Novel Food Information - Herbicide Tolerant Maize – MON 87419

Health Canada has notified Monsanto Canada Inc. that it has no objection to the food use of Herbicide Tolerant Maize - MON 87419. 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 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 has developed Herbicide Tolerant Maize - MON 87419 using recombinant DNA techniques to introduce two coding sequences: dmo and pat, which encode a dicamba mono-oxygenase (DMO) derived from Stenotrophomonas maltophilia and a phosphinothricin N-acetyltransferase (PAT) derived from Streptomyces viridochromogenes, 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 MON 87419 was developed; how the composition and nutritional quality of MON 87419 compared to non-modified varieties; and the potential for MON 87419 maize to be toxic or cause allergic reactions. Monsanto Canada Inc. has provided data that demonstrates that MON 87419 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 28). Food use of Herbicide Tolerant Maize - MON 87419 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 Herbicide Tolerant Maize - MON 87419 and the molecular biology data that characterize the genetic change, which results in a tolerance to dicamba and glufosinate herbicides. This phenotype was achieved by transformation of the conventional soybean variety A3555 with a transgenic expression cassette containing the novel cry1A.105 and cry2Ab2 genes and their associated regulatory elements.

Herbicide Tolerant Maize - MON 87419 was genetically modified using Agrobacterium-mediated transformation of the inbred corn line LH244 with the the plasmid PV-ZMHT507801. The plasmid PV-ZMHT507801 carried two separate transfer DNA (T-DNA I and T-DNA II) sequences, each comprised of an expression cassette, one containing the dmo and pat coding sequences (T-DNA I) and one containing the cp4 epsps selectable marker gene. The selectable marker gene encodes an A. tumefaciens strain CP4 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme. The CP4 EPSPS enzyme exhibits a decreased affinity for the herbicide glyphosate, thereby conferring increased tolerance to the herbicidal agent upon expression. Initial transformants were selected for glyphosate tolerance. Subsequent conventional breeding followed by molecular analysis were used to select plants containing only the intended T-DNA I insert containing the dmo and pat expression cassettes and lacking the cp4 epsps selectable marker gene (i.e. lacking the T-DNA II insert).

The dmo and pat expression cassette contains the following genetic elements: the promoter for a ubiquitin gene (Ubq) derived from Andropogon geradii (big bluestem grass) that initiates and directs transcription; the 5¢ untranslated region (UTR) leader sequence for the ubiquitin gene (Ubq) derived from A. geradii that is involved in regulating gene expression; the intron sequence of the ubiquitin gene (Ubq) derived from A. geradii that is involved in regulating gene expression; the coding sequence for the phosphinothricin N-acetyltransferase (PAT) protein derived from S. viridochromogenes that confers tolerance to glufosinate; the 3¢ UTR sequence of the RA5B precursor gene encoding an alpha-amylase/trypsin inhibitor (Ara5) derived from Oryza sativa (rice) that directs polyadenylation of the mRNA; the promoter for the full length transcript (FLt) derived from Peanut Chlorotic Streak Caulimovirus (PClSV) that directs transcription in plant cells; the 5¢ UTR leader sequence from chlorophyll a/b binding (CAB) protein derived from Triticum aestivum (wheat) that is involved in regulating gene expression; the intron and flanking UTR sequence of the act1 gene derived from O. sativa encoding rice Actin1 protein that is involved in regulating gene expression; the targeting and 5¢ UTR leader sequence of the ShkG gene derived from Petunia hybrida encoding the EPSPS transit peptide region that directs the protein to the chloroplast; the codon-optimized coding sequence for the dicamba mono-oxygenase (DMO) protein derived from S. maltophilia that confers tolerance to dicamba; and the 3¢ UTR sequence from a heat shock protein, Hsp17, derived from T. aestivum that directs polyadenylation of the mRNA.

The cp4 epsps expression cassette contained the following genetic elements: the promoter of the act1 gene derived from O. sativa encoding rice Actin1 protein that directs transcription in plant cells; the leader sequence of the act1 gene derived from O. sativa encoding rice Actin1 protein that is involved in regulating gene expression; the intron and flanking UTR sequence of the act1 gene derived from O. sativa encoding rice Actin1 protein that is involved in regulating gene expression; the targeting sequence of the ShkG gene derived from Arabidopsis thaliana encoding the EPSPS transit peptide region that directs the protein to the chloroplast; the coding sequence of the aroA gene derived from Agrobacterium sp. strain CP4 encoding the CP4 EPSPS protein that confers tolerance to glyphosate; and the 3¢ UTR sequence of the nopaline synthase (nos) gene derived from A. tumefaciens pTi encoding NOS that directs polyadenylation of the mRNA. Using traditional methods of breeding, the T-DNA II transfer DNA was subsequently removed from the maize genome, producing the MON 87419 genetic event containing only dmo and pat expression cassette (T-DNA I).

