Novel food information - Herbicide-Tolerant MZHG0JG Corn

Health Canada has notified Syngenta Canada Inc. that it has no objection to the food use of Herbicide-Tolerant MZHG0JG corn (henceforth referred to as MZHG0JG corn). The Department conducted a comprehensive assessment of this corn event 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 confidential business information.

1. Introduction

Syngenta Canada Inc. has developed a genetically modified corn event using recombinant DNA techniques. This corn event contains the mepsps-02 gene, which encodes a double-mutant 5-enolpyruvylshikimate-3-phosphate synthase (mEPSPS) derived from Zea mays, and the pat-09 gene, which encodes a phosphinothricin N-acetyltransferase (PAT) derived from Streptomyces viridochromogenes. Expression of these two proteins confers tolerance to both glyphosate and glufosinate herbicides, respectively.

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 this corn event was developed; how the composition and nutritional quality of this event compared to non-modified corn varieties; and the potential for this corn event to be toxic or cause allergic reactions. Syngenta Canada Inc. has provided data that demonstrate that MZHG0JG 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). MZHG0JG corn 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 MZHG0JG corn and the molecular biology data that characterize the genetic change, which results in tolerance to glyphosate and glufosinate herbicides. This phenotype was achieved by Agrobacterium-mediated transformation of the conventional corn variety NP2222 using the transformation plasmid pSYN18857 which contains a transgenic (T-DNA) expression cassette containing two genes: mepsps-02, which encodes a modified variant of a 5-enolpyruvylshikimate-3-phosphate synthase (mEPSPS) derived from Zea mays (corn), and pat-09, which encodes a phosphinothricin N-acetyltransferase (PAT) derived from Streptomyces viridochromogenes.

The native corn EPSPS is involved in the biosynthesis of essential aromatic amino acids. The mEPSPS protein was generated by introducing two amino acid substitutions. These two modifications result in a lower affinity for the herbicide glyphosate compared to the native EPSPS enzyme and allow MZHG0JG corn to continue to thrive in the presence of glyphosate. The PAT protein is a phosphinothricin N-acetyltransferase (PAT) derived from S. viridochromogenes. The PAT protein acetylates glufosinate, rendering the herbicidal compound non-toxic to the plant. The expression of these two proteins thus confers tolerance to both glyphosate and glufosinate herbicides, respectively.

The T-DNA expression cassette contains the following genetic elements: the FMV-05 enhancer region derived from the figwort mosaic virus (FMV), which increases gene expression; the CaMV 35S-05 enhancer region derived from the cauliflower mosaic virus (CaMV), which activates heterologous core promoters; the Ubi158092 promoter derived from Z. mays (corn), which drives constitutive expression of the mepsps-02 gene; the TMV-03 enhancer region derived from the tobacco mosaic virus (TMV), which is a translational enhancer in plants; the optimized transit peptide (OTP-02) derived from Helianthus annus (sunflower), which directs the mEPSPS protein to the chloroplast; the mepsps-02 gene derived from Z. mays (corn), which encodes the modified mEPSPS protein; the Ubi158-02 terminator derived from Z. mays (corn), which is a transcriptional terminator; the 35S-19 promoter derived from the CaMV, which drives constitutive expression of the pat-09 gene; the pat-09 gene derived from S. viridochromogenes; and the NOS-05-01 terminator derived from Agrobacterium tumefaciens, which is a transcriptional terminator and provides a site for polyadenylation.

3. Characterization of the Modified Plants

Southern blot analysis and DNA sequencing of MZHG0JG corn confirmed a single copy of the T-DNA insert located at a single locus in the MZHG0JG corn genome. No plasmid backbone sequences were detected. A comparison of the T-DNA insert with the transformation plasmid pSYN18857 showed that the T-DNA insert was intact, with no rearrangements or base pair changes. Some truncation occurred at the right and left border regions of the T-DNA insert during the transformation process however these deletions have no effect on the functionality of the insert.

Basic Local Alignment Search Tool for translated nucleotides (BLASTX) analyses comparing the genomic sequences flanking the T-DNA insert with sequences in the public databases indicated that the insert does not disrupt any known endogenous corn gene. Analysis also indicated that no potential open reading frames (ORFs) equal or greater than 30 amino acids in length (based on the presence of start and stop codons) span the junctions between the corn genome and the T-DNA insert.

Generational stability of the T-DNA insert was determined across multiple generations of MZHG0JG corn. Genomic DNA from four consecutive generations was analyzed by Southern blot analyses and the presence of the insert was confirmed in each generation.

