insert copy number, organization of the inserted DNA, and to confirm the absence of any unintended plasmid sequences The sequencing reads obtained by SbS were compared to the intended insertion sequence, the plasmid sequences, and to the endogenous maize genome to identify unique junctions attributable to inserted DNA.

Genomic DNA from ten plants from the T1 generation of DP-51291-2 was analyzed by SbS, along with DNA from a plant from the non-GM PHR03 line as a control. Positive control samples were generated using PHR03 DNA spiked with each of the seven plasmids used for development of DP-51291-2. The sequencing reads obtained by SbS were compared to the intended insertion sequence, the plasmid sequences, and to the endogenous maize genome to identify unique junctions attributable to inserted DNA. Sanger sequencing of overlapping PCR fragments was used to sequence the insert and flanking maize genomic regions.

Ten plants from the T1 generation of DP-51291-2 analyzed by SbS consisted of 5 transgenic and 5 null segregant plants. SbS analysis of each of the 5 transgenic plants yielded sequencing reads that aligned to the intended insertion, and identified two unique genome-insertion junctions, indicating that a single copy of the intended insertion, with the intended organization, was integrated into the genome of DP-51291-2. No junctions were detected in either the control or in the 5 null segregant plants.

The SbS analysis used a set of hybridization probes covering the backbone sequences for all seven plasmids used in the transformation process. Alignment of NGS reads from the controls or DP-51291-2 to all plasmid sequences confirmed there was no integration of backbone sequences into DP-51291-2, including any antibiotic resistance genes.

The SbS analysis indicated that DP-51291-2 contains a single copy of the intended insertion, with the expected organization, and no unintended sequences or rearrangements.

Sanger sequencing of overlapping PCR fragments covering the insert and flanking maize sequences of DP-51291-2 confirmed that the insertion is 12,203 bp in length and is derived from plasmids PHP50742 and PHP74638. Comparison of the insert sequence with the landing pad from PHP50742 and the recombination fragment region from PHP74638 confirmed that the insert has the expected sequence and organization, with the exception of small deletions in the left and right border regions. Compared to the intended insertion, the insert begins at bp 23 and ends at bp 12,225, meaning that 22 bases from the right border and 13 bases from the left border are not present in DP-51291-2. Such changes during T-DNA insertion are common during Agrobacterium-mediated plant transformation due to double-stranded break repair mechanisms (Anderson et al., 2018^{Footnote 3}). Given that these sequences are not part of the gene cassettes it is unlikely they would affect the function of the inserted genetic elements.

Bioinformatics analyses were performed to assess the potential toxicity, allergenicity, or biological activity of the putative peptides encoded by the 5' and 3' inserted T-DNA/genomic DNA junctions. All stop-codon bracketed reading frames of ≥ 8 amino acids in length spanning the 5' and 3' insert-flank junctions of DP-51291-2, or contained within the insert itself, were translated in silico from stop codon to stop codon (TGA, TAG, TAA) in all six reading frames. A total of 771 theoretical open reading frames (ORFs) ≥ 8 aa were identified and queried against allergen and toxin databases.

The version of the Comprehensive Protein Allergen Resource (COMPARE) (January 2022; https://comparedatabase.org) used contained 2,463 sequences. A FASTA search algorithm (v35.4.4) (Pearson and Lipman, 1988^{Footnote 4}) was used to identify alignments between the query sequences and the COMPARE database, using a BLOSUM50 scoring matrix and an E-value threshold of 100. Only matches with a linear identity of greater than 35% over 80 amino acids were considered. In addition, a search for ≥eight contiguous amino acid matches to the allergens from the COMPARE database was performed using EMBOSS FUZZPRO.

To assess potential toxicity, an internal toxin database (updated in January 2022) was generated by filtering the UniProtKB/Swiss-Prot database using keywords relating to potential toxicity. The ORFs were also queried against the NCBI non-redundant (nr) protein database. For both the toxin and nr protein database searches, a BLASTP algorithm with a BLOSUM62 scoring matrix and an E-value threshold of 0.0001 was used.

Comparison of 771 putative peptides against the COMPARE allergen database identified five ORFs that had ≥35% identity with nine allergens over an 80 amino acid sliding window. No alignments were found between the 771 putative proteins and any of the toxins in the in-house toxin database. Twelve alignments were found between the ORFs and the NCBI nr protein database, but none of these proteins were related to any known toxic proteins.

