Novel Food Information - Insect Resistant Corn Event MIR 162

Health Canada has notified Syngenta Seeds Canada Inc. that it has no objection to the sale of food derived from Insect Resistant Corn Event MIR 162. The Department conducted a comprehensive assessment of this corn event hybrid 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 Seeds Canada and the evaluation by Heath Canada and contains no confidential business information.

1. Introduction

Syngenta Seeds Canada developed Insect Resistant Corn Event MIR 162 using recombinant DNA techniques to introduce the insecticidal vip3Aa20 coding sequence derived from the common soil bacterium Bacillus thuringiensis strain AB88. The introduction of this gene confers resistance to lepidoptran corn pests. This event was also modified to the phosphomannose isomerase (pmi) coding sequence from Escherichia coli, permitting cells producing PMI to use mannose as a primary carbon source. This gene was introduced for use as a selectable marker.

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 Insect Resistant Corn Event MIR 162 was developed; how the composition and nutritional quality of Insect Resistant Corn Event MIR 162 compared to non-modified varieties; and what the potential is for Insect Resistant Corn Event MIR 162 to be toxic or cause allergic reactions. Syngenta has provided data which demonstrates that Insect Resistant Corn Event MIR 162 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 Insect Resistant Corn Event MIR 162 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

1. 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 Insect Resistant Corn event MIR 162 and molecular biology data that characterizes the genetic change which confers resistance to lepidoptran pests. Corn embryo tissue from variety NP2500 x NP2499 was genetically modified using an Agrobacterium tumefaciens transformation. The transforming plasmid pNOV1300 carried a transfer DNA (T-DNA) sequence comprised of two gene expression cassettes, one containing the vip3Aa19 sequence and one containing the pmi sequence.

The Vip3Aa19 protein used to produce event MIR 162 is a version of the native insecticidal Vip3Aa1 protein from B. thuringiensis (Bt) strain AB88 that has been modified to provide preferred codon usage in corn. The Vip3Aa19 protein differs from the native Vip3Aa1 protein by a single amino acid substitution, Lys-to-Gln, at position 284.

The vip3Aa19 expression cassette contained the promoter of the polyubiquitingene from Zea mays (ZmUbilnt), a corn optimized vip3Aa19 sequence from the B. thuringiensis (vip3Aa19), intron 9 of the phosphoenolpyruvate carboxlase gene of Zea mays (IPEPC9), and the terminator sequence from the cauliflower mosaic virus (CaMV35S).

The pmi expression cassette contained the promoter of the polyubiquitingene from Zea mays (ZmUbilnt), the pmi sequence from E. coli (pmi), and the terminator sequence from the nopaline synthase gene from A. tumefaciens (NOS).

3. Characterization of the Modified Plant

Southern blot analysis of Insect Resistant Corn event MIR 162 demonstrated the insertion of a single copy of the T-DNA, comprising both expression cassettes, into the corn genome at a single locus. Southern blot analysis also demonstrated the integrity of both the vip3Aa19 and the pmi genes and their associated regulatory elements. Southern blot analysis also demonstrated, as expected, the absence of any plasmid derived sequences outside the T-DNA region, such as the spectinomycin resistance gene found in the plasmid backbone. The elements contained in both the vip3Aa19 and the pmi expression cassettes have been shown to be stable with no rearrangements through Southern and PCR analysis. These analyses of the insert show that the cassettes are entirely integrated into the genome and that all the elements are intact.