3. Characterization of the Modified Plant

A combination of Next Generation Sequencing (NGS) and Junction Sequence Analysis (JSA), in addition to Southern blot analysis and DNA sequencing of Herbicide Tolerant Maize - MON 87419 demonstrated the presence of a single copy of the dmo and pat expression cassette in the maize genome at a single locus. Both NGS/JSA and Southern blot analysis confirmed the absence of any plasmid backbone DNA in Herbicide Tolerant Maize - MON 87419.

The stability of the inserted dmo and pat expression cassette was evaluated from the progeny of five different generations. The results of NGS/JSA and Southern blot analysis and segregation data demonstrated the stability of Herbicide Tolerant Maize - MON 87419 at the genomic level.

4. Product Information

Herbicide Tolerant Maize - MON 87419 differs from its traditional counterpart by the addition of the dmo and pat genes and their associated regulatory elements. The insertion of these gene results in expression of the novel DMO and PAT proteins in MON 87419. Expression of these two proteins confers tolerance to dicamba and glufosinate herbicides.

The DMO protein is a dicamba mono-oxygenase derived from S. maltophilia. The DMO protein rapidly demethylates dicamba into inactive metabolites, 3,6 dichlorosalicylic acid (DCSA) and formaldehyde. The PAT protein is a phosphinothricin N-acetyltransferase derived from S. viridochromogenes. The PAT protein acetylates glufosinate, rendering the herbicidal compound non-toxic to the plant.

The host organism, maize (Zea mays), has a long history of safe use for food in Canada and is not associated with toxic or allergenic concerns. As well, the donor organisms S. maltophilia and S. viridochromogenes are 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 DMO and PAT proteins in MON 87419. This study used plant samples from five field trial locations conducted in the United States during the 2013 growing season: Iowa, Indiana, Kansas, Nebraska, and Pennsylvania. All locations are relevant maize-growing regions and represent a range of environmental conditions typically encountered in the production of maize. At each field site, four replicated plots of MON 87419 were planted using a randomized complete block field design. Tissues of MON 87419 that were collected include: over season leaf (OSL) 1, over season root (OSR) 1, seed, and forage. The quantities of DMO and PAT proteins were determined by enzyme-linked immunosorbent assay (ELISA). The across-site means, standard deviations (SDs), and ranges for DMO and PAT protein levels in maize OSL 1, OSR 1, seed, and forage tissues were reported on a mg/g dry weight (dwt) basis.

Mean levels of DMO and PAT proteins were both very low in all tissues, but the highest levels were observed in OSL 1 (26 and 11 mg/g for DMO and PAT, respectively), while the lowest levels of expression were observed in seed (0.19 and 0.93 mg/g for DMO and PAT, respectively).

5. Dietary Exposure

It is expected that Herbicide Tolerant Maize - MON 87419 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 MON 87419.

Based on the maize intake values reported in the Canadian Community Health Survey, in the worst case exposure scenario, for children 2-3 years of age, the subgroup with the greatest intakes of maize and maize products, the margins of exposure (MOEs) between the highest dose tested in acute toxicity studies (see Chemistry/Toxicology below), where there was no evidence of toxicity, and the estimated dietary intakes for acute dietary intake estimated to be 1.6×105 for the DMO protein and 1.2×105 for the PAT protein. In absence of any evidence of toxicity, these MOEs are considered sufficient to protect consumers.

6. Nutrition

The nutritional and anti-nutritional components of Herbicide-Tolerant Maize - MON 87419 were measured and compared with the conventional control variety NL6169.

For maize compositional analysis, MON 87419 and NL6169 were grown at five locations in the United States: Iowa, Indiana, Kansas, Nebraska, and Pennsylvania during the 2013 growing season. These areas were representative of the major growing areas for maize. All plants were grown under normal agronomic field conditions for their respective geographic regions. Further, within any given individual site, these agronomic treatments were performed uniformly across all plots.

