Novel Food Information: Insect Resistant and Herbicide Tolerant Corn - MON 87411

Health Canada has notified Monsanto Canada Inc. that it has no objection to the sale of food derived from Insect Resistant and Herbicide Tolerant Corn MON 87411. 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.


The following provides a summary of the notification from Monsanto Canada Inc. and the evaluation by Heath Canada. This document contains no confidential business information.

1. Introduction

Monsanto Canda Inc., developed Insect Resistant and Herbicide Tolerant Corn MON 87411 (hereafter MON 87411) to be resistant to corn rootworm (Diabrotica spp.) pests and be tolerant to glyphosate based herbicides. Using recombinant DNA techniques Monsanto introduced three distinct expression cassettes.

The first of these cassettes results in the production of a double stranded RNA (dsRNA) molecule. This dsRNA is designed to match to the Snf7 sequence of western corn rootworm. This dsRNA is consumed by Diabrotica spp. and is recognised by the rootworm’s natural RNA interference (RNAi) machinery. Recognition by the RNAi machinery results in down regulation of the targeted Diabrotica Snf7 (DvSnf7) gene leading to mortality. The second expression cassette confers resistance to corn rootworm through the expression of the Cry3Bb1 protein from Bacillus thuringiensis subspecies kumamotoensis. The expression of this protein protects the plant against larval feeding through the well characterised pathway common to all Bt Cry proteins. Finally, tolerance to the herbicide glyphosate was conferred through the insertion of the third expression cassette containing the 5-enolpyruvylshikimate-3-phosphate (cp4-epsps) coding sequence derived from Agrobacterium sp. strain CP4. This sequence expresses the CP4-EPSPS protein, which acts in place of the plants natural EPSPS and is resistant to glyphosate.

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 reflects international guidance documents in this area (eg., Codex Alimentarius). The assessment considered: how MON 87411 was developed; how the composition and nutritional quality of MON 87411 compared to non-modified corn varieties; and what the potential is for MON 87411 to be toxic or cause allergic reactions. Monsanto Canada Inc. has provided data which demonstrates that MON 87411 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 Division 28 of Part B of the Food and Drug Regulations (Novel Foods). Foods derived from MON 87411 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 MON 87411 and molecular biology data that characterize the genetic change that results in insect and herbicide resistance. MON 87411was produced using Agrobacterium tumefaciens (A.tumefaciens) mediated transformation of corn variety LH244 with the transformation vector PV-ZMIR10871. This transformation vector was constructed to contain three gene cassettes, one for the DvSnf7 fragment, one for the cry3Bb1 gene and one for the herbicide tolerance gene cp4-epsps.

The first gene cassette contains the coding region that results in the production of the double stranded DvSnf7 RNA. This coding region consists of an inverted repeat of a 240 bp fragment of the Diabrotica virgifera virgifera Snf7 gene (DvSnf7). This gene encodes the SNF7 subunit of the endosomal sorting complex required for the transport III (ESCRT-III) protein.   When transcribed, this inverted repeat fragment will generate a double stranded RNA which, when consumed by the corn rootworm (CRW), will act through the endogenous CRW RNA interference mechanisms to suppress expression of the SNF7 subunit, leading to  mortality in the pest. 

The second gene cassette contains the coding regions for the insecticidal Cry3Bb1 protein. The integration of this gene cassette in the corn genome confers a second mode of tolerance to Diabrotica species of corn pests. Cry3Bb1 is active in the insect mid-gut and achieves insect mortality through the well-established mechanism of all Cry proteins.

The third gene cassette contains the coding regions for the CP4-EPSPS protein, which confers tolerance to the herbicide glyphosate. The CP4-EPSPS protein is originally derived from Agrobacterium sp. strain CP4, a common soil-borne bacterium. The CP4-EPSPS protein is functionally identical to the endogenous plant EPSPS enzymes, including that naturally found in corn, with the exception of comparatively reduced affinity to glyphosate. In conventional plants, glyphosate binding to EPSPS enzymes results in the blockage of synthesis of the 5-hydroxyl of shikimate-3-phosphate, a precursor of essential aromatic amino acids and other aromatic metabolites necessary for growth and development. In the case of the CP4-EPSPS, the reduced affinity to glyphosate results in continued production of aromatic amino acids.

