Novel Food Information – Maize Event MON 87403

Health Canada has notified Monsanto Canada Inc. that it has no objection to the food use of increased ear biomass maize event MON 87403. 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 Monsanto Canada Inc. and the evaluation by Heath Canada and contains no confidential business information.

1. Introduction

Monsanto Canada Inc. has developed maize event MON 87403 to have increased ear biomass at the early reproductive phase compared to conventional control corn grown under comparable environmental conditions. Recombinant DNA techniques were used in the development of MON 87403 maize. Early reproductive stages in maize are a critical period of maize growth at which the maximum ear biomass (sink size) is determined by a combination of genetics and environmental conditions. Dry matter produced by the plant during reproductive stages is allocated to the ear for its growth after the sink size is determined. Thus, ear biomass, which is set during early reproductive stages, is considered an important determinant of reproductive success and a larger ear biomass at early reproductive stages is associated with increased grain yield at harvest. MON 87403 will be combined with other authorized biotechnology-derived traits through traditional breeding methods to create commercial products with increased yield as well as protection against maize pests and tolerance to multiple 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 maize event MON 87403 was developed; how the composition and nutritional quality of maize event MON 87403 compared to non-modified varieties; and the potential for maize event MON 87403 to be toxic or cause allergic reactions. Monsanto Canada Inc. has provided data that demonstrates that maize event MON 87403 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 increased ear biomass maize event MON 87403 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

9. 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 MON 87403 maize and data that characterize the genetic modification which results in the increased ear biomass through the expression of the ATHB17 protein from Arabidopsis thaliana.

MON 87403 maize was developed by Agrobacterium-mediated transformation of immature maize embryos using the plasmid PV-ZMAP5714. The T-DNA found in MON 87403 contains the coding region of the full length Arabidopsis thaliana ATHB17 gene, which is a member of the HD-zip family of plant transcription factors. In MON 87403, maize-specific splicing of the ATHB17 transcript results in a truncated protein, ATHB17Δ113, which is missing the first 113 N-terminal amino acids that is expressed in Arabidopsis. The ATHB17Δ113 protein retains the ability to form homo- and hetero-dimers and bind to target DNA sequences like the full length protein. ATHB17Δ113 is unable to function as a transcriptional repressor because the protein lacks a functional repression domain. By a dominant-negative mechanism, ATHB17Δ113 can alter the activity of endogenous maize HD-Zip II proteins, which are predominantly expressed in ear tissue. Thus, the ATHB17Δ113 protein likely modulates HD-Zip II regulated pathways in the ear, which likely leads to increased ear growth at the early reproductive phase. This increased ear growth is associated with improved partitioning of dry matter from the source (vegetative) tissue to the sink tissue (ear) in MON 87403 compared to control plants grown under comparable environmental and agronomic conditions.

3. Characterization of the Modified Plant

Southern blot analysis was used to determine 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 the T-DNA inserted at a single site and at a single locus of the maize genome, and a single functional copy of the T-DNA was integrated. MON 87403 maize genomic DNA probed for backbone sequences showed no detectable hybridization bands, thus MON 87403 maize does not contain backbone sequences from the transformation vector PV-ZMAP5714.

In addition to Southern blot analysis, genomic DNA extracted from MON 87403 maize was subjected to next-generation sequencing analysis. This analysis confirmed the Southern blot results that MON 87403 maize contains a single insert copy at a single locus. The organization of the elements within the MON 87403 insert was confirmed using PCR and DNA sequence analysis. The complete sequence of the single DNA insert from PV-ZMAP5714, the adjacent flanking DNA, and the 5' and 3' insert-to-flank junctions was determined. The analysis confirmed that the sequence and organization of the DNA is identical to the corresponding region in the vector T-DNA. The sequencing analysis shows that MON 87403 contains only a single DNA insert with no unintended fragments, which also confirms that no vector backbone or other unintended plasmid sequences are present in MON 87403. Also, the genomic organization at the insertion site was assessed by comparing the sequences flanking the T-DNA insert in MON 87403 to the sequence of the insertion site in conventional maize. This analysis determined that no DNA rearrangement occurred at the insertion site.

Southern blot analysis was performed using multiple generations of MON 87403 maize. The digested genomic DNA of MON 87403 maize showed the expected banding pattern across the generations tested. The segregation data further confirmed the stability of the insert, showing that it segregates according to Mendelian inheritance principles. These results were also consistent with the molecular characterization data that indicated a single genomic insertion site.

4. Product Information

One new protein is expected to be expressed in MON 87403 maize based on the characterization of the inserted genetic material. The ATHB17 gene expressed in MON 87403 maize encodes a 22.4 kDa protein, ATHB17Δ113.

Characterization of the ATHB17Δ113 in MON 87403 was conducted using SDS-PAGE and Western blot analysis, and identity confirmed using protein sequence analysis by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The protein analysis demonstrated that the plant produced ATHB17Δ113 was equivalent to the microbial (E.coli) produced protein that was used in the toxicology studies.

