Novel Food Information: Insect-resistant and herbicide-tolerant DP910521 maize

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 DP910521 maize (DP910521). The Department conducted a comprehensive assessment of this maize 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 Pioneer and the evaluation by Health Canada. This document contains no confidential business information.

Introduction

Pioneer has developed a novel maize (Zea mays L.) variety, DP910521 that exhibits resistance to insect pest pressure from lepidopteran pests as well as tolerance to glufosinate herbicides.

DP910521 was developed through the introduction of three gene expression constructs for the expression of an insecticidal Cry protein, Cry1B.34, a phosphinothricin N-acetyltyltransferase (PAT), and a phosophomannose isomerase (PMI). Expression of the Cry1B.34 and PAT protein confer the insect resistance and glufosinate herbicide tolerance, respectively. The PMI protein was used as a selectable marker to help with transformation event selection.

The safety assessment performed by 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 DP910521 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 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 DP910521 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

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

Development of the Modified Plant

DP910521was developed through site-specific integration of a DNA sequence that encodes the expression cassettes of the desired novel characteristics. The site-specific integration was achieved using two sequential transformation steps: (1) insertion of a 'landing pad' sequence at a specific location in the plant genome, achieved using biolistics and CRISPR-Cas9-mediated insertion, and (2) insertion of the intended expression cassettes from the PHP79620 plasmid into the previously integrated maize landing pad. This second step was accomplished by microprojectile co-bombardment of multiple plasmids.

Characterization of the Modified Plant

The number of insertions, intactness of insertions, and the presence/absence of unintentional insertions in the DP910521 genome were determined using a form of Whole Genome Sequencing (WGS) called Southern-by-Sequencing (SbS). The SbS approach uses capture probes that are homologous to the transformation plasmids to first isolate genomic DNA that hybridizes to the probe sequences. This captured genomic DNA is then sequenced using WGS and the resulting sequence data is analyzed using bioinformatics.

The SbS analysis of DP910521 demonstrated a single, intact copy of the intended DNA insert and no other inserts (e.g., plasmid backbone sequence) are present in the host genome.

A bioinformatics analysis was performed looking for open reading frames (ORFs) both within the DNA insert and that which could be generated across the insert-genome junctions. These searches looked for sequences that encode proteins of at least 8 amino acids (AA) which share sequence similarity to known toxins or allergens. Nine hundred and twenty-five (925) putative ORFs greater than or equal to 8 AA were identified for DP910521. Overall, the analysis of the DNA Insert through the border junctions has demonstrated that there are likely no amino acid sequence similarities to known allergens or toxins.

Southern blot analysis was performed on five generations of DP910521 to determine genetic stability of the DNA insert, which includes the cry1B.34, mo-pat, and pmi gene cassettes. The results of the analysis demonstrated event-specific bands across generations had consistent genomic border regions, as evidenced by the consistent signal sizes across generations, and therefore the DNA insert was stably inherited across all 5 generations.

To evaluate the mode of inheritance, 100 plants from each of five generations of DP910521 were evaluated using genotyping and phenotyping. A genotypic study used qPCR to evaluate for the presence/absence of the DP910521 cry1B.34, mo-pat, and pmi genes. The phenotypic analysis used visual herbicide injury to confirm the presence/absence of tolerance to glufosinate-ammonium in each plant. The phenotypic results were compared to genotypic results to evaluate the co-segregation of genotype with phenotype. 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 phenotypes co-segregate with the DNA insert, and that the DNA insert segregates according to Mendelian rules of inheritance.

Based on the available data provided, the Bureau of Microbial Hazards (BMH) has no safety concerns regarding DP910521 from a molecular perspective.

Product Information

DP910521 differs from its conventional counterpart by the expression of an insecticidal Cry protein, Cry1B.34, a phosphinothricin N-acetyltyltransferase (PAT), and a phosophomannose isomerase (PMI).

The Cry1B.34 protein is encoded by the cry1B.34 gene, a chimeric gene comprised of sequences from a cry1B-class gene, the cry1Ca1 gene, and the cry9Db1 gene, all derived from Bacillus thuringiensis. The expressed Cry1B.34 protein is effective against certain susceptible lepidopteran pests by causing disruption of the midgut epithelium. The Cry1B.34 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 PAT protein, encoded by a maize-optimized version of the phosphinothricin acetyltransferase (mo-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 DP910521 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 phosphomannose isomerase (PMI) protein, encoded by the pmi gene from Escherichia coli, serves as a selectable marker in plant tissue during transformation which allows for tissue growth using mannose as the carbon source. The PMI protein is found in several approved events that are currently in commercial use.

Tissue samples were harvested at six different sites during the 2020 growing season between the United States (US) and Canada. Tissue samples were collected for leaf (V6, V9, R1, and R4 growth phases), root (V9, R1, and R4 growth phases), pollen (R1 growth phase), stalk (R1 growth phase), forage (R4 growth phase), and grain (R6 growth phase). Expression levels for Cry1B.34, PAT, and PMI proteins were determined using enzyme-linked immunosorbent assay (ELISA).

For human food use, only maize grain is expected to be used. Cry1B.34 protein was observed at 5.5 (+ 2.8) ng/mg dry weight. PAT protein was observed at 10 (+ 3.3) ng/mg dry weight. PMI protein was observed at 5.2 (+ 1.8) ng/mg dry weight.

Dietary exposure

It is expected that DP910521 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 DP910521.

Nutrition

The petitioner provided compositional data for DP910521, non-GM near-isoline control maize (control), and four 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 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 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 DP910521 were noted (P < 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.