From sequence analysis, a transformation-induced change was detected in the vip3Aa1 coding sequence that was introduced into corn to produce event MIR 162. Two nucleotide changes were identified within the coding sequence of the vip3Aa gene in event MIR 162. The first of these nucleotide changes resulted in an amino acid change of Met-to-Ile at position 129 of the Vip3Aa19 protein. The second nucleotide change within the vip3Aa coding sequence did not result in an amino acid change. The modified vip3Aa gene resulting from event MIR 162 was given the designation vip3Aa20, and the encoded protein was designated Vip3Aa20.Therefore, the Vip3Aa20 protein is a variant having two amino acid changes from the native insecticidal Vip3Aa1 protein from B. thuringiensis (Bt) strain AB88. The Lys-to-Gln change at position 284 in both Vip3Aa19 and Vip3Aa20 proteins is a conservative substitution, as both are polar amino acids with a molecular weight of 146. There is no apparent effect of this amino acid substitution on Vip3Aa protein function. The Met-to-Ile(129) conversion found only in the Vip3Aa20 protein, is inconsequential with respect to insecticidal activity because it is contained within the N-terminal portion of the protein that is lost during proteolytic processing in the insect gut.

The stability of the insertedexpressioncassettes was evaluated in three generations of corn. The results of Southern blot analysis and segregation data demonstrated the stability ofInsect Resistant Corn event MIR 162 at the genomic and phenotypic levels.

Confirmation of the vip3Aa20protein identity was generated using Western blot, SDS-PAGE, MALDI-ToF, N-terminal sequence analysis and glycosylation analysis. The protein analysis demonstrated that the plant-produced vip3Aa20is equivalent to the E. coli produced protein that was in the toxicology studies and that no glycosylation of the protein occurs.

Confirmation of the PMIprotein identity was generated using Western blot, SDS-PAGE, and functional activity. The protein analysis demonstrated that the plant-produced PMIis equivalent to the E. coli produced protein that was in the toxicology studies. Although glycosylation analysis was not performed by the petitioner, a comparison of the molecular mass of the proteins indicates that no glycoslyation occurs.

4. Product Information

Insect Resistant Corn Event MIR 162 differs from conventional corn by the insertion of two novel genes; vip3Aa20 and pmi and their associated regulatory elements. The insertion of these genes results in the expression of two novel proteins in MIR 162; vip3Aa20 and PMI. The expression of vip3Aa20 confers resistance to lepidopteran pests. The expression of PMI in MIR162, confers the ability to use mannose as a primary carbon source.

5. Dietary Exposure

It is expected that Insect Resistant Corn event MIR 162 will be used in applications similar to that derived from other corn varieties. 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 use of the food product from dry milling is for brewing.

6. Nutrition

Compositional analysis of Event MIR162 and near-isogenic non-transgenic control maize hybrids were compared using a complete randomized block design with three replicates per block at six field sites across the United States. A total of 18 samples each of test and control maize were harvested between mid-October and November 2005 and were analysed between December 2005 and April 2006. ANOVA was used for statistical analysis and an F-test probability level of p<0.05 was used to detect any statistically significant differences.

The components analyzed included 9 proximates (moisture, ash, protein, fat, carbohydrate, starch, total dietary fibre, acid detergent fibre, and neutral detergent fibre), 9 minerals (calcium, copper, iron, magnesium, manganese, phosphorous, potassium, zinc, selenium), 9 vitamins (beta-carotene, cryptoxanthin, alpha-tocopherol, thiamin, riboflavin, niacin, pantothenic acid, pyridoxine, and folic acid), 18 amino acids (aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, cystine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, arginine, and tryptophan), 9 fatty acids (palmitic, palmitoleic, stearic, oleic, linoleic, linolenic, arachidic, eicosenoic, and behenic), 4 sterols (cholesterol, campesterol, stigmasterol, and β-sitosterol), 4 secondary metabolites (ferulic acid, ρ-coumaric acid, furfural, and inositol), and 3 anti-nutrients (phytic acid, raffinose, trypsin inhibitor).

A total of 16 statistically significant differences were detected between test and control hybrids in forage and grain. The differences in grain included: a higher Ca, P, and Fe content in MIR 162 compared with control; a lower beta-carotene, vitamin E and vitamin B6 content compared with control; and in two fatty acids (a higher linolenic and a lower linoleic content in MIR 162 compared with control). There were no differences in total fat, protein, fibre, or amino acids, or any of the other components listed in the previous paragraph. The differences noted were not consistent between the forage and the grain and were not consistent across locations. The reported statistical differences were considered nutritionally insignificant, as they were small (<15%) and the average and range of results were within reported natural variation as reported by OECD (2002) and ILSI (2006).

