Novel food information: Enhanced yield and herbicide tolerant maize DP-202216-6

Background:

Health Canada has notified Pioneer Hi-Bred Canada Company that it has no objection to the food use of enhanced yield and herbicide tolerant maize DP-202216-6. The Department conducted a comprehensive assessment of this variety according to its Guidelines for the Safety Assessment of Novel Foods. These Guidelines are based upon internationally accepted principles for establishing the safety of foods with novel traits.

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

1. Introduction

Pioneer Hi-Bred Canada Company developed maize event DP-202216-6 to enhance grain yield potential and impart tolerance to glufosinate-ammonium, the active ingredient in phosphinothricin 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 DP-202216-6 maize was developed; how the composition and nutritional quality of DP-202216-6 maize compared to non-modified varieties; and the potential for DP-202216-6 maize to be toxic or cause allergic reactions. Pioneer Hi-Bred Canada Company provided data that demonstrates that DP-202216-6 maize is as safe and of the same nutritional quality as traditional maize varieties used 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 the Food and Drug Regulations (Division B.28). Food use of DP-202216-6 maize is considered 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

(i) the plant, animal or microorganism exhibits characteristics that were not previously observed in that plant, animal or microorganism.”

2. Development of the modified plant

The petitioner has provided information describing the methods used to develop maize DP-202216-6 and data that characterize the genetic modification that results in the enhanced grain yield potential and herbicide tolerance through the expression of the zmm28 and mo-pat gene cassettes. Maize DP-202216-6 was produced through Agrobacterium mediated transformation of immature maize embryos of the proprietary inbred line PH17AW with the plasmid PHP40099 containing two gene cassettes. The first expression cassette contains the zmm28 gene from Zea mays encoding the ZMM28 protein, a MADS-box transcription factor, controlled by the promoter region from the Zea mays translation initiation factor gos2 (zm-gos2) gene along with the intron region from the Zea mays ubiquitin gene 1 (ubiZM1) and the terminator region from the potato proteinase inhibitor II (pinII) gene. The second cassette contains a maize-optimized version of the PAT gene (mo-pat) from Streptomyces viridochromogenes controlled by the promoter region from the ubiZM1 gene, including the 5’ untranslated region (UTR) and intron, with the terminator being a second copy of the pinII gene.

3. Characterization of the modified plant

Southern blot, PCR and DNA sequence analysis were performed in order to characterize the number of insertion sites and copies of the integrated T-DNA as well as the presence or absence of plasmid backbone sequence. This analysis demonstrated a single intact copy of the T-DNA at a single locus in maize DP-202216-6 and no vector backbone sequences are present.

The stability of the T-DNA was demonstrated using plants from five generations. The event specific PCR results showed the expected genotype ratios based on Mendelian segregation principles. The results confirm the stability of the insert across multiple generations of maize DP-202216-6.

4. Product information

One new protein, PAT, and one native protein, ZMM28, are expressed in maize DP-202216-6 based on the characterization of the inserted genetic material. The PAT protein produced by the insertion of the mo-pat cassette was characterized. The PAT protein expressed in this maize line was of the expected molecular weight as the PAT reference standard protein and the PAT protein in previously approved maize events 1507 and 59122 as shown using Western blot analysis. Amino acid sequence analysis also confirmed that the PAT protein found in maize DP-202216-6 is the same as the PAT protein present in the previously approved events.

Amino acid sequence and Western blot analyses confirmed the native ZMM28 transcription factor protein was identical to the introduced ZMM28 protein in maize DP-202216-6. This maize event has increased and extended expression of this transcription factor protein with expression beginning in the early plant growth stages (V2-V5) as opposed to the V6 growth stage for the native ZMM28 protein. The earlier expression of the transcription factor improves plant vigor and photosynthetic capacity, and enhances nutrient utilization.

Expression levels of ZMM28 and PAT proteins were examined using ELISA or Western blot analysis. Maize tissue samples were collected from leaf, pollen, root, forage, whole plant, and grain at developmental stages that were representative of commercial maize production practices. The expression of the PAT protein was above the assay LLOQ in all tissues except the R6 leaf. The ZMM28 protein was higher in all tissues of maize DP-202216-6 given that a native protein also exists. The concentrations of the ZMM28 protein remained in the parts per billion range as with the levels of the native ZMM28 in the control variety.

5. Dietary exposure

The genetic modification of maize DP-202216-6 is not intended to alter any of its nutritional aspects when compared to conventional non-genetically modified varieties. It is expected that foods derived from maize DP202216 will be used in applications similar to those from conventional maize and thus no change in the food use of maize is anticipated.

6. Nutrition

To evaluate if there were any unanticipated consequences of the genetic modifications to maize DP-202216-6, the nutritional and anti-nutritional components of maize DP-202216-6 were analyzed and compared to non-genetically modified (non-GM), near-isoline controls as well as non-GM commercial reference lines. This was done as part of a field trial conducted in 2017 at 8 different sites in the US (one site in Iowa, Illinois, Indiana, Missouri, Nebraska, Pennsylvania and Texas) and Canada (one site in Ontario). The field trial was a randomized complete block design with four blocks at each site and each block containing maize DP-202216, control maize and non-GM reference lines.