The elements contained in the PV-ZMIR10871 T-DNA were as follows: the 3’ untranslated region of the rbcS gene family of Pisum sativum (E9), the partial coding sequence of the Snf7 gene encoding the SNF7 subunit of the ESCRT-III complex from Diabrotica virgifera virgifera (DvSnf7), the intron and flanking sequence of the hsp70 gene encoding heat shock protein 70 of Zea mays (Hsp70), the e35S promoter from Cauliflower Mosaic Virus (CaMV)  to direct transcription of Snf7(e35S), the Promoter sequence from the physical impedance induced protein of Zea mays (pIIG), the synthetic, the 5’ untranslated region from the chlorophyll a/b binding (CAB) protein of Triticum aestivum (Cab), the intron and flanking untranslated region of the act1 gene from Oryza sativa encoding rice Actin 1 protein (Ract1), the codon-optimised coding sequence for the Cry3Bb1 protein from Bacillus thuringiensis subspecies kumamotoensis (cry3Bb1),the 3’ untranslated region from the gene encoding heat shock protein 17 of Triticum aestivum (Hsp17), the 5’ untranslated region leader and intron sequences from the OsTubeA gene of Oryza sativa (TubA), the targeting sequence of the ShkG gene encoding the EPSPS transit peptide from Arabidopsis thaliana (CTPT2), the  codon optimised coding sequence of the cp4-epsps gene encoding the CP4 EPSPS protein from Agrobacterium sp. strain CP4 (cp4-epsps) and the 3’ untranslated region sequence from the OsTubeA gene of Oryza sativa (TubA).

Immature embryos from line LH244 were aseptically removed from 10 - 13 day post- pollination ears and transformed, using a disarmed strain of Agrobacterium tumefaciens, with the T-DNA from plasmid vector PV-ZMIR10871. After co-culturing with the Agrobacterium carrying the vector, the embryos were placed on selection medium containing glyphosate, and carbenicillin, to inhibit the growth of untransformed plant cells and excess Agrobacterium, and to permit the development of callus tissue. Resulting callus was then placed in a medium that supported shoot regeneration and root development. Rooted plants (generation R0) with normal phenotypic characteristics and tolerance to glyphosate were selected and transferred to soil for growth and further assessment.

R0 plants generated through the transformation process described in were self-pollinated over six (R1 through R6) generations in order to produce homozygous lines. At each generation, the progeny were evaluated for desirable molecular and phenotypic characteristics. MON87411 was selected as the lead event, based on its insert integrity, glyphosate tolerance, efficacy against CRW larval damage and superior phenotypic characteristics. Seed from the R4 and R5 generations was used in trait integration and further commercial development through crosses with conventional inbred lines HCL645 and LH287, respectively. These crosses resulted in the R4F1 and R5F1 generations. R4F1 was further bred through subsequent backcrosses with the non-modifed parent F1266Z to produce the generations BC1F1 and BC2F1. BC2F1 was then further bred through an additional backcross to produce BC3F1 and, alternatively, through self-pollination to produce BC2F2.

