Novel food information - Herbicide-tolerant and insect-protected corn event MZIR098

Herbicide-tolerant and insect-protected corn event MZIR098

Health Canada has notified Syngenta Canada Inc. that it has no objection to the food use of herbicide-tolerant and insect-protected corn event MZIR098. 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.

Background:

The following provides a summary of the notification from Syngenta Canada Inc. and the evaluation by Heath Canada and contains no confid

1. Introduction

Syngenta Canada Inc. has developed MZIR098 corn to provide dual modes of action for corn rootworm control in a single event and also to be tolerant to glufosinate-ammonium herbicides. Recombinant DNA techniques were used in the development of MZIR098 corn to introduce the insecticidal proteins eCry3.1Ab and mCry3A, and the phosphinothricin acetyltransferase (PAT) protein which was used as a selectable marker in the development of MZIR098 corn.

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 MZIR098 corn was developed; how the composition and nutritional quality of MZIR098 corn compared to non-modified varieties; and the potential for MZIR098 corn to be toxic or cause allergic reactions. Syngenta Canada Inc. has provided data that demonstrates that MZIR098 corn is as safe and of the same nutritional quality as traditional corn 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 28). Foods derived from MZIR098 corn 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

(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 MZIR098 corn and data that characterize the genetic modification which results in the herbicide-tolerant and insect-protected corn event through the expression of the insecticidal proteins eCry3.1Ab and mCry3A, and the PAT protein.

MZIR098 corn was developed by Agrobacterium-mediated transformation of immature embryos of proprietary variety NP2222 utilizing the vector pSYN17629.  The vector contains three gene expression cassettes: ecry3.1Ab, mcry3A, and pat-08.  The pat-08 gene was used as a selectable marker during the transformation process.

The ecry3.1Ab expression cassette consists of the ecry3.1Ab coding region regulated by a CMP promoter from cestrum yellow leaf curling virus (CMP-04) and the nopaline synthase (NOS) terminator sequence from A. tumefaciens (NOS-05-01), as well as the NOS enhancer sequence (NOS-02).  The mcry3A expression cassette consists of the mcry3A coding region regulated by a corn ubiquitin promoter (Ubi1-18) and NOS terminator (NOS-20).  The pat-08 expression cassette consists of the pat-08 coding region regulated by the 35S promoter from cauliflower mosaic virus (35S-04) and the NOS terminator (NOS-05-01).

The mCry3A protein produced by MZIR098 corn is a modified Cry3A protein from the soil bacterium Bacillus thuringiensis subsp. Tenebrionis.  The modification enhances insecticidal activity against western corn rootworm (Diabrotica virgifera virgifera) and other related coleopteran pests.  The engineered protein eCry3.1Ab is a chimera of mCry3A and Cry1Ab.  The Cry1Ab protein is from B. thuringiensis subsp. Kurstaki. The pat-08 transgene was derived from the soil bacterium Streptomyces viridochromogenes.  PAT acetylates glufosinate-ammonium which inactivates it and confers tolerance to glufosinate-ammonium containing herbicides.

3. Characterization of the Modified Plant

Southern blot analysis was performed to characterize the insertion of the T-DNA into the corn genome.  The restriction enzymes HindIII, XcmI, and BmtI were used to cut within the insert or within the flanking corn genomic DNA sequence. The use of three overlapping probes spanning the T-DNA sequence resulted in hybridization patterns that indicated a single copy of T-DNA present at a single locus in MZIR098 corn.

MZIR098 genomic DNA digested with a combination of restriction enzymes was hybridized with two probes spanning the backbone sequence of the vector and produced no detectable hybridization bands.  This indicates that MZIR098 corn does not contain any detectable backbone sequence from the transformation vector, and only the intended T-DNA was inserted.

The organization of the elements within the MZIR098 insert was confirmed using PCR and DNA sequence analysis.  The complete sequence of the single DNA insert from pSYN17629 and the adjacent flanking DNA was determined.  The analysis confirmed that the sequence and organization of the DNA is identical to the corresponding region in the vector T-DNA with no rearrangements or base-pair changes.  Some truncation occurred at the right and left border ends of the T-DNA during the transformation process in MZIR098 corn.  The right border along with 10 base pairs of non-coding sequence were truncated and 10 base pairs from the left border were truncated. Truncations of the border sequences are not uncommon for Agrobacterium-mediated transformation events. The sequencing analysis of the MZIR098 insertion site demonstrated that 24 bp from the corn genomic sequence were deleted during the integration of the MZIR098 insert.