The five ORFs which produced alignments with allergens from the COMPARE database were identified using a highly conservative approach and, collectively, these alignments either lacked upstream promoters, lacked methionine residues (start codons), or possessed start codons in positions only a short distance from a stop codon, meaning that either transcription or translation of these ORFs would be highly unlikely. The risk of allergenic proteins with relevance to human safety being produced by these ORFs is negligible.

The genetic stability of the T-DNA insert in DP-51291-2 was assessed by Southern blot analysis. Leaf-derived genomic DNA from five generations of DP-51291-2 (T1, T2, T3, T4 and T5) was used. Genomic DNA from the non-GM parental line PHR03 served as a control, and PHR03 DNA spiked with plasmid PHP74638 served as a reference substance to confirm probe hybridization. The results showed equivalent bands of the expected sizes across all five generations. The consistency of these results confirms that the inserted DNA is maintained stably in DP-51291-2.

The pattern of inheritance of the T-DNA insert in DP-51291-2 was demonstrated through segregation analysis of five generations (T1, T2, T3, T4, and T5). Plants from each generation were evaluated by both quantitative and qualitative PCR assays. Plants were also examined at a phenotypic level by observing plant survival after exposure to glufosinate. A Pearson's chi-square (χ²) analysis was used to compare the observed segregation ratios of the DP-51291-2 T-DNA insert to the expected ratios. The results of the χ² analysis of the segregating progeny of DP-51291-2 indicated no statistically significant difference between the observed and expected segregation ratios of the DP-51291-2 T-DNA insert. These results support the conclusion that the DP-51291-2 T-DNA insert resides at a single locus within the maize genome and is inherited according to Mendelian principles of inheritance. These results are consistent with the molecular characterization data indicating that DP-51291-2 contains a single, intact copy of the T-DNA inserted at a single locus in the maize genome.

Based on the available data provided, the Bureau of Microbial Hazards (BMH) has no safety concerns regarding DP-51291-2 from a molecular perspective.

Product Information

DP-51291-2 differs from its conventional counterpart by the expression of a IPD072Aa protein, a maize-optimized version of a phosphinothricin N-acetyltransferase (PAT) protein, and a phosphomannose isomerase (PMI) protein.

The IPD072Aa protein, encoded by the ipd072Aa gene from Pseudomonas chlororaphis strain SS143D5, disrupts midgut epithelial cells of corn rootworm larvae, leading to insect death. The IPD072Aa protein present in DP-51291-2 is identical to the corresponding protein found in insect resistant and herbicide tolerant maize – DP-023211-2, previously assessed and approved by Health Canada (2021).

The PAT protein, encoded by the codon-optimized mo-pat gene synthesized from the native pat gene from Streptomyces viridochromogenes, strain Tü494, confers tolerance to the herbicidal active ingredient glufosinate-ammonium by acetylating phosphinothricin to an inactive form. The PAT protein present in DP-51291-2 is identical to the corresponding protein found in a number of events across several different crops previously assessed and approved by Health Canada including dicamba and glufosinate-tolerant maize – MON 87419, herbicide tolerant HT4 maize – MON 87429, glufosinate-tolerant maize – T25, insect-resistant and glufosinate-tolerant maize – TC 1507, insect-resistant and glufosinate tolerant maize – DAS-59122, herbicide-tolerant and pest-resistant maize – 4114, glufosinate-tolerant soybean A5547-127, insect resistant and herbicide tolerant maize – DP-023211-2, and herbicide tolerant sugar beet - KWS20-1.

PMI protein, encoded by the pmi gene from Escherichia coli, strain K-12, serves as a selectable marker in plant tissue during transformation which allows for tissue growth using mannose as the carbon source. The PMI protein present in DP-51291-2 is identical to the corresponding protein found in a number of events across several different crops previously assessed and approved by Health Canada including insect resistant maize - MIR162, Golden Rice, insect resistant maize MIR604, insect resistant maize - 5307, alpha-amylase maize - 3272, insect resistant and herbicide tolerant maize – DP-023211-2, and herbicide-tolerant maize - DP910521.