3. Characterization of the Modified Plant

The molecular characterisation of MON87411 incorporated the use of eSoutherns a technique that applies Next Generation Sequencing (NGS) and Junction Sequence Analysis (JSA) together with bioinformatics to determine the number of inserts. Total genomic DNA from grain of verified MON87411 and the untransformed parent was sequenced using Illumina NGS technology. Reference DNA was also used from the plasmid vector PV-ZMIR10871. As a positive control, plasmid DNA was spiked into LH244 DNA at a single copy genome equivalent ratio and 1/10 copy genome equivalent ratio. The DNA was sheared into approximately 325bp fragments, processed for deep sequencing enriched through ten cycles of PCR and then sequenced using Illumina HiSeq technology that produces short-sequence reads approximately 100bp long. The 100-mer sequence reads from all samples were analysed to determine the effective depth of coverage by mapping all reads to a known single-copy endogenous corn gene. The analysis showed an effective depth of coverage of >107 times for each sample. An in silico analysis using the BLAST algorithm then followed, in which only those 100-mer reads containing sequence similarity to the plasmid PV-ZMIR10871 were selected. Using Bowtie short sequence alignment software, non-duplicated reads of ≥30 nt were collected and were aligned to the whole plasmid PV-ZMIR10871 sequence in order to find junction region sequences. Reads were also aligned against the control genome in order to remove those reads sourced from endogenous homologues. From this analysis only two unique junction sequence classes, both containing portions of T-DNA and flanking sequence were detected. This result indicates that the T-DNA region is inserted at a single locus of the genome.

The petitioner provided a molecular characterisation using traditional Southern blots to confirm the number of copies of each functional element within the insert. To do this the petitioner used a variety of restriction enzymes and nine different probes, each specific for a region of the T-DNA. The analysis of the Southern blots provided by the petitioner confirmed that at single copy of the T-DNA is present in MON87411, containing all three gene cassettes.

In addition to the Southern blots used to characterise the insert, the petitioner performed a series of Southern blots to demonstrate the absence of any plasmid backbone sequences. Using four overlapping probes, the petitioner probed MON87411 genomic DNA after digestion with one of two restriction enzymes. From the evidence presented by the petitioner no plasmid backbone has been transferred into MON87411.

Similar to the characterisation the petitioner provided the results of eSoutherns to demonstrate the genetic stability of the insert. Using the same procedure used to identify the number of inserts through NGS/JSA the petitioner tested the R4, R5, R6, R4F1 and R5F1 generations of MON87411. This analysis demonstrated the presence of the same two junction sequences identified in the insert number analysis. The petitioner found no other junction sequences in these generations. This consistency in the junction sequences across generations confirms the genetic stability of the insertion.

The petitioner undertook testing on the BC2F1, BC2F2 and BC3F1 generations to demonstrate that the insert is inherited in the expected manner for an insert at a single locus. Using real-time TaqMan PCR to analyze the generations, the petitioner tested for the presence of the T-DNA. Based on this analysis segregation ratios were determined and tested against the expected. These compared ratios were then analysed using Chi square to test the hypothesis of Mendalian inheritance. Based on the segregation ratios from the PCR, and confirmed by the Chi square analysis, the trait is inherited in the expected Mendelian manner.

The petitioner completed an analysis of any potential opening reading frames created by the insertion into the genomic DNA. The analysis was done using bioinformatics to assess the homology between any potential reading frames and databases of known toxins and allergens. Open Reading Frames (ORFs) spanning the 5' flanking sequence DNA-inserted DNA junctions, the 3' flanking sequence DNA-inserted DNA junctions and the inserted T-DNA sequence present in MON 87411 were translated in all six reading frames. The extent of structural relatedness was evaluated by detailed visual inspection of the alignment, the calculated percent identity, and the E-score. An E-score of 1e-5 (1x10-5) was set as an initial high cut-off value for alignment significance. Although all alignments were inspected visually, any aligned sequence that yielded an E-score less than or equal to 1e-5 was analyzed further to determine if such an alignment represented significant sequence homology. The petitioner has stated that there is no analytical data to indicate that any of these putative proteins is produced in MON87411. 

Using the FASTA algorithm to search the TOX_2013 database and the PRT_2013 database found no alignments with any of the query sequences generated an E-score of less than or equal to 1e-5 for any of the ORFs at the junctions. These results indicate that these putative ORFs are unlikely to be a toxin or a protein that would result in an adverse biological effect.

