Novel Food Information - Insect Resistant Corn 5307

Health Canada has notified Syngenta Seeds Canada Inc. that it has no objection to the sale of food derived from Insect Resistant Maize 5307. 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 Seeds Canada and the evaluation by Heath Canada and contains no confidential business information.

1. Introduction

Syngenta Seeds Canada developed Insect Resistant Maize 5307 using recombinant DNA techniques to introduce the coding sequence for a synthetic insecticidal protein (ecry3.1Ab). This coding sequence was engineered using the coding sequences from two naturally occurring Cry proteins (Cry3A and Cry1Ab) from Bacillus thuringiensis. The sequence codes for the eCry3.1Ab protein which is an insecticidal agent for corn rootworm (Diabrotica spp.) pests. The introduction of this gene confers resistance to coleopteran 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 Maize 5307 was developed; how the composition and nutritional quality of Insect Resistant Maize 5307 compared to non-modified varieties; and what the potential is for Insect Resistant Maize 5307 to be toxic or cause allergic reactions. Syngenta has provided data which demonstrates that Insect Resistant Maize 5307 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 Maize 5307 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 Maize 5307 and molecular biology data that characterize the genetic change that confers insect resistance. Maize 5307 was produced using Agrobacterium mediated transformation of proprietary, maize variety NP2222 with the transformation vector pSYN12274.

The transformation vector pSYN12274 was constructed to contain a single T-DNA cassette, which contained the coding sequences and regulatory elements for both ecry3.1Ab and pmi. The T-DNA cassette contains the following elements, the promoter for the Cestruim Yellow Leaf Curling virus (CMP), the coding region for the insecticidal protein eCry3.1Ab (ecry3.1Ab), the terminator sequence from the nopaline synthase gene of A. tumefaciens (nos), the promoter from the maize polyubiquitin gene containing the first intron (ZMubilnt), the coding sequence of phosphomannose isomerase (pmi), and the terminator sequence from the nopaline synthase gene of A. tumefaciens (nos). The integration of this cassette results in both insect resistance and the ability to use mannose as a carbon source.

Immature maize embryos of the non-transgenic maize variety, NP2222, were transformed using vector pSYN12274 transferred via Agrobacterium tumefaciens. Plants positive for the genes of interest and negative for plasmid backbone elements were transferred to the greenhouse for further propagation and were then backcrossed with NP2222 to produce NP2222 x 5307 plants. These plants were then crossed with NP2460, a non-transgenic proprietary maize line to generate F1 plants. The F1 plants were then backcrossed with NP2460 for 5 successive generations to reach the BC5 generation. The petitioner then inbred BC5 for two generations producing BC5F2 and BC5F3. BC5F3 was then crossed with NP2171, a third proprietary, non-transgenic maize line, to produce NP2171 x BC5F3. This final generation was used in all the following studies to characterize the 5307 maize line.

3. Characterization of the Modified Plant

Southern blot analysis of Insect Resistant Maize 5307 demonstrated the insertion of a single copy of the T-DNA cassette in the maize genome at a single locus. Analysis also verified the absence of any extraneous sequences related to the pSYN12274 plasmid backbone.

The petitioner conducted further analysis of the insertion through PCR and sequencing. Using PCR primers, two overlapping PCR products encompassing the inserted cassette and the flanking genomic DNA were generated. The PCR products were sequenced to confirm that no sequence alterations had occurred in the inserted cassette. Based on the sequencing, the functional elements contained in the T-DNA cassette integrated into the maize genome matched the sequences contained in the plasmid, with no alteration in the sequence. The petitioner did note that a single change was present in a non-coding region of the insert 48 bp upstream of the CMP promoter. The petitioner also noted that the entire right boarder (RB) and the 3 bp immediately adjacent were truncated, as well as 8 bp of the left boarder (LB). These changes have no impact on any of the functional elements of the cassette, and the RB and LB truncations are common occurrences generated in A. tumefaciens mediated transformations.