An open reading frame analysis of the sequences spanning the junctions between the corn genome and the T-DNA insert in MZIR098 corn was conducted.  The bioinformatics analysis identified a single potential new open reading frame based on a nucleotide sequence that spanned the genomic DNA and insert DNA which was contained between a start codon (ATG) and a stop codon (TAG, TAA, or TGA).  This hypothetical open reading frame was shown to share no biologically relevant amino acid sequence similarities to known putative allergens or toxins.

The heritability and stability of the T-DNA insert in MZIR098 was demonstrated to be stable across five generations using Southern blot analysis.

MZIR098 corn segregation data was collected over three generations and analyzed using chi-square analysis.  The observed segregation ratios for ecry3.1Ab, mcry3A, and pat-08 were as expected for a gene at a single locus inherited according to Mendelian principles.

4. Product Information

Three new proteins would be expected to be expressed in MZIR098 corn based on the characterization of the inserted genetic material.  The ecry3.1Ab gene encodes a 73.7 kDa protein, the mcry3A gene encodes a 67.7 kDa protein, and the pat-08 gene encodes a 20.5 kDa protein.

Characterization and equivalency of the proteins produced in MZIR098 to proteins produced in previously approved products was conducted using a comparison of deduced amino acid sequence alignment, peptide mass coverage, western blot analysis, and glycosylation analysis.

The eCry3.1Ab protein produced in MZIR098 corn was shown to be identical in amino acid sequence to the eCry3.1Ab produced in 5307 corn.  The mCry3A protein produced in MZIR098 corn was shown to be identical in amino acid sequence to the mCry3A produced in MIR604 corn.

Peptide mass coverage analysis was used to provide further evidence for the identity of purified eCry3.1Ab and of purified mCry3A from MZIR098 corn extract by liquid chromatography-tandem mass spectrometry (LC-MS/MS).  The collective analysis of three proteolytic digests of the purified eCry3.1Ab from MZIR098 yielded a coverage of 85.9% of the total predicted eCry3.1Ab amino acid sequence.  The collective analysis of three proteolytic digests of the purified mCry3A from MZIR098 yielded a coverage of 91.6% of the total predicted eCry3.1Ab amino acid sequence.

Western blot analysis showed that the apparent molecular weight of eCry3.1Ab produced in MZIR098 corn was consistent with the predicted molecular weight of 73.7 kDa and that the apparent molecular weight of mCry3A in MZIR098 was consistent with the predicted molecular weight of 67.7 kDa.  A glycosylation analysis demonstrated the absence of post-translational glycosylation of eCry3.1Ab and mCry3A produced in MZIR098 corn; therefore, no post-translational glycosylation of the proteins occurs in MZIR098 corn.

The nucleotide sequence of pat-08 encoding PAT in MZIR098 corn was shown to be identical to the nucleotide sequence of pat encoding PAT in Bt11 corn.  The deduced amino acid sequence of the PAT protein in MZIR098 corn and BT11 corn is identical. The collective analysis of three proteolytic digests of the purified PAT from MZIR098 yielded a coverage of 96% of the total predicted PAT protein amino acid sequence.  Western blot analysis showed the protein was cross-reactive with the PAT-specific antibody and the apparent molecular weight of PAT in MZIR098 corn was consistent with the predicted molecular weight of 20.5 kDa.  A glycosylation analysis demonstrated the absence of post-translational glycosylation of PAT produced in MZIR098.

The expression levels of the eCry3.1Ab, mCry3A and PAT protein in various corn tissues was determined by enzyme-linked immunosorbent assay (ELISA).  The tissues analyzed were leaves and roots (V6, R1, R6, and senescence), whole plants (V6, R1, R6), kernels (R6 and senescence), and pollen (R1). Kernels from MZIR098 corn are the most likely tissue to enter the food supply, as either grain or grain by-products.  Corn is generally consumed at the senescent stage of development with the levels of protein as follows in senescent stage kernels: average eCry3.1Ab 2.08 µg/g dry weight, average mCry3A 11.21 µg/g dry weight, PAT of below the LOD for the assay (0.025 µg/g dry weight).

5. Dietary Exposure

The use of MZIR098 corn, and products derived from it, will be similar to traditional corn varieties and is not anticipated to result in a change in the dietary intake of corn and corn-derived products.