Similar analysis of the putative ORFs in the insert found alignments in frames 2 and 3 that were below or equal to threshold alignment in both the TOX_2013 database and the PRT_2013 database. However, inspection of these alignments revealed that, although yielding below or equal to threshold E-scores, these alignments required either numerous gaps to optimize the alignment, were punctuated with numerous stop codons, or were not annotated to be associated with any known toxicity or untoward biological activity. As a result, in the unlikely event that an unexpected translation product from the insert was produced, it is not expected that these polypeptides would be a known protein that displays adverse biological activity.

For allergenicity the petitioner applied the conservative approach recommend by Codex in which related protein sequences were identified as potentially cross-reactive if linear identity is 35% or greater in an 80 amino acid overlap. Using the FASTA algorithm to search the AD_2013 database, no alignments with any of the query sequences generated an E-score of less than or equal to 1e-5. Likewise, no alignment met or exceeded the Codex FASTA alignment threshold for potential allergenicity of 35% identity over 80 amino acids. In addition to structural similarity, the query sequences were screened for short polypeptide matches using a pair-wise comparison algorithm. In these analyses, eight contiguous and identical amino acids were defined as immunologically relevant, where eight represents the typical minimum sequence length likely to represent an immunological epitope. No alignments of eight or more consecutive identical amino acids with were found between any query sequence and the AD_2013 database. As a result, the putative polypeptides are unlikely to contain any cross-reactive IgE binding epitopes with known allergens.

The petitioner conducted the toxicological assessment using Cry3Bb1 and CP4-EPSPS proteins expressed in and purified from Escherichia coli. To ensure that the results of the toxicological studies are applicable to the proteins expressed in MON 87411, equivalence studies (i.e., SDS-PAGE, western blot analysis, glycoprotein staining, MALDI-TOF MS, N-terminal amino acid sequence analysis and specific activity) were conducted to confirm that the protein produced in E.coli used for toxicology studies is representative of the protein produced in the modified corn. Based on the results of these studies, the proteins were determined to be equivalent with respect to their physical properties, immunological staining properties and sequencing. The petitioner has also demonstrated that the microbially produced proteins are equivalent to or are from the same batch as used in previously submitted toxicology studies.

Similarly, because the levels of DvSnf7 RNA in MON 87411 are very low, RNA was produced by in vitro transcription (identified as DvSnf7_968 RNA) in order to generate sufficient quantities of DvSnf7 RNA for safety assessments. As a result the petitioner undertook studies to demonstrate the equivalence of DvSnf7 RNA in MON 87411 and DvSnf7_968 RNA. DvSnf7 RNA in MON 87411 was characterized by sequencing its transcript, by identifying the presence of the dsRNA region which has been shown to be the active region of DvSnf7, and through a functional activity assay. The equivalence of the DvSnf7 RNA in MON 87411 and DvSnf7_968 RNA was assessed by comparing their sequences, the presence and size of dsRNA regions in each,  the results of a northern blot analyses and by comparing their functional activity. Based on the evidence provided by the petitioner the plant and in vitro produced DvSnf7 RNA are equivalent.

4. Product Information

Insect Resistant and Herbicide Tolerant Corn MON 87411differs from conventional corn through the introduction of the coding sequences for the double stranded RNA molecule designed to match the Snf7 sequence of western corn rootworm, the Cry3Bb1 protein from Bacillus thuringiensis subspecies kumamotoensis, and the CP4-EPSPS protein derived from Agrobacterium sp. strain CP4.

Field trials were initiated during the 2011 planting season to generate protein expression data for MON 87411 (R4F1) at the following maize growing locations in Argentina: Pergamino, Buenos Aires; Hunter, Buenos Aires; Pergamino, Buenos Aires; Sarasa, Buenos Aires; and Salto, Buenos Aires. These field sites were representative of maize producing regions suitable for commercial production. The identity harvested grain from each site was confirmed using event-specific polymerase chain reaction (PCR). There were four replicated plots at each site planted in a randomised complete-block design. The MON87411 plots were sprayed, at the 2-4 leaf stage with glyphosate herbicide. Samples were taken at various stages of growth. The levels of DvSnf7 were measured using a validated Quantigene Plex Assay 2.0 and the levels of Cry3Bb1 and CP4-EPSPS proteins were determined for each sample type using a validated enzyme linked immunosorbent assays (ELISA).