Generational stability of the single insert was determined across four generations of Insect Resistant Maize 5307 (F1, BC6, BC7 and NP2171xBC5F3). Southern blot analysis was presented for each generation confirming the presence of the T-DNA cassette and its stability across generations.

The petitioner also provided the results of segregation analysis from four generations (F1, BC6, BC7 and NP2171xBC5F3) to demonstrate that the trait is inherited in the expected Mendelian fashion. Using real-time PCR, the petitioner assayed plants for the presence of the T-DNA cassette. The data provided showed no differences from the expected ratios and therefore the segregation is considered to occur in the expected manner.

Due to the low levels of protein expressed in plants, the petitioner has conducted the toxicological assessment using eCry3.1Ab and PMI proteins expressed in and purified from E. coli. To ensure that the results of the toxicological studies are applicable to the protein expressed in 5307 maize, equivalence studies (i.e., SDS PAGE, Western blot analysis, glycoprotein staining, MALDI-TOF MS, and N-terminal amino acid sequence analysis) were conducted to confirm that the protein produced in E. coli used for toxicology studies is representative of the protein produced in the modified maize plant. 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.

4. Product Information

Insect Resistant Maize 5307 differs from conventional corn by the insertion of two novel genes; ecry3.1Ab and pmi and their associated regulatory elements. The insertion of these genes results in the expression of two novel proteins for this maize variety; eCry3.1Ab and PMI. The expression of ecry3.1Ab confers resistance to corn rootworm pests. The expression of PMI in Maize 5307, confers the ability to use mannose as a primary carbon source.

The ecry3.1Ab coding region that confers insect resistance produces the eCry3.1Ab protein. This protein is a engineered protein based on two separate, naturally occurring Cry proteins produced by Bacillus thuringiensis species. Because Cry proteins share structural similarities, domains within them can be exchanged to create novel proteins. eCry3.1Ab was generated by fusing the 5' end of a synthetic Cry3A and the 3' end of a synthetic Cry1Ab. These synthetic versions of the genes were generated using the wild type sequences from B. thuringiensis subsp. tenebrionsis and subsp. kurstaki strain HD-1, respectively. In both cases the synthetic versions of the coding sequence have been codon optimized for maize. Both Cry3A and Cry1Ab have been incorporated into crops previously assessed and authorized for food use in Canada.

B. thuringiensis is a ubiquitous, Gram-positive, spore forming bacterium that forms crystals during the stationary phase. The parasporal crystals, or Cry proteins, were identified as insect pathogens and over 100 different crystal protein genes have been sequenced. B. thuringiensis is indigenous to many environments, with strains being isolated from soil, insects, stored products dust and tree leaves. B. thuringiensis is one of the mostly widely used biologically controlled pesticides and has been used in agriculture applications from more than 40 years. Both subspecies of Bt used in the generation of the eCry3.1Ab protein coding sequence have been used as source organisms on multiple occasions.

The second coding sequence encodes for the enzyme phosphomannose isomerase (PMI). This enzyme permits the utilization of mannose as a primary carbon source, allowing proliferation of cells containing the enzyme on mannose-based cell culture medium. This coding region was introduced for use as a selectable marker. The coding region, pmi, represents the manA gene from Escherichia coli. This coding sequence has been used in numerous previous applications as a selectable marker and its safety has been assessed on all these occasions.

5. Dietary Exposure

It is expected that Insect Resistant Maize 5307 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.

The petitioner has provided data to demonstrate the level of expression of both the eCry3.1Ab and PMI in the altered maize line. This study used plant samples from four sites in 2008 in the American Midwest. Data were presented as mean data points, and as a range, over all sites for whole plants at four time points (Whorl, Anthesis, Maturity and Senescence). In addition, the petitioner provided range values for leaves, kernels, root and pollen protein concentrations at the same four sites.