Expression of the IPD072Aa, PAT, and PMI proteins in DP-51291-2 was analyzed in various tissues including leaf (V9, R1, and R4 growth stages), root (V6, V9, R1, and R4 growth stages), pollen (R1 growth stage), forage (R4 growth stage), and grain (R6 growth stage). Plants were grown at 5 sites in the United States and 1 site in Canada during the 2021 growing season, with four replicate plots per site. Samples from both glufosinate-treated and non-treated DP-51291-2 were collected from each replicate at each site, resulting in a total of 24 samples for each tissue. Protein concentrations were quantified for each sample type using a validated enzyme-linked immunosorbent assay (ELISA).

IPD072Aa was detected in glufosinate-treated DP-51291-2, with the highest expression on a dry weight (DW) basis at the early reproductive stage in root tissue (200 ng/mg DW), which is the target tissue for corn rootworm larvae consumption. IPD072Aa was detected in the grain at a very low level compared to root tissue (3.8 ng/mg DW), and pollen had the lowest level of IPD072Aa expression (1.0 ng/mg DW). Similar levels of IPD072Aa were detected in DP-51291-2 not treated with glufosinate.

PAT was detected in glufosinate-treated DP-51291-2, with the highest expression in pollen in the early reproductive stage (R1 – 68 ng/mg DW). By maturity (R6), PAT was detected in the grain (5.8 ng/mg DW). Similar levels of PAT expression were detected in DP51291 not treated with glufosinate.

PMI was detected in glufosinate-treated DP-51291-2, with the highest expression in leaf at the R4 growth stage (31 ng/mg DW). By maturity (R6), PMI is detected in the grain (3.7 ng/mg DW). Similar levels of PMI expression were detected in DP-51291-2 not treated with glufosinate.

Dietary exposure

It is expected that DP-51291-2 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 DP-51291-2.

Nutrition

The petitioner provided compositional data for DP-51291-2, a near-isoline control maize (control), and twenty conventional reference varieties collected from eight field trials in the United States and Canada during the 2021 growing season. In each trial, four replicates of the novel and control cultivar were planted in a randomized complete block design (n=32 per cultivar). Typical commercial agriculture production practices were used for the field trials.

Grain samples were harvested at physiological maturity and analyzed 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 in the Organisation for Economic Co-Operation and Development (OECD) Consensus Document on Compositional Considerations for New Varieties of Maize (Zea mays): Key Food and Feed Nutrients, Anti-nutrients and Secondary Plant Metabolites (2002).

When statistically significant differences between the conventional control and DP-51291-2 were noted (P-value <0.05), the nutritional relevance of these differences was further examined by comparing the results to expected ranges for conventional maize 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 DP-51291-2 and the conventional control were observed for several fatty acids (i.e., palmitic acid, oleic acid, linoleic acid, eicosenoic acid, lignoceric acid), copper, and trypsin inhibitor. However, the DP-51291-2 values were within the expected ranges for conventional maize in all cases.

The Bureau of Nutritional Sciences has not identified any nutritional concerns related to the proposed food use of DP-51291-2.

Chemistry

Chemical contaminant residue data have not been provided, nor have any unique contaminant considerations been identified with respect to DP-51291-2. 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 Sections 4(1)(a) and (d) of the Food and Drugs Act, which state 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.

Toxicology

The host organism (maize), novel proteins (IPD072Aa, PAT, and PMI) and assays (e.g., heat stability, simulated gastric fluid assay, simulated intestinal fluid assay, acute toxicity assays) were the same as those previously evaluated in insect resistant and herbicide tolerant maize – DP-023211-2 which was authorized, indicating no safety concerns.

Updated dietary exposure estimates and amino acid homology searches were considered for this maize but did not modify the previous conclusions.

Therefore, the Pre-market Toxicology Assessment Section (PTAS) has not identified any toxicological safety concerns with the proposed food use of DP-51291-2.

Allergenicity

The host organism (maize), novel proteins (IPD072Aa, PAT, and PMI) and assays (e.g., heat stability, simulated gastric fluid assay, simulated intestinal fluid assay, acute toxicity assays) were the same as those previously evaluated in insect resistant and herbicide tolerant maize – DP-023211-2 which was authorized, indicating no safety concerns.

Updated dietary exposure estimates and amino acid homology searches were considered for this maize but did not modify the previous conclusions.

Therefore, the PTAS has not identified any allergenic safety concerns with the proposed food use of DP-51291-2.

Conclusion

Health Canada's review of the information presented in support of the use of DP-51291-2 does not raise concerns related to food safety.

Health Canada's opinion refers only to the food use of DP-51291-2. 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