For DvSnf7 mean levels were lowest in grain (0.104 x10-3 ug/g dry weight) and highest in whole plant (84.8x10 x10-3 ug/g dry weight). For both Cry3Bb1 and CP4-EPSPS, mean levels were lowest in the grain (4.0 and 1.9 µg/g dry weight, respectively). The highest levels of both proteins were in the whole plant samples at the V3 - V4 stages (340 and 63 µg/g dry weight, respectively) as would be expected from the high levels in both the leaves and roots at this stage.

5. Dietary Exposure

Insect Protected and Herbicide Tolerant Corn MON87411 is expected to be used in similar applications as traditional corn varieties by the food industry. The petitioner has indicated that the greatest use of yellow dent corn in food is the production of starch and sweetener products through wet milling. Dry milling is also used to produce corn grits, flour and meal, although the greatest food product from dry milling is in brewing. As the uses of MON87411 corn is not expected to change, no increase in dietary exposure to corn is expected to occur.

6. Nutrition

A field trial was conducted at eight sites in Buenos Aires, Argentina during the 2011/12 growing season. At each of the eight field sites, MON87411, conventional control (NL6169) and 20 conventional reference varieties were planted in a randomized complete block design with four replicates at each of the eight field sites. All plants were grown under normal agronomic conditions for their respective geographic regions; including treatment with glyphosate to generate samples under the intended conditions of use. Further, within any given individual site, these agronomic treatments were performed uniformly across all plots (test, control, and references).  

The compositional analytes measured in MON87411 and conventional control maize grain were: proximates (ash, moisture, protein, fat, and carbohydrates by calculation), total dietary fibre, acid detergent fiber (ADF), neutral detergent fibre (NDF) , amino acids, fatty acids (C8-C22), vitamins [β-carotene (referred to as vitamin A), B1, B2, B6, E (α-tocopherol), niacin and folate], minerals (calcium, copper, iron, manganese, phosphorus, potassium, sodium, and zinc), anti-nutrients (phytic acid and raffinose) and secondary metabolites (furfural, ferulic acid and p-coumaric acid). Proximates, , ADF, NDF, calcium and phosphorus were measured in forage. These analytes were selected according to the OECD Revised Consensus Document on Compositional Considerations for New Varieties of Maize: Key Food and Feed Nutrients, Antinutrients, and Secondary Plant Nutrients.

Sixteen analytes in grain with more that 50% of observations below the assay limit of quantification (LOQ) were excluded from statistical analysis. These analytes included caprylic acid, capric acid, lauric acid, myristic acid, myristoleic acid, pentadecanoic acid, pentadecenoic acid, palmitoleic acid, heptadecanoic acid, heptadecenoic acid, gamma linolenic acid, eicosadienoic acid, eicosatrienoic acid, arachidonic acid, sodium and furfural.

With the exception of 12 components, no statistically significant differences were observed in the combined site analysis between MON87411 and the conventional control. In the 11 components in grain and one in forage, where the combined-site analysis showed a statistically significant overall treatment effect, all the differences were considered to be of a small magnitude. These differences were, in grain: protein (+4.2%), neutral detergent fiber (-0.1%), histidine (+3.7%), tyrosine (+5.0%), oleic acid (+2.8%), vitamin B1 (-3.4%), vitamin B3 (-7.7%), , zinc (+15.2%) copper (-5.7%), iron (+3.0%) and manganese (+2.8%).   The difference in forage was:  ash (-6.8%).

All combined-site mean values for MON87411 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 for MON87411 components, including those that were significantly different, were within the range for commercial maize composition published in the OECD consensus document, scientific literature and/or available in the ILSI Crop Composition Database. 