The expression levels of the ATHB17Δ113 protein in over-season leaf (OSL1-OSL4), over-season root (OSR1-OSR4), forage root, senescent root, over season whole plant (OSWP1-OSWPR), forage, stover, pollen, grain, and silk were examined. The mean ATHB17Δ113 protein level in MON 87403 across all sites was highest in OSWP1 at 0.015 ug/g dry weight and lowest in OSR4, forage root, senescent root, pollen and grain at less than the limit of detection (<0.00065 ug/g dry weight).

5. Dietary Exposure

The genetic modification of MON 87403 maize is not intended to alter any of its nutritional aspects. Therefore, the use of MON 87403 maize and products derived from it will be similar to traditional maize varieties. The introduction of MON 87403 maize will only replace a fraction of current maize varieties and is not anticipated to result in a change in the dietary intake of maize and maize-derived products.

6. Nutrition

A crop composition study with MON 87403 maize grain samples, conventional control, and reference hybrid lines was grown in 2012 at eight sites located in the United States. Several of these sites were located at similar latitudes and conditions to the corn-growing regions of Canada. Starting seeds were planted in a randomized complete block design with four replicates at each site. MON 87403, conventional control, and reference hybrids were grown under normal agronomic field conditions for their respective geographic regions. Furthermore, within any given individual site, these agronomic treatments were performed uniformly across all plots (test, control, and references).

The compositional analytes measured in the MON 87403 and control maize seeds were: proximates (ash, moisture, protein, and fat), carbohydrates by calculation, acid detergent fiber (ADF), neutral detergent fiber (NDF), total dietary fiber (TDF), amino acids, fatty acids (C8-C22), vitamins (β-carotene [referred to as vitamin A], B1, B2, B6, E[α-tocopherol], niacin, and folic acid), minerals ( calcium, copper, iron, magnesium, manganese, phosphorus, potassium, sodium, and zinc), anti-nutrients (phytic acid and raffinose), and secondary metabolites (furfural, ferulic acid, and p-coumaric acid). Proximates, carbohydrates by calculation, ADF, NDF, calcium, and phosphorus were measured in forage. The key nutrients and anti-nutrients were selected according to the Revised Consensus Document on Compositional Considerations for New Varieties of Maize [Zea mays]: Key Food and Feed Nutrients, Anti-nutrients, and Secondary Plant Nutrients (2012).

No statistically significant differences were observed in the combined site analysis between MON 87403 and the conventional control for any of the 60 components analysed. All combined-site mean values of MON 87403 were within the 99% tolerance intervals established from the conventional commercial reference varieties grown in the same trial. Furthermore, all combined-site mean values and ranges of MON 87403 for all nutrient components, were within the context of natural variability of commercial maize composition published in the OECD maize document, scientific literature, and/or available in the ILSI Crop Composition Database.

MON 87403 maize has similar composition compared to its non-transgenic control, and conventional maize varieties.

7. Toxicology

The donor organism, A. thaliana, is not known to cause human toxicity. Health Canada has previously approved other genetically modified crops (e.g., corn, canola, rice, lentils, sunflower and wheat) that express transgenic proteins derived from A. thaliana. The ATHB17Δ113 protein expressed in MON 87403 is similar to proteins from other plants, like turnip, napa, cabbage and Brussel sprouts. All of these plants are part of the normal human diet and are not associated with adverse effects. It was not expected that the ATHB17Δ113 protein expressed in MON 87403 would pose a health concern.

The expression level of ATHB17Δ113 proteins in MON 87403 maize grain were below the limit of detection (ELISA, LOD = 0.00028 µg/g tissue d.w.; < 0.001% of total grain protein) and was indistinguishable from that of non-transgenic maize controls. The estimated intake in the most sensitive subpopulation, children 2-3 years of age (95^th percentile), is expected to be very low (1.6 ng/kg b.w./day).

An acute oral toxicity study was performed with ten male and ten female CD-1 mice administered 1335 mg of microbial derived ATHB17Δ113/kg b.w. by gavage in two oral doses given four hours apart. Animals were observed for 14 days post-treatment and then sacrificed. The mice showed no treatment-related mortality, changes in clinical observations, body weights, body weight gains, food consumption or gross pathology. A NOEL of 1335 mg/kg b.w. was determined.

The conservative estimate for ATHB17Δ113 intake in the subpopulation with the greatest exposure to corn and corn products, children 2-3 years of age, was calculated to be six orders of magnitude less than the reported NOEL. Based on the large difference between the acute study NOEL and the exposure estimate, it is unlikely that ATHB17Δ113 will cause adverse toxicological effects when consumed by Canadians, including the greatest consumers of corn and corn products.

The simulated gastric fluid (pepsin; pH 1.2; 37 ºC) and simulated intestinal fluid assays (pancreatin; pH 7.5; 37 ºC) performed on microbial produced ATHB17Δ113 determined that the novel protein was fully digested within 2 and 5 minutes, respectively. It is likely that ATHB17Δ113 protein from MON 87403 will be readily digested by enzymes of the gastrointestinal tract and will not be absorbed in the intestinal mucosa as functionally active protein.