Statistically significant differences compared to the control were observed for the following components (control vs DP910521): phytic acid (0.972 vs 0.927 % dry weight), and moisture (20.4 vs 21.1 %). In both cases, the composition of DP910521 was within the reference ranges provided by the petitioner and the expected range for conventional maize. There were no differences in the fibre, fatty acid, amino acid, vitamin, mineral, or secondary metabolite content between DP910521 and the control maize.

The Bureau of Nutritional Sciences (BNS) has not identified any nutritional concerns related to the proposed use of DP910521.

Chemistry

Chemical contaminant residue data have not been provided, nor have any unique contaminant considerations been identified with respect to DP910521. 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 maize, 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 Bureau of Chemical Safety (BCS) evaluated the safety of DP910521 by assessing the potential toxicity of the Cry1B.34, PAT, and PMI proteins.

Bioinformatics analysis conducted by the petitioner confirmed the absence of significant similarity of the novel Cry1B.34 protein to known toxins that are harmful to humans.

Experimental data using a surrogate protein produced in a bacterial expression system provided by the petitioner suggests that the Cry1B.34 protein is heat sensitive at temperatures ≥ 75°C and the protein is expected to be susceptible to in vivo digestive processes, similar to other dietary proteins.

An acute oral toxicity study in mice was conducted that followed the Organisation for Economic Co-operation and Development (OECD) test guidelines and was compliant with Good Laboratory Practices (GLP). No test substance-related mortality or clinical observations were observed at the limit dose of 5000 mg/kg body weight (bw).

The BMH confirmed that the PAT protein expressed in DP910521 is identical to the PAT protein expressed in maize DP-915635 ^{Footnote 1}, previously approved by Health Canada. The petitioner provided updated bioinformatic analyses confirming the absence of significant similarity of the PAT protein to known toxins that are harmful to humans.

To support the safety of the PAT protein, the petitioner used a weight of evidence approach based on its equivalence to PAT proteins studied in published peer-reviewed scientific literature. Hérouet et al. (2005)^{Footnote 2} reported that enzyme activity is inactivated after 10 minutes at temperatures ≥ 55°C and that the protein is digested quickly (less than 0.5 minutes) in a simulated gastric fluid assay, similar to other dietary proteins. No acute toxicity was observed with intravenous administration of 1 or 10 mg PAT/kg bw to female OF1 mice (5/group), that were subsequently observed for 15 days for clinical signs and changes in body weight. Health Canada has reported low acute oral toxicity for the PAT protein (LD50 5000 mg/kg bw) in maize lines approved for food use ^{Footnote 3Footnote 4}.

The BMH confirmed that the PMI protein expressed in DP910521 is identical to the PMI protein expressed in maize DP-915635, previously approved by Health Canada. The petitioner provided updated bioinformatic analyses confirming the absence of significant similarity of the PMI protein to known toxins that are harmful to humans.

The petitioner provided evidence that the PMI protein is rapidly degraded in simulated gastric fluid and simulated intestinal fluid (Reed et al., 2001) ^{Footnote 5}, thus the protein is expected to be susceptible to in vivo digestive processes, similar to other dietary proteins. The PMI protein was found to have low acute oral toxicity. No clinical signs of toxicity or effects on body weight gain were identified, and no gross abnormalities were observed at necropsy, when tested at 3030 mg/kg bw in mice (Reed et al., 2001).

The PMI protein has been used as a selectable marker in several different crops authorized by Health Canada for food use (e.g., MIR162, Golden Rice, MIR604 maize, 5307, maize, 3272 maize).

The dietary exposures for the Cry1B.34, PAT and PMI proteins are expected to be low, and are likely overestimates because it is assumed that all corn products eaten would be derived from this variety, and furthermore did not account for any reductions from food processing.

Based on the available toxicity data, the BCS has not identified any toxicological food safety concerns with use of DP910521 as proposed.

Allergenicity

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 from the Health and Environmental Sciences Institute (HESI). Searches included an eighty amino acid sliding window search to identify alignments greater than 35% identity; and an eight amino acid exact match.

No significant homology matches were identified for the Cry1B.34 protein, suggesting that the protein is unlikely to cross react with known allergens. The novel Cry1B.34 protein is sensitive to heat at temperatures of ≥ 75°C, suggesting that the protein would be denatured during the processing of some corn products. Experimental data demonstrated that the Cry1B.34 protein is expected to be susceptible to in vivo digestive processes, similar to other dietary proteins.

No significant homology matches were identified for the PAT protein. This is consistent with previous bioinformatics analysis (Hérouet et al., 2005). The PAT protein is inactivated under elevated temperatures (10 minutes at ≥ 55°C) and the protein is digested quickly in simulated gastric fluid, similar to other dietary proteins.

No sequence similarity greater than 35% over 80 amino acids was identified for the PMI protein. One contiguous 8-amino acid match was identified for the CAC83047.1 alpha-parvalbumin frog allergen. An acceptable scientific rationale was provided for this match as being a false positive. In a previously assessed corn event containing the identical PMI protein, an exact match of 8 contiguous amino acids for alpha-parvalbumin from frog was also identified. Immunoglobulin E (IgE) binding using the serum of the single individual known to have this allergy was determined to be negative for cross-reactivity, indicating that serum IgE does not recognize any portion of the PMI protein as an allergenic epitope. Furthermore, the petitioner presented evidence that the PMI protein is expected to be susceptible to in vivo digestive processes, similar to other dietary proteins.

Based on the information provided, the BCS has not identified any allergenic concerns with the use of DP910521 as proposed.

Conclusion

Health Canada's review of the information presented in support of the use of DP910521 does not raise concerns related to food safety.

Health Canada's opinion refers only to the food use of DP910521. 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