7. Toxicology

Cry3Bb1 and CP4-EPSPS proteins have been evaluated previously by Health Canada and have a history of safe use in genetically-modified food. The Cry3Bb1 and CP4-EPSPS expressed by MON 87411 have both been demonstrated to be identical in amino acid sequence to those proteins produced in a previously approved genetically modified corn. Further, newly submitted safety data submitted under this petition serve to support the conclusions previously established regarding these proteins. These data included updated bioinformatics assessing the homology of each novel protein to known protein toxins, and a study assessing the effect of heat treatment on Cry3Bb1 functional activity. Expression of Cry3Bb1 and CP4-EPSPS proteins in MON 87411 is not considered to represent a toxicological concern.

Bioinformatics analyses revealed that the novel siRNA molecules are unlikely to result in off-target gene suppression in humans, should the molecules be absorbed. A match length of 21 nucleotides was used in bioinformatics analyses to represent the minimal length of a dsRNA molecule that is necessary to mediate RNAi. No sequence homology matches of 21 nucleotides in length were identified between the novel RNA and the human transcriptome.

Toxicology studies were submitted and provided an assessment of the potential for either the novel dsRNA or MON 87411 maize to cause physiological effects in a mammalian system following oral ingestion. When administered as a daily gavage dose over 28 days, an in vitro transcribed surrogate of the novel dsRNA molecule resulted in no toxicity to mice (10/sex/group) up the NOAEL of  100 mg/kg bw/day (the highest dose tested). This NOAEL was more than a 100 million-fold greater than human dietary exposure to the dsRNA, which was conservatively estimated for the most sensitive sub-population. The margin of exposure was considered sufficient from a safety perspective.

In another study, rats (16/sex/group) were fed with MON 87411 grain incorporated at 33% into a standard diet for 90 days. A limitation of this study was that the level of the novel siRNA molecules in the test diet was not measured. These levels could be estimated, however, based on the expression analysis of MON 87411 grain. The estimated level of intake of the siRNA molecules by rats in the study is comparable to the estimated dietary intake of the siRNAs in human adults and infants.  The test diet administered to the rats was well tolerated and no adverse effects due to treatment were observed.

It should be noted that dietary exposure estimates of dsRNA and siRNA uptake in humans are considered to be conservative, and are likely to be overestimated. These estimates assume that all consumed corn commodities are derived solely from MON 87411 grain, when in fact the commodities are blended. Further, these estimates assume the novel RNA molecules would be intact and not impacted by processing or cooking. It is likely that the novel RNA molecules would be degraded or removed to some extent by the processing applied to the majority of the consumed maize commodities.

The potential impacts of the novel molecular products on the allergencity of MON 87411 were also considered. Cry3Bb1 and CP4-EPSPS proteins have a history of safe use in genetically-modified food, and both proteins expressed by MON 87411 are identical in amino acid sequence to Cry3Bb1 and CP4-EPSPS produced in a previously approved genetically modified corn. New bioinformatics data were submitted which demonstrate that neither protein has significant homology to known allergenic proteins. The suppression sequence coding for novel RNA molecules does not express any protein products and therefore will not impact the allergenicity of MON 87411.


Health Canada’s review of the information presented in support of the food use of Insect Resistant and Herbicide Tolerant Corn MON 87411concluded that derived food products do not raise concerns related to safety. Health Canada is of the opinion that Insect Resistant and Herbicide Tolerant Corn MON 87411is similar to conventional corn in terms of being an acceptable food source.

Health Canada's opinion deals only with the human food use of Insect Resistant and Herbicide Tolerant Corn MON 87411. Issues related to the environmental safety of Insect Resistant and Herbicide Tolerant Corn MON 87411 in Canada and its use as livestock feed have been addressed separately through existing regulatory processes in the Canadian Food Inspection Agency.

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

Report a problem or mistake on this page
Please select all that apply:

Thank you for your help!

You will not receive a reply. For enquiries, contact us.

Date modified: