ARCHIVED - Novel Food Information

Alpha-amylase Corn Event 3272

Health Canada has notified Syngenta Seeds Canada, Inc. that it has no objection to the food use of alpha-amylase corn event 3272. 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, Inc. and the evaluation by Heath Canada and contains no confidential business information.

1. Introduction

Alpha-amylase corn event 3272 was developed through genetic modification techniques so that this corn produces a thermostable AMY797E alpha-amylase for use in the dry-grind ethanol production from corn grain. While event 3272-derived field corn lines are anticipated to be used mainly in fuel ethanol production in the United States, they may also be used like other field corn lines as feed for livestock animals and in the production of processed food products or ingredients, such as beverage alcohol and corn syrup.

The safety assessment performed by Food Directorate evaluators was conducted according to Health Canada's Guidelines for the Safety Assessment of Novel Foods. The assessment considered: how corn event 3272 was developed; how the composition and nutritional quality of corn grain derived from plants containing this event compare to non-modified corn; and what the potential is for food products derived from plants containing this event to be toxic or cause allergic reactions.

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 alpha-amylase corn event 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

Corn event 3272 was produced via transformation of immature corn embryos with Agrobacterium tumefaciens harbouring the transformation vector pNOV7013. The transforming vector carried a transfer DNA (T-DNA) sequence comprised of: 1) the amy797E gene encoding the thermostable AMY797E alpha-amylase protein and 2) the pmi (manA) gene from Escherichia coli, which encodes the enzyme phosphomannose isomerase (PMI), used as a plant selectable marker.

The alpha-amylase enzyme expressed in event 3272 is a chimeric enzyme derived from three wild-type alpha-amylases isolated from the archaeal order Thermococcales. Gene reassembly was performed using the three wild-type genes as a parental sequence to produce chimeric alpha-amylase genes. A screening strategy was designed to identify a chimeric alpha-amylase with increased thermostability and activity during the high temperatures required for starch hydrolysis in dry-grind ethanol production from corn grain. The introduction of the thermostable alpha-amylase gene into corn will replace the need to add supplemental microbially-produced amylase in the production of ethanol derived from corn.

The phosphomannose isomerase gene (pmi) from Escherichia coli was employed as a selectable marker during the transformation process. Corn cells producing PMI can utilize mannose as a primary carbon source, whereas cells lacking PMI fail to proliferate in a mannose-based culture medium. PMI enzymes are naturally found in mammals, insects, nematodes, bacteria, fungi, and many plant species with a history of safe use as human food such as pine, walnut, soybeans and other legumes.

3. Characterization of the Modified Plant

Southern blot analysis was used to determine the insert copy number, intactness of both the amy797E and pmi coding regions, intactness of the entire introduced T-DNA, and to confirm the absence of vector backbone sequences in event 3272. This analysis demonstrated a single site of insertion of the pNOV7013 T-DNA in the genome of event 3272. This insertion is composed of single intact copies of the AMY797E and PMI protein expression cassettes, including the amy797E and pmi genes and their respective promoter sequences, GZein and ZmUbiInt, were present in the insertion. Event 3272 does not contain any of the backbone sequences from the transformation plasmid pNOV7013.

The stability of the inserted amy797E and pmi genes was evaluated in multiple generations of corn grown at six separate locations. The results of Southern blot analysis and segregation data demonstrated the stability of corn event 3272 at the genomic level in different environments. The stability of the inserted DNA and levels of expression AMY797E and PMI protein were demonstrated over five generations.

4. Product Information

Corn event 3272 differs from its traditional counterparts by the expression of a chimeric alpha-amylase enzyme derived from three wild-type alpha-amylases isolated from the archaeal order Thermococcales. The chimeric alpha-amylase protein is directed specifically to the endosperm tissue of the corn kernel by the corn gamma-zein promoter. As expected, relatively high levels of AMY797E protein were measured in kernels of corn hybrids derived from event 3272. Mean AMY797E alpha-amylase levels in mature kernels of hybrids ranged from 0.89 - 1.73 mg/g dry weight. AMY797E alpha-amylase was found at low levels in other plant tissues.

Corn event 3272 also differs from its traditional counterparts by the addition of the pmi gene from Escherichia coli. The PMI enzyme serves as a selectable marker for post transformation selection on mannose media and does not supply any agronomic function in field grown plants. PMI protein was detected at low levels in most of the event 3272-derived plant tissues analysed. Mean PMI levels measured in kernels ranged from <0.5-1.8 μg/g dry weight.

5. Dietary Exposure

Event 3272-derived corn lines will be mainly utilized in fuel ethanol production in the United States. The petitioner is not proposing to limit the food use of products derived from event 3272 corn hybrids, although the anticipated human dietary exposure in these products is likely to be minimal. Field corn hybrids will be developed from corn event 3272 which are mainly used in animal feed. However, some human food uses are relevant for field corn. Event 3272 based corn hybrids could be either dry- or wet-milled into various processed corn products (e.g. high fructose corn syrup, starch or oil, grits and flour).

6. Nutrition

Test and control corn plants were grown at multiple locations in the United States in 2003 and 2004. Test and control grain samples were analysed for the following: proximates (moisture, protein, fat, ash, carbohydrate, starch, acid detergent fibre, neutral detergent fibre, and total dietary fibre), 18 amino acids, fatty acids (palmitic, stearic, oleic, linoleic and linolenic), minerals (Ca, Cu, Fe, Mg, P, K, Zn, Se) vitamins (B1, B2, B3, B6, beta-carotene, E (alpha and gamma tocopherol), and crypto-xanthin), secondary metabolites and anti-nutrients (inositol, raffinose, furfural, p-coumaric, ferulic acid, phytic acid and trypsin inhibitor). Based on the assessment of the compositional data it was concluded that corn event 3272 grains are equivalent to the non-modified and commercial controls.

7. Chemistry/Toxicology

The weight of evidence suggests that corn event 3272 expressing a thermostable alpha-amylase and phosphomannose isomerase does not contain novel toxins or novel allergens. This conclusion is based on the observations that neither protein causes acute oral toxicity when administered to mice at amounts more than three orders of magnitude greater than those anticipated to be consumed by the population. There is no significant amino acid sequence homology between these proteins and known toxins and allergens. Both proteins are readily degraded in a simulated gastric fluid system and consequently the systemic exposure to the intact protein is considered negligible.

Conclusion:

Health Canada’s review of the information presented in support of the food use of alpha-amylase corn event 3272 determined that there are no safety or nutritional concerns associated with this corn event. Health Canada is of the opinion that products from alpha-amylase corn event 3272 are as safe and nutritious as those available from commercial field corn varieties.

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

Tunney's Pasture

Ottawa, Ontario K1A 0L2

Telephone: (613) 941-5535

Facsimile: (613) 952-6400