The compositional analytes measured in maize DP-202216-6 and non-GM control maize grain were: protein, fat, total dietary fibre, ash, minerals (calcium, phosphorous, copper, potassium, iron, sodium, magnesium, zinc, and manganese), vitamins (B1, B2, B3, B5, B6, B9, vitamin E, beta-tocopherol, gamma-tocopherol, delta-tocopherol, and total tocopherols), beta-carotene, fatty acids (lauric acid, stearic acid, eicosadienoic acid, myristic acid, oleic acid, behenic acid, palmitic acid, linoleic acid, erucic acid, palmitoleic acid, alpha-linolenic acid, lignoceric acid, heptadecanoic acid, arachidic acid, heptadecenoic acid, and eicosenoic acid), amino acids (alanine, lysine, histidine, arginine, methionine, isoleucine, aspartic acid, phenylalanine, leucine, cysteine, proline, tryptophan, glutamic acid, serine, tyrosine, glycine, threonine, valine), p-coumaric acid, phytic acid, ferulic acid, trypsin inhibitor, and raffinose.

Of the analytes measured, the following showed a statistically significant treatment effect in the maize DP-202216-6 compared to the control: serine (higher in DP-202216-6 than in control), threonine (higher), glycine (higher), methionine (higher), vitamin B1 (higher), vitamin B3 (lower), and p-coumaric acid (higher). These differences between maize DP-202216-6 and controls were acceptable as the analyte levels remained within the tolerance intervals, literature, reference, and/or Organization for Economic Co-operation and Development (OECD) ranges. The remaining analytes were not statistically significantly different in maize DP-202216-6 compared to control.

Based on the information provided on the composition of maize DP-202216-6, there are no nutritional safety concerns with the sale of foods derived from maize DP-202216-6.

7. Chemistry/toxicology

The safety of maize grain derived from event DP-202216-6 is supported by the results of submitted toxicity data including equivalence studies on the expressed proteins ZMM28 and PAT, an updated bioinformatics search for the PAT protein, as well as, references that describe the established low toxicity and heat sensitivity of the PAT protein.

The expressed proteins were assessed for equivalence to the native ZMM28 protein and the known PAT protein. Equivalence is supported by the similarity in amino acid sequence, molecular weight, and immunoreactivity.

The source of the zmm28 gene is maize. The increased and extended expression of the native ZMM28 protein in maize DP-202216-6 is within the limits of natural crop variation, as demonstrated by measurements of the amount of ZMM28 protein in other types of maize. DP-202216-6 maize showed substantial equivalence to non-genetically modified maize, with regard to nutrient composition and agronomic characteristics, which indicates there are no apparent unintended effects. Commonly consumed sweet corn kernels contain higher levels of ZMM28 protein than DP-202216-6 maize grain, and given this history of safe food use, no bioinformatics search or dietary exposure was performed.

A bioinformatics analysis of the amino acid sequence of the PAT protein was conducted, according to European Food Safety Authority (EFSA) guidelines, using the UniProtKB/Swiss-Prot database (searched January 16, 2018). There were no biologically relevant matches for PAT with any proteins associated with toxicity or adverse health effects.

The source of the PAT gene is non-pathogenic bacteria. At the levels present in food, PAT protein is considered safe by Health Canada and is approved in several different crops. An acute oral (gavage) mouse toxicity study, conducted according to OECD guidelines, determined the No Observed Adverse Effect Level (NOAEL) is greater than 5000 mg/kg bw. PAT enzyme activity is sensitive to heat and the protein is easily digested in the stomach, so exposure to the PAT protein is expected to be negligible.

Estimated dietary exposures of PAT protein were calculated by the petitioner (with the assumption that no loss or degradation occurs). The highest exposure (0.045 mg/kg bw per day) occurs in children 1-2 years, which are users at the 95th percentile. To calculate the margin of exposure (MOE) for PAT protein, the petitioner used a NOAEL of 5000 mg/kg bw per day (obtained from an acute toxicity study in mice) and the dietary exposure in children. This conservative MOE of 1.12 x 10⁵ is considered sufficient from a safety perspective.

An allergenicity assessment for ZMM28 protein was not conducted, as this protein is a natural constituent in the diet and not associated with allergenicity.

PAT protein is a novel protein, which quickly degrades within 30 seconds in the simulated gastric fluid assay. Proteins which pose an allergenic risk are typically resistant to degradation in the digestive system. The bioinformatics analysis of the amino acid sequence of the PAT protein did not detect any sequence homology matches with known allergens. Searches were conducted according to EFSA guidelines, using the Comprehensive Protein Allergen Resource (COMPARE) 2018 database (February, 2018).

Maize DP-202216-6 would not be expected to pose a toxicological safety concern and is not expected to pose an allergenic safety concern.

Conclusion:

Health Canada’s review of the information presented in support of the food use of maize DP-202216-6 does not raise concerns related to food safety. Health Canada is of the opinion that food derived from maize DP-202216-6 is as safe and nutritious as food from current commercial maize varieties.

Health Canada's opinion deals only with the food use of DP-202216-6. Issues related to its environmental release and use as animal feed have been addressed separately through existing regulatory processes in the CFIA.

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.

(Également disponible en français)

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

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Date modified:: 2022-03-15

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