Inheritance of the mepsps-02 and pat-09 genes in progeny plants was examined using segregation analysis. Real-time PCR analysis was used to detect both the mepsps-02 and pat-09 genes present in the T-DNA insert. Observed segregation patterns were compared to the expected segregation patterns and these data were compared using a chi-squared (c2) distribution analysis. A c2 value of ³0.05 was treated as the cut-off for statistical support of a 1:1 (positive: negative) ratio for all three generations (i.e. BC1F1, BC2F1, and BC3F1). The c2 values obtained confirmed that both genes are inherited according to Mendelian principles, further supporting the conclusion that the T-DNA insert is stably integrated into a single locus in the MZHG0JG genome.

4. Product Information

MZHG0JG corn differs from its traditional counterpart by the addition of the mepsps-02 and pat-09 genes and their associated regulatory elements. The insertion of these genes results in the expression of the novel mEPSPS and PAT proteins in MZHG0JG corn. Expression of these two proteins confers tolerance to glyphosate and glufosinate herbicides.

The mEPSPS protein is a double-mutant 5-enolpyruvylshikimate-3-phosphate synthase derived from Zea mays. The native corn EPSPS is involved in the biosynthesis of essential aromatic amino acids. The mEPSPS protein has a lower affinity for the herbicide glyphosate compared to the native EPSPS enzyme and allows MZHG0JG corn to continue to thrive in the presence of glyphosate. The PAT protein is a phosphinothricin N-acetyltransferase (PAT) derived from Streptomyces viridochromogenes. The PAT protein acetylates glufosinate, rendering the herbicidal compound non-toxic to the plant. The expression of these two proteins thus confers tolerance to both glyphosate and glufosinate herbicides, respectively.

The host organism, Z. mays (corn), has a long history of safe use for food in Canada and is not associated with toxic or allergenic concerns. Furthermore, the donor organism S. viridochromogenes is a microorganism that is ubiquitous in the environment and not known to commonly cause pathogenic or allergenic concerns.

The petitioner has provided data to demonstrate the level of expression of the mEPSPS and PAT proteins in MZHG0JG corn. Protein expression levels of mEPSPS and PAT in various tissues (i.e., whole plant, root, leaf, and kernel) of MZHG0JG corn were determined using enzyme-linked immunosorbent assay (ELISA). Plant material was produced at four field locations in the United States and harvested over the entire growing season of 2013. Each trial had two replicated plots of MZHG0JG corn and one plot of non-transgenic, near-isogenic corn planted in a randomized complete-block design. One of the MZHG0JG corn plots received a single post-emergent spray application of the trait-specific herbicide glufosinate at the V3/V4 growth stage. The same plot also received a single post-emergent spray application of the trait-specific herbicide glyphosate at the V5 growth stage.

Mean levels of mEPSPS protein expression were extremely low in all tissues, with the highest level of expression observed in R1 leaf tissue (1816 mg/g dry weight for herbicide-treated samples, 1934 mg/g dry weight for samples not treated with herbicide), and lowest in kernel tissue at R6 and senescence growth stages (61.14 and 42.20 mg/g dry weight, respectively for herbicide-treated samples, and 58.23 and 36.89 m/g dry weight, respectively for samples not treated with herbicide).

Mean levels of PAT protein expression were extremely low in all tissues, with the highest level of expression observed in R1 leaf tissue (10.22 mg/g dry weight for herbicide-treated samples, 9.95 mg/g dry weight for samples not treated with herbicide), and lowest in kernel tissue at R6 and senescence growth stages which were below the Limit of Detection (0.025 mg/g dry weight).

5. Dietary Exposure

MZHG0JG corn is the result of a genetic modification to confer tolerance to glyphosate and glufosinate herbicides, with no intention to significantly alter nutritional parameters in the food. It is expected that MZHG0JG corn will be used in applications similar to conventional corn varieties. The petitioner does not anticipate a significant change in the food use of corn with the introduction of MZHG0JG corn.

6. Nutrition

The petitioner provided a compositional study that measured and compared key food nutrients, anti-nutrients, and secondary metabolites of grain from MZHG0JG corn and a non-transgenic, near-isogenic control corn variety.