The quantity of eCry3.1Ab protein was determined by a validated enzyme-linked immunosorbent assay (ELISA). Protein quantities for the tissues were calculated on a microgram (µg) per gram (g) fresh weight (fwt) basis. The mean eCry3.1Ab protein concentration across locations for whole plants at the Whorl, Anthesis, Maturity and Senescence time points were 15.78 (11.41-28.64), 8.11 (3.10 - 13.12), 8.86 (3.36 - 21.96), and 3.60 (1.70 - 10.65) µg/g fwt, respectively. Additionally, the petitioner provided the following range values for leaves, kernels, root and pollen eCry3.1Ab concentration, < Limit of Quantification (LOQ) to 71.21, 1.60 to 7.29, 0.40 to 9.29, and <LOQ to 0.09 µg/g fwt, respectively.

The quantity of PMI protein was determined by a validated enzyme-linked immunosorbent assay (ELISA). Protein quantities for the tissues were calculated on a microgram (µg) per gram (g) fresh weight (fwt) basis. The mean PMI protein concentration across locations for whole plants at the Whorl, Anthesis, Maturity and Senescence time points were 0.62 (0.34-1.13), 0.93 (0.44 - 2.13), 0.96 (0.41 - 1.57), and 0.43 (0.15 - 0.71) µg/g fwt, respectively. Additionally, the petitioner provided the following range values for leaves, kernels, root and pollen PMI concentration, < Limit of Detection (LOD) to 1.66, 0.50 to 2.38, <LOQ to 1.07, and 5.16 to 6.06 µg/g fwt, respectively.

The petitioner also provided an analysis of the protein concentrations in the processed food and feed fractions. From this analysis the petitioner provided mean concentration values for each of the following fractions: grain, wet milled products (gluten, starch and dried germ) and dry milled products (flour, germ). For grain, the petitioner determined the mean eCry3.1Ab and PMI concentrations were 4.98 and 1.31 ug/g, respectively. For the wet milled products, concentrations for all the fractions tested were below the limit of detection. For flour, the determined concentrations were 1.06 and 0.20 ug/g for eCry3.1Ab and PMI, respectively. For germ, concentrations of 19.33 and 3.97 ug/g for eCry3.1Ab and PMI respectively, were determined. Higher levels of protein concentration are expected in the dry milled product than grain, as the protein concentration in those fractions is greater than in whole grain.

Based on available consumption patterns for corn flour, if a 70 kg adult consumes 8.8 g of corn flour per day containing 1.06 µg/g eCry3.1Ab and 0.20 µg/g PMI, the exposure to these proteins would equal 0.13 µg/kg bw/day and 0.025 µg/kg bw/day, respectively. If a 70 kg individual was to consume 100 g of corn kernels containing 4.98 µg/g of eCry3.1Ab and 1.31 µg/ g of PMI, the exposure would be approximately 7.1 µg/kg bw/day for eCry3.1Ab and 1.9 µg/kg bw/day for PMI.

6. Nutrition

The nutrient data for this submission was obtained from test (event 5307) and control maize grown in 2008 at six locations in a randomized complete block design with three plots for both test and control.

Test and control were analyzed for nutrients, anti-nutrients and secondary metabolites, in grain, for Proximates (moisture, crude protein, crude fat, ash, carbohydrates; plus acid detergent fibre (ADF), neutral detergent fibre (NDF), total dietary fibre and starch), Minerals (calcium, copper, iron, magnesium, manganese, phosphorus, potassium, selenium, sodium and zinc), Fatty Acids (C8 to C22), Amino Acids (aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, cysteine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, arginine, and tryptophan), Vitamins (folate, vitamin A (beta-carotene), vitamin B1, vitamin B2, vitamin B3, vitamin E (a-tocopherol)), Anti-Nutrients (phytic acid, raffinose, trypsin inhibitor) and Secondary Metabolites (ferulic acid, furfural, inositol, p-coumaric acid).

For the combined locations, statistical differences (5307 vs. control) were noted in 7 analytes as follows: eicosenoic acid, linolenic acid, palmitic acid, stearic acid, vitamin A, vitamin B6 and folic acid. However, for all analytes where a statistical difference was determined, all mean values were within reported literature ranges.

All field trial experiments used to test event 5307 maize were acceptable. All analysis of test, control and reference varieties were done using approved scientific and appropriate statistical methods.