6. Nutrition

For the compositional analysis, Syngenta Inc. used conventional agronomic practices to plant, maintain, and harvest MZIR098 corn (test), MZIR098 treated with the trait specific herbicide (test-TSH), and the corresponding non-transgenic, near-isogenic control corn (control) in a randomized complete block design with four replicates at each of eight field locations in the United States in 2013. In addition, at each location, six non-transgenic corn reference lines were also grown as reference material. The field trials were conducted at two sites in Iowa, three sites in Illinois, and one site in each of Nebraska, Kansas, and Pennsylvania. The field locations selected by Syngenta Inc. were representative of the normal agronomic field conditions for their respective geographic regions.

Grain and forage were harvested at physiological maturity (R6 growth and R5 growth stages, respectively), dried, subsampled, stored and transported for compositional analysis.  Appropriate AOAC or other methods were used for analysis.

Grain and forage were analyzed for various nutritional and anti-nutritional components which were selected based on recommendations in the OECD Consensus Document on Compositional Considerations for New Varieties of Maize (2002). The analytes measured included: Grain: Proximates (moisture, protein, fat, ash, carbohydrates, total dietary fibre (TDF), starch); Minerals: calcium, copper, iron, magnesium, manganese, phosphorus, potassium, selenium, sodium and zinc; Vitamins: A (β-carotene), B1 (thiamine), B2 (riboflavin), B3 (niacin), B6 (pyridoxine), B9 (folic acid) and E (α-tocopherol). Amino acids: alanine, lysine, arginine, methionine, aspartic acid, phenylalanine, cysteine, proline, glutamic acid, glycine, histidine, serine, threonine, tryptophan, isoleucine, tyrosine, leucine and valine; Fatty acids: 8:0 caprylic,  18:0 stearic, 10:0 capric,18:1 oleic, 12:0 lauric, 18:2 linoleic, 14:0 myristic, 18:3 gamma linolenic, 14:1 myristoleic, 18:3 alpha-linolenic, 15:0 pentadecanoic,  20:0 arachidic,  15:1 pentadecenoic, 20:1 eicosenoic, 16:0 palmitic,  20:2 eicosadienoic, 16:1 palmitoleic,  20:3 eicosatrienoic, 17:0 heptadecanoic, 20:4 arachidonic, 17:1 heptadecenoic and 22:0 behenic; Secondary metabolites: ferulic acid, furfural, inositol, p-coumaric acid ;  Antinutrients: phytic acid, raffinose, trypsin inhibitor. Forage: Proximates (moisture, protein, fat, ash carbohydrates, acid detergent fibre (ADF), Neutral detergent fibre (NDF) and minerals (calcium and phosphorus).

The compositional comparison between MZIR098 corn and the corresponding non-transgenic, near-isogenic control corn was based on compositional data combined across all field locations (the combined-location analysis). Statistically significant differences were identified at the 5% level (α = 0.05).

Of the 73 components measured by the petitioner in grain, 15 components had values below the level of quantification and were excluded from statistical analysis.

Of all the analytes measured, 12 showed statistically significant differences in the MZIR098 corn (treated and untreated) compared to the corresponding control corn. Eight components (calcium, copper, potassium, β-carotene, vitamin E, heptadecanoic acid, linoleic acid and stearic acid) were higher in MZIR098 corn (treated and/or untreated) than control, whereas four components (starch, lysine, oleic acid and arachidic acid) were higher in the control than MZIR098 corn (treated and/or untreated).

It was noted that the statistically significant differences were not consistent for test vs. test-TSH MZIR098. Four analytes (lysine, calcium, copper and vitamin E) were observed to be different in test vs. control, but not test-TSH vs. control, while one analyte (starch) was different in test-TSH vs. control but not test vs. control.

The magnitude of the differences observed in the test means when compared to the control means were low (max ~6%). The statistically significant but small differences observed in these analytes do not raise any nutritional concerns as some components (e.g., minerals) vary considerably as result of environmental and agronomic factors.

It was noted that ferulic acid levels were higher in both the control and MZIR098 grain than in the reference lines.  However, the levels of all components analysed in grain and forage were within the range reported for the conventional control, the commercial reference lines or values reported in the ILSI-CCDB (2014).

Overall, the composition of the MZIR098 corn was similar to control and commercial varieties of corn. The small differences measured for some analytes would not impact nutritional safety.