7. Toxicology

B. thuringiensis toxins are widely used a supplement or alternative to chemical means of pest control and the bacterium is a key source of genes for transgenic expression to provide pest resistance in plants. Its insecticidal toxins are not known to be pathogenic to mammals or other vertebrates. Phosphomannose isomerase, the selectable marker, is a ubiquitous enzyme allowing the utilization of mannose as a carbon source. It may always have been present in the human diet at low levels.

In an acute oral (gavage) toxicity study, male and female mice (5/sex) administered 1250 mg Vip3Aa20/kg bw showed no adverse effects other than the diarrhea induced by the corn oil vehicle. In another study, no clinical signs of toxicity or other treatment-related effects were observed when PMI was administered by gavage to 6 or 7 mice/sex at a dose of 3080 mg/kg bw. In contrast, protein toxins often exert acute effects at relatively low doses. Therefore, neither novel protein would be anticipated to exert toxic effects.

Kernels from insect resistant corn event MIR162 are the most likely tissue to comprise food stuffs. The average Vip3Aa20 concentration in kernels from this event represented less than 0.004% of total protein. Similarly, PMI concentration in kernels from this event represented less than 0.0002% of total protein. In contrast, most major food allergens that sensitize via the gastrointestinal tract constitute at least 1% of the total protein content of the food.

Both the Vip3Aa20 and PMI proteins are readily degraded in simulated mammalian gastric fluid and are inactived by heat. Neither protein showed any evidence of glycoslyation.

In order to assess whether either protein has amino acid sequence similarity to any known or putative toxins, the amino acid sequences of these proteins were compared with the amino acid sequences in the latest posting of the National Center for Biotechnology Information (NCBI) Entrez Protein Database. Neither protein was found to have amino acid sequence homology with any known or putative toxin.

Two studies were conducted to determine whether the Vip3Aa20 or PMI proteins had significant amino acid sequence homology with any known allergens. In the first study, neither protein was shown to have an amino acid identity of 35% or greater over any block of 80-amino acids (considered evidence of homology) with the sequence of any known allergens. In the second study, the proteins were screened to identify short, local regions of amino acid identity (8 amino acids or greater) that might indicate the presence of common IgE-binding epitopes. Vip3Aa20 was shown not to have any such regions of homology with any allergen. One such region of homology was identified in PMI and a known allergen, alpha-parvalbumin, from Rana species.

In accordance with current guidance from the Codex Alimentarius Commission's document Guidelines for the Conduct of Food Safety Assessment of Foods Derived from Recombinant-DNA plants, the petitioner conducted additional testing on the potential allergenicity of the PMI protein. Using standard immunoblotting techniques, the petitioner examined the potential cross-reactivity between PMI and IgE from a sensitive individual's serum. The petitioner's study was limited to sera obtained from a single individual, as worldwide only a single individual has been reported to be sensitive to alpha-parvalbumin. The petitioner's study revealed no cross-reactivity between PMI and the IgE from this serum, indicating that the serum IgE does not recognize any portion of the PMI protein as an allergenic epitope. Based on the overall data presented, and even though the number of sensitive individual's sera tested is limited to one, the probability that these novel proteins are allergenic is considered remote.

Conclusion

Health Canada's review of the information presented in support of the food use of Insect Resistant Corn Event MIR 162 concluded that derived food products do not raise concerns related to safety. Health Canada is of the opinion that Insect Resistant Corn Event MIR 162 is similar to regular conventional commodity corn in terms of being an acceptable food source.

Health Canada's opinion deals only with the human food use of Insect Resistant Corn Event MIR 162. Issues related to the environmental safety of Insect Resistant Corn Event MIR 162 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
novelfoods-alimentsnouveaux@hc-sc.gc.ca