The information submitted by the petitioner was reviewed in accordance to Health Canada's Guidelines for the Safety Assessment of Novel Foods Derived from Plants and Microorganisms (Health Canada, 2006) and the OECD Consensus Document on Compositional Considerations for New Varieties of maize (Zea mays); Key Food and Feed Nutrients, Anti-nutrients and Secondary Metabolites (Organization for Economic Co-operation and Development, 2002).

Specifically, the compositional data was obtained from untreated (i.e. no post-emergent spray application of herbicides were performed) MZHG0JG corn (test) and non-transgenic, near-isogenic corn (control), and six non-transgenic commercial corn varieties (reference lines) grown in 2013 at eight locations in the United States in a randomized complete block design with four replicates per entry. A comparison was also done between MZHG0JG corn treated with trait-specific herbicides (i.e. glyphosate and glufosinate) (test + TSH) and the control corn.

Analytical data from the grain samples were combined across all sites and the mean levels were compared using appropriate statistical methods. Moreover, to determine the biological significance of any observed statistical differences, mean levels of analytes were compared non-statistically with ranges from the non-transgenic corn reference lines, and with the ranges for conventional corn reported in the 2014 International Life Sciences Institute (ILSI) Crop Composition Database.

The nutritional and compositional analytes measured in MZHG0JG corn (treated and untreated with trait-specific herbicides) and the non-transgenic, near-isogenic control corn were: proximates (moisture, protein, fat, ash, and carbohydrates), starch, fiber (acid detergent fiber, neutral detergent fiber, and total dietary fiber), minerals (calcium, copper, iron, magnesium, manganese, phosphorus, potassium, selenium, sodium, and zinc), vitamins (A [b-carotene], B1 [thiamine], B2 [riboflavin], B3 [niacin], B6 [pyridoxine], B9 [folic acid], and E [a-tocopherol]), amino acids (alanine, arginine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), fatty acids (8:0 caprylic, 10:0 capric, 12:0 lauric, 14:0 myristic, 14:1 myristoleic, 15:0 pentadecanoic, 15:1 pentadecenoic, 16:0 palmitic, 16:1 palmitoleic, 17:0 heptadecanoic, 17:1 heptadecenoic, 18:0 stearic, 18:1 oleic, 18:2 linoleic, 18:3 gamma linolenic, 18:3 linolenic, 20:0 arachidic, 20:1 eicosenoic, 20:2 eicosadienoic, 20:3 eicosatrienoic, 20:4 arachidonic, and 22:0 behenic), secondary metabolites (ferulic acid, furfural, inositol, and p-coumaric acid), and anti-nutrients (phytic acid, raffinose, and trypsin inhibitor).

Statistical differences were noted in 12 out of 73 (16.4%) analytes tested in grain of untreated test compared to untreated control: neutral detergent fiber (-4.35%), copper (-15.0%), iron (-4.15%), b-carotene (+16.55%), vitamin B6 (-5.62%), a-tocopherol (-3.31%), asparagine (-4.04%), arginine (-3.03%), tyrosine (-2.79%), 17:0 heptadecenoic (+3.84%), 18:3 linolenic (+1.69%), and p-coumaric acid (+14.5%).

When comparing the treated test and the untreated control, 14 out of 73 analytes in grain were statistically significantly different: copper (-14.6%), iron (-3.11%), b-carotene (+22.8%), thiamin (-5.31%), a-tocopherol (-4.13%), asparagine  (-4.63%), lysine (-3.72%), tyrosine (-2.44%), 16:0 palmitic (-1.40%), 17:0 heptadecanoic (+2.88%), 18:3 linolenic (+2.25%), p-coumaric acid (+12.2%), and inositol (+9.06%).

Under both test conditions (herbicide treated and untreated), where a difference was determined to be statistically significant, the values were within the 99% tolerance intervals generated from the reference lines and/or the ILSI Crop Composition Database range of values. Therefore, these differences were not considered to be biologically relevant or nutritionally important. The analytes with the largest magnitude of change (> ±10%) such as copper, b-carotene, and p-coumaric acid are not expected to impact dietary intakes and pose a nutritional concern. The use of the trait-specific herbicides did not have any observed effect on the composition of MZHG0JG corn.

7. Chemistry/Toxicology

The mEPSPS protein was generated by modifying an endogenous gene in corn. There is no safety concern associated with using corn as a donor organism of genetic material. Further, the petitioner stated that an identical mEPSPS protein is expressed in another commercially available corn variety (GA21 ) that has been sold in the Canadian market since 1999 without apparent adverse effects in consumers.