7. Toxicology

The safety of MZIR098 corn was evaluated by assessing the potential toxicity of the novel mCry3A (mcry3A), eCry3.1Ab (ecry3.1Ab) and PAT (pat-08) proteins, derived from B. thuringiensis and S. viridochromogenes. The mean expression levels of mCry3A, eCry3.1Ab and PAT proteins in MZIR098 senescent corn grain were 11.21, 2.08 µg/g tissue d.w. and below the limit of detection (0.025 µg/g tissue d.w.), respectively.

The novel expression of PAT protein derived from S. viridochromogenes (strain Tü494) has been previously approved by Health Canada in various crop varieties intended for human consumption.  The safety of S. viridochromogenes PAT protein is well established and therefore does not require re-evaluation in this submission.

The safety of mCry3A and eCry3.1Ab, identical in deduced amino acid sequence to those expressed in MZIR098, was previously established for corn varieties MIR604 (NF-134) and corn event 5307 (NF-236) which were approved by Health Canada in 2007 and 2013, respectively.

Syngenta Canada Inc. used bridged data from the previous approvals to support the safety of mCry3A and eCry3.1Ab.  The data included acute oral toxicity studies performed with mice (5 mice/sex/group), as well as simulated gastric fluid, thermostability and bioinformatics assays.

The results of these studies showed that mCry3A and eCry3.1Ab would not be expected to pose a toxicological concern to consumers as neither protein shared amino acid sequence homology with putative toxins and no adverse effects were reported in treated mice.

Further, both proteins were found to be readily degraded and digested under conditions normally found during food preparation and in the gastrointestinal tract.  As such, intact and functional mCry3A and eCry3.1Ab protein are not expected to be absorbed in humans following consumption with food, and would not result in systemic exposure.

Although the expression levels for mCry3A and eCry3.1Abproteins differ between MZIR098 corn and the previously approved corn varieties, the new margins of exposure (i.e., the difference between the predicted levels of exposure and the no-observable-effect level reported in the animal acute study) calculated for the greatest consumers (90th percentile of consumers aged 2-3 years old; eater’s only) were four orders of magnitude and are considered sufficiently large to be protective of consumer safety.

Based on the body of evidence and previous decisions, the consumption of mCry3A, eCry3.1Ab and PAT proteins from MZIR098 would not be expected to pose a toxicological human health concern.

8. Allergenicity

The safety of MZIR098 corn was evaluated by assessing the potential allergenicity of the novel mCry3A (mcry3A), eCry3.1Ab (ecry3.1Ab) and PAT (pat-08) proteins, derived from B. thuringiensis and S. viridochromogenes.

The novel expression of PAT protein derived from S. viridochromogenes (strain Tü494) has been previously approved by Health Canada in various crop varieties intended for human consumption.  The safety of S. viridochromogenes PAT protein is well established and therefore does not require re-evaluation in this submission.

The safety of mCry3A and eCry3.1Ab, identical in deduced amino acid sequence to those expressed in MZIR098, was previously established for corn varieties MIR604 (NF-134) and corn event 5307 (NF-236) which were approved by Health Canada in 2007 and 2013, respectively.

The petitioner used bridged data from the previous approvals to support the safety of mCry3A and eCry3.1Ab.  The data included simulated gastric fluid, thermostability and bioinformatics assays.

The results of these studies showed that mCry3A and eCry3.1Ab would not be expected to pose an allergenic concern to consumers as neither protein shared amino acid sequence homology with putative allergens.  Further, both proteins were found to be readily degraded and digested under conditions normally found during food preparation and in the gastrointestinal tract.  As such, intact and functional mCry3A and eCry3.1Ab protein are not expected to be absorbed in humans following consumption with food, and would not result in systemic exposure.

Based on the body of evidence and previous decisions, the consumption of mCry3A, eCry3.1Ab and PAT proteins from MZIR098 would not be expected to pose a safety concern from an allergy perspective.

Conclusion:

Health Canada’s review of the information presented in support of the food use of herbicide-tolerant and insect-protected corn event MZIR098 does not raise concerns related to food safety. Health Canada is of the opinion that food derived from corn event MZIR098 is as safe and nutritious as food from current commercial corn varieties.

Health Canada's opinion deals only with the food use of corn event MZIR098. Issues related to its use as animal feed have been addressed separately through existing regulatory processes in the Canadian Food Inspection Agency (CFIA).  From their assessment, the CFIA concluded that there are no concerns from an environmental and feed safety perspective. This perspective is applicable to the food and feed products derived from corn event MZIR098 destined for commercial sale.

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
novelfoods-alimentsnouveaux@hc-sc.gc.ca

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