The key nutritional and compositional analytes measured in the MON 87419 and control variety NL6169 seed were: proximate content (ash, protein, total fat, and carbohydrate by calculation), fibre (acid detergent fibre, neutral detergent fibre, and total dietary fibre), minerals (calcium, iron, magnesium, manganese, phosphorus, potassium, sodium, and zinc), 18 amino acids, 22 fatty acids, vitamins (folic acid, niacin, vitamin A, B1, B2, B6, and E), anti-nutrients (phytic acid and raffinose), and secondary metabolites (ferulic acid and p-coumaric acid). The key nutritional and compositional analytes measured in the MON 87419 and control variety NL6169 forage were: proximate content (ash, protein, total fat, and carbohydrate by calculation), fibre (acid detergent fibre, and neutral detergent fibre), and minerals (calcium and phosphorus).

Of the 61 (53 in grain, 8 in forage) components statistically assessed for MON 87419 there were no significant differences in 60 components. Only one component (manganese in grain) showed a significant difference (i.e. p<0.05) between MON 87419 and the conventional control. For this component, the mean difference in the component values between MON 87419 and the conventional control was less than the range value of the conventional control. The MON 87419 mean component value and range were also within the range of values observed in the literature and the International Life Sciences Institute Crop Composition Database (ILSI-CCDB). All combined-site mean values and ranges of MON 87419 for all nutrients, anti-nutrients, and secondary metabolites were also within the range of values observed in the literature and the ILSI-CCDB.

The petitioner has sufficiently demonstrated that Herbicide-Tolerant Maize - MON 87419 grain and forage have similar compositions compared to their non-transgenic controls and therefore would not pose an increased nutritional risk to consumers.

7. Chemistry/Toxicology

Dicamba-tolerant soybean - MON 87708 and dicamba and glufosinate-tolerant cotton - MON 88701 contain DMO and/or PAT proteins that share high amino acid sequence similarity with the DMO and PAT proteins in MON 87419. The DMO and/or PAT proteins in MON 87708 and MON 88701 have been previously assessed for their acute oral toxicity, digestibility, heat stability, and bioinformatics data and have been shown to be safe to consumers and are approved by Health Canada for food use.

The modes of action for both the DMO and PAT proteins are the inactivation of specific herbicidal compounds. The proteins themselves do not function as toxins, and their reaction metabolites would not be expected to pose a health concern. Furthermore, bioinformatics analysis demonstrated that both the DMO and PAT proteins do not have amino acid sequence similarities to known toxins or allergens, indicating these newly expressed proteins would not be expected to pose a toxicological or allergenic concern.

The DMO and PAT proteins were shown to be rapidly degraded in simulated gastric fluid and/or simulated intestinal fluid, indicating functionally active DMO and PAT proteins are not expected to be absorbed into the small intestine or have any systemic adverse effects.

The functional activities of the DMO and PAT proteins were rapidly inactivated by temperatures above 60 °C, indicating their lack of stability under processing and cooking conditions. Typical cooking conditions are more extreme than this condition. Therefore, there would be no exposure to the active protein, and the denatured protein would be expected to be digested like any other dietary proteins. As a result, the DMO and PAT proteins would not be expected to pose a toxicological or allergenic concern.

The levels of the DMO protein in MON 87419 grain at the mature plant stage range from 0.14 to 0.31 mg/g dry weight, equivalent to about 0.00012% to 0.00027% of the total protein in MON 87419 grain. The levels of the PAT protein in MON 87419 grain at the mature plant stage range from 0.56 to 1.60 mg/g dry weight, equivalent to about 0.0005% to 0.0014% of the total protein in MON 87419 grain. In both cases, the amount of protein indicates a very low human dietary exposure, which is uncharacteristic of a food allergen.

Based on the weight of evidence, it was concluded that MON 87419 is as safe for consumption as conventional maize varieties.

Conclusion:

Health Canada's review of the information presented in support of the food use of Herbicide-Tolerant Maize - MON 87419 does not raise concerns related to food safety. Health Canada is of the opinion that food derived from MON 87419 is as safe and nutritious as food from current commercial maize varieties.

Health Canada's opinion deals only with the food use of Herbicide-Tolerant Maize - MON 87419. 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 Herbicide-Tolerant Maize - MON 87419 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 Herbicide-Tolerant Maize - MON 87419 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.

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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