Many corn products are devoid of protein (e.g., sugars, oil, starch, etc.) and others are highly processed and require exposure of the corn grain to very high temperatures during the manufacturing process (e.g., corn meal, flour, etc.). It is likely that some protein degradation occurs during corn processing and cooking and it will further reduce the amount of active ATHB17Δ113 in the human diet. Taken together, it is unlikely that functional ATHB17Δ113 protein will be absorbed in humans, and the actual exposure will be significantly less than the estimated exposure.

A bioinformatics search was conducted using the amino acid sequence of ATHB17Δ113 and the amino acid sequences of toxins and total proteins retrieved from three databases (AD_2013, TOX_2013 and PRT_2013, respectively) using the FASTA sequence comparison algorithm. ATHB17Δ113 did not share ≥ 35% amino acid identity (over a sequence of 80 amino acids) with known toxins or other biologically active proteins that may be harmful to human health.

Monsanto Canada Inc. also provided evidence ¹ demonstrating that the introduction of a modified transcription factor, ATHB17Δ113 (and thus the modification of global HD-Zip transcriptional activity), in MON 87403 resulted in very subtle changes in maize ear transcript expression profiles. The few mRNA transcripts that were differentially expressed were not candidate toxins based on BLAST analysis. Further, there was no evidence of any alterations in the pathways that could lead to the production of toxins. This suggests that off-target, toxicological effects in MON 87403 are unlikely.

The novel ATHB17Δ113 protein expressed in MON 87403 will not pose a toxicological concern to consumers based on data demonstrating very low levels of protein exposure, absence of acute oral toxicity in treated mice, rapid digestion of ATHB17Δ113 by gastrointestinal enzymes, lack of amino acid homology between ATHB17Δ113 and known toxins, and evidence demonstrating very minor changes to the transcriptome of MON 87403 that are unrelated to the formation of toxins.

The donor organism, A. thaliana, is not known to cause human allergy. Health Canada has previously approved other genetically modified crops (e.g., corn, canola, rice, lentils, sunflower and wheat) that express transgenic proteins derived from A. thaliana. It is not expected that ATHB17Δ113, derived from A. thaliana, will pose an allergenic concern when expressed in MON 87403. Similar proteins from other plants, like turnip, napa, cabbage and Brussel sprouts are part of the normal human diet and are not associated with any allergy.

The simulated gastric fluid (pepsin; pH 1.2; 37 ºC) and simulated intestinal fluid assays (pancreatin; pH 7.5; 37 ºC) performed on microbial produced ATHB17Δ113 determined that the novel protein was fully digested within 2 and 5 minutes, respectively. It is likely that ATHB17Δ113 protein from MON 87403 will be readily digested by enzymes of the gastrointestinal tract and will not be absorbed in the intestinal mucosa as functionally active protein.

Many corn products are devoid of protein (e.g., sugars, oil, starch, etc.) and others are highly processed and require exposure of the corn grain to high temperatures during the manufacturing process (e.g., corn meal, flour, etc.). It is likely that some protein degradation occurs during corn processing and cooking and it will further reduce the amount of active ATHB17Δ113 in the human diet. Taken together, it is unlikely that functional ATHB17Δ113 protein will be absorbed in humans, and the actual exposure will be significantly less than the estimated exposure.

A bioinformatics search was conducted using the amino acid sequence of ATHB17Δ113 and the amino acid sequences of allergens and total proteins retrieved from three databases (AD_2013, TOX_2013 and PRT_2013, respectively) using the FASTA sequence comparison algorithm. ATHB17Δ113 did not share ≥ 35% amino acid identity (over a sequence of 80 amino acids) with known allergens. Further, ATHB17Δ113 does not contain potential allergen epitopes as determined by an 8 amino acid segment analysis. The bioinformatics search suggests that ATHB17Δ113, based on protein sequence alone, is not similar to any known allergen.

Monsanto Canada Inc. also provided evidence ¹ demonstrating that the introduction of a modified transcription factor, ATHB17Δ113 (and thus the modification of global HD-Zip transcriptional activity), in MON 87403 resulted in very subtle changes in maize ear transcript expression profiles. The few mRNA transcripts that were differentially expressed were not candidate allergens based on BLAST analysis. Further, there was no evidence of any alterations in the pathways that could lead to the production of allergens. This suggests that off-target, allergenic effects in MON 87403 are unlikely.

The novel ATHB17Δ113 protein expressed in MON 87403 will not pose an allergenic concern to consumers based on data demonstrating rapid digestion of ATHB17Δ113 by gastrointestinal enzymes, lack of amino acid homology between ATHB17Δ113 and known allergens, and evidence demonstrating very minor changes to the transcriptome of MON 87403 that are unrelated to the formation of allergens.

CONCLUSION:

Health Canada's review of the information presented in support of the food use of increased ear biomass maize event MON 87403 does not raise concerns related to food safety. Health Canada is of the opinion that food derived from maize event MON 87403 is as safe and nutritious as food from current commercial maize varieties.

Health Canada's opinion deals only with the food use of maize event MON 87403. 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 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.

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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