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.

The PMI protein was previously evaluated in several products which were authorized for food use by Health Canada. Those toxicological assessments of PMI protein were based on its lack of acute oral toxicity in mice, in silico evidence that it has no amino acid sequence homology with known toxins, its degradation when exposed to high temperatures, its rapid digestion in simulated mammalian gastric fluids and the low level of exposure from its proposed use. Based on those previous toxicological evaluations and the low level of exposure from this novel food, additional safety studies were not considered warranted.

The potential toxicity of eCry3.1Ab protein was assessed in an acute oral toxicity study, where mice (5 animals/sex) were administered the protein as a single dose of 2,000 mg/kg bw. Animals were observed for 14 days after administration, then euthanized and necropsied. The parameters that were recorded included mortality, signs of clinical toxicity, body weight, food consumption, as well as macroscopic and histological examination were conducted at termination. The test substance was well tolerated and did not induce any treatment-related or adverse effects.

An in-vitro digestibility assay for the eCry3.1Ab protein was carried out in simulated gastric fluid (SGF) containing the mammalian digestive enzyme pepsin. An SDS-PAGE analysis, densitometry analysis and Western blot analysis was performed to detect the eCry3.1Ab after 0-15 minutes of digestion. All three assays demonstrated that eCry3.1Ab is readily digested in SGF within 30 seconds, with approximately only 3% of the protein remaining after 15 seconds. This suggests the intact protein is unlikely to enter the bloodstream and cause systemic toxicity.

A heat stability study with the eCry3.1Ab protein was conducted at temperatures ranging from 25ºC to 95ºC for 30 minutes. The presence of the eCry3.1Ab protein after heat treatment was determined by measuring its insecticidal activity (LC50 and % mortality) on the Colorado potato beetle larvae. The results from this study demonstrated that the eCry3.1Ab protein becomes denatured and inactivated, and therefore an active protein is unlikely to be consumed from products that are processed normally (e.g. cooking).

A bioinformatic search was conducted using an extensive protein database (National Centre for Biotechnology Information, 2012) to compare the amino acid sequence from the eCry3.1Ab protein to known or putative toxins. Other than matches with other Cry-related insecticidal proteins, one similarity was found between an open reading frame sequence in the insert and a hypothetical protein from A. tumefaciens. Neither match was considered toxicologically significant, since no actual protein was made. In summary, no relevant matches were made comparing eCry3.1Ab protein to known or putative toxins.

The petitioner provided an assessment on the similarity of the eCry3.1Ab protein to known or putative allergens using the most up to-date version of the FARRP database (2012). Two searches were performed: one for sequence similarity for the entire eCry3.1Ab protein with at least 35% shared identity over 80 or more amino acids, and a second search for matches of 8 or more contiguous amino acids with the sequences in the FARRP protein database. The results of these searches found that no significant similarity was observed between the eCry3.1Ab protein and any entry in the FARRP database using the above mentioned criteria. This updated study supports the conclusion that there are no meaningful amino acid sequence similarities between the eCry3.1Ab protein and any known allergens.

The margin of exposure (MOE) for eCry3.1Ab between the No Observed Adverse Effect Level of the acute oral toxicity study in mice and the estimated daily intake (EDI) of eCry3.1Ab from the consumption of corn flour, was estimated to be more than 15,000,000 [15,384,615= 2,000 mg per kg bw/ 0.00013 mg per kg bw per day]. This MOE value is sufficiently large to support the safety of eCry3.1Ab at the proposed level of use.

Based on the low levels of expected exposure, their rapid degradation when exposed to SGF, denaturation at high temperatures and the absence of homology with known toxins and allergens, no toxicity or allergenicity concerns with 5307 maize expressing eCry3.1Ab and PMI were identified.

Conclusion

Health Canada's review of the information presented in support of the food use of Insect Resistant Maize 5307 concluded that derived food products do not raise concerns related to safety. Health Canada is of the opinion that Insect Resistant Maize 5307 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 Maize 5307. Issues related to the environmental safety of Insect Resistant Maize 5307 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.

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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