To determine if the mEPSPS protein had any similarity to known or putative toxins, the petitioner conducted a BLASTP alignment (E-value of 1×10-5) of the mEPSPS amino acid sequence with that of known and putative toxins (NCBI Entrez Protein database 2015; 57,851,050 sequences; "toxins" search, 15,588 sequences). It was determined that mEPSPS did not share ³35% amino acid identity (over a sequence of 80 amino acids) with known toxins or other biologically active proteins. Therefore, no significant identity was observed between the amino acid sequence of mEPSPS and any known or putative toxins.

To supplement the safety data that was provided in the submission, Health Canada evaluators found and reviewed data reported in the scientific literature. Hérouet-Guicheney et al. (2009)  performed an acute toxicity study with five female OF1 mice administered 2000 mg of microbially-derived mEPSPS/kg body weight or control material (bovine serum albumin) by gavage as a single dose. Animals were observed for 15 days post-treatment. No treatment-related mortality, signs of clinical toxicity were observed. Additionally, no differences were observed between groups with respect to body weights, body weight gain or macroscopic observations at necropsy. A No Observable Effect Level (NOEL) of 2000 mg/kg body weight was determined.

Hérouet-Guicheney et al. (2009) conducted digestion assays with simulated gastric fluid (SGF) (pepsin; pH 1.2; 37 °C) and simulated intestinal fluid (SIF) (pancreatin; pH 7.5; 37 °C) assays performed on microbially-derived mEPSPS. The results of the assays determined that the novel protein is fully digested within 30 seconds. This finding suggests that the mEPSPS protein would be readily digested by enzymes of the gastrointestinal tract. Therefore, its consumption would not be expected to result in systemic exposure and would not pose a health concern.

PAT protein derived from Streptomyces viridochromogenes has been evaluated by Health Canada numerous times and its toxicological safety is well established. Health Canada has previously approved various crop varieties expressing S. viridochromogenes-derived PAT including soybean, cotton, sugar beet, canola, and corn lines intended for human consumption.

8. Allergenicity

To determine if the mEPSPS protein had any similarity to known or putative allergens, the petitioner conducted a FASTA (version 3.45) alignment of the mEPSPS amino acid sequence with that of known and putative allergens (AllergenOnline database 2015; 1,897 sequences). It was determined that mEPSPS did not share ³35% amino acid identity (over a sequence of 80 amino acids) with known allergens. Further, mEPSPS does not contain potential allergen epitopes as determined by an 8-amino acid segment analysis. The bioinformatics search suggests that mEPSPS, based on protein sequence alone, is not similar to any known allergen.

Many corn products are devoid of protein (e.g. sugars, oil, starch, etc.) and others are highly processed (e.g. corn meal, flour, etc.) and require exposure of the corn grain to very high temperatures during the manufacturing process (e.g. 80-150 °C). The petitioner demonstrated that microbially-derived mEPSPS lost immunoreactivity when incubated at temperatures of ³65 °C for 30 minutes, as determined by ELISA. It is likely that the cooking or processing of corn will remove, degrade and/or denature the mEPSPS protein in the final food product. These actions will result in a reduced amount of active mEPSPS in the human diet. Taken together, it is unlikely that functional mEPSPS protein will be absorbed by humans.

Hérouet-Guicheney et al. (2009) conducted digestion assays with SGF and SIF using microbially-derived mEPSPS protein. The results of the assays determined that the novel protein is fully digested within 30 seconds. This finding suggests that the mEPSPS protein would be readily digested by enzymes of the gastrointestinal tract. Therefore, its consumption would not be expected to result in systemic exposure and would not pose a health concern.

PAT protein derived from Streptomyces viridochromogenes has been evaluated by Health Canada numerous times and its lack of allergenicity is well established. Health Canada has previously approved various crop varieties expressing S. viridochromogenes-derived PAT including soybean, cotton, sugar beet, canola, and maize lines intended for human consumption.

Conclusion:

Health Canada's review of the information presented in support of the food use of MZHG0JG corn does not raise concerns related to food safety. Health Canada is of the opinion that food derived from this event to be as safe and nutritious as food from current commercial corn varieties.

Health Canada's opinion deals only with the food use of MZHG0JG corn. Issues related to its use as animal feed have been addressed separately through existing regulatory processes in the Canadian Food Inspection Agency (CFIA). The CFIA evaluated information provided on the environmental, animal, and human health safety of MZHG0JG corn with the intended use in animal feed.  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 MZHG0JG corn destined for commercial sale.