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Lepidopteran insect-protected corn event MON 89034

Health Canada has notified Monsanto Canada Inc. that it has no objection to the sale of food derived from corn lines containing the transformation event MON 89034 , which provides protection against feeding damage caused by Lepidopteran insect pests such as the European corn borer (ECB), the Southwestern corn borer (SWCB), the corn earworm (CEW), and the fall armyworm (FAW). 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 application from Monsanto Canada Inc. and the evaluation by Heath Canada and contains no confidential business information.

1. Introduction

Genetically modified MON 89034 corn was developed using recombinant DNA techniques to introduce two Bacillus thuringiensis (Bt) derived novel genes: the cry1A.105 sequences encoding Cry1A.105, a synthetic chimeric protein; and the cry2Ab2 gene encoding the Cry2Ab2 protein. The Cry1A.105 protein is a chimeric protein comprised of various domains from Cry1Ac, Cry1Ab, and Cry1F. The Cry2Ab2 protein as expressed in MON 89034 corn differs by a single amino acid from the wild type protein equivalent. All cry sequences are originally derived from Bt subsp. kurstaki with the exception of domain III sequences for cry1F, which are originally derived from Bt subsp. aizawai

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 MON 89034 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 corn lines containing event MON 89034 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 MON 89034 was developed through Agrobacterium mediated transformation of corn embryo derived tissue. The transforming plasmid PV-ZMIR245 uses a two transfer DNA (T-DNA) system. Two gene expression cassettes, one containing cry1A.105 sequences and one containing cry2Ab2 sequences, are located between the right and left borders of T-DNA I. The expression cassette containing the antibiotic resistance selectable marker gene nptII is located between a separate set of right and left border sequences in T-DNA II. The above two T-DNAs are independently inserted into separate loci in the corn genome. Regenerated plants containing both the T-DNA I and T-DNA II inserts are selected for subsequent conventional breeding. Resulting progeny are genetically selected for the presence of the cry1A.105 and cry2Ab2 cassettes of T-DNA I, and the absence of the nptII cassette of T-DNA II.

The final transgenes remaining in corn event MON 89034 consist of the following components flanked by the right and left border sequences of T-DNA I: The cry1A.105 gene cassette is composed of the promoter and 9 bp leader for the cauliflower mosaic virus (CaMV) 35S RNA containing the duplicated enhancer region (P-e35S), the 5' untranslated leader of the wheat chlorophyll a/b-binding protein gene (L-Cab), the rice actin intron sequence (I-ract1), the Bt derived coding sequences for the chimeric Cry1A.105 protein codon optimized for monocots (CS-cry1A.105), and the 3' nontranslated region of the coding sequence for wheat heat shock protein 17.3 (T-Hsp17). The cry2Ab2 gene expression cassette is composed of the Figwort Mosaic Virus 35S promoter (P-FMV), the first intron sequences from the maize heat shock protein 70 gene (I-Hsp70), the target sequence for the chloroplast transit peptide region of maize ribulose 1,5-bisphosphate carboxylase small subunit (TS-SSU-CTP), the Bt derived coding sequences for the Cry2Ab2 protein with modified codon usage (CS-cry2Ab2), and the 3' nontranslated region of the nopaline synthase gene (T-nos) from Agrobacterium tumefaciens.

3. Characterization of the Modified Plant

Southern blot analysis of MON 89034 demonstrated the insertion of a single copy of T-DNA I in the maize genome at a single locus. Southern blot analysis also demonstrated the integrity of the genetic elements, as well as the absence of any plasmid derived sequences outside the T-DNA region, such as the aad gene conferring resistance to spectinomycin and streptomycin found in the plasmid backbone. The elements contained in the cry1A.105 and cry2Ab2 expression cassettes have been shown to be stable with no rearrangements through Southern blot, PCR, and sequence analysis. Sequence analysis has also shown that both expression cassettes were entirely integrated into the corn genome and that all the elements are intact and free of rearrangements.

The stable inheritance of the insert in MON 89034 is demonstrated by segregation analysis of progeny across six generations. ELISAs were used to detect the Cry1A.105 and Cry2Ab2 proteins, while PCR was used to detect the cry1A.105 and cry2Ab2 DNA sequences. Statistical significance is determined using Chi square analysis and all generations segregated as expected for a single copy dominant allele according to Mendelian inheritance. Additionally, Southern blot analysis of plants spanning seven generations demonstrated stability at the genotypic level for the entire T-DNA I insert.

4. Product Information

Corn event MON 89034 differs from conventional corn by the insertion of two B.t. derived novel genes expressing two novel proteins that confer resistance to an expanded range of Lepidopteran insect pests including the European corn borer (ECB), the Southwestern corn borer (SWCB), the corn earworm (CEW), and the fall armyworm (FAW). The cry1A.105 gene expressed in corn event MON 89034 encodes a 133 kDa protein (Cry1A.105) consisting of 1177 amino acids. The cry2Ab2 gene expressed in corn event MON 89034 encodes 71 kDa protein (Cry2Ab2) consisting of 637 amino acids. On a dry weight basis, the levels of Cry1A.105 average 5.9 µg/g of grain, while the levels of Cry2Ab2 protein average 1.3 µg/g of grain.

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

In North America, corn derived food products for human consumption are found mainly in the form of processed foods containing maize derived ingredients such as high fructose corn syrup, starch, oil, and flour. This product would not change this pattern of consumption. The potential consumption of MON 89034 corn is assumed to be equivalent to that of normal corn consumption. Therefore, assuming a 60 kg person, the intake of these two proteins will be 0.87 µg/kg bw/day of Cry1A.105 and 0.19 µg/kg bw/day of Cry2Ab2, respectively, provided no protein is lost during processing.

6. Nutrition

Corn MON 89034 and the control (LH198 x LH172) substances were planted in a randomized complete block design with three replicates per block, at each of five field sites across the U.S. during the 2004 field season. In addition, 15 conventional, commercial corn hybrids (three different varieties in each site) were also included as references. The 15 conventional corn reference hybrids were included to provide data for the development of a 99% tolerance interval for each component analyzed. This interval is expected to contain, with 95% confidence, 99% of the values obtained from the population of commercial references. The compositional data from the five field sites were statistically analysed separately (individual site analyses) and combined across all sites (combined site analyses) using a mixed model analysis of variance.

The components analysed included protein, fat, ash, moisture, acid detergent fibre (ADF), neutral detergent fibre (NDF), total dietary fiber (TDF), carbohydrates (by calculation), 18 amino acids (alanine, arginine, aspartic acid, cystine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophane, tyrosine and valine), 22 fatty acids (C8:0 caprylic, C10:0 capric, C12:0 lauric, C14:0 myristic, C14:1 myristoleic, C15:0 pentadecanoic, C15:1 pentadecenoic, C16:0 palmitic, C16:1 palmitoleic, C17:0 heptadecanoic, C17:1 heptadecenoic, C18:0 stearic, C18:1 oleic, C18:2 linoleic, C18:3 gamma linolenic, C18:3 linolenic, C20:0 arachidic, C20:1 eicosenoic, C20:2 eicosadienoic, C20:4 arachidonic, C20:3 eicosatrienoic and C22:0 behenic acid), 9 minerals (calcium, copper, iron, magnesium, manganese, phosphorus, potassium, sodium and zinc); 2 anti-nutrients (phytic acid and raffinose) and 3 secondary plant metabolites (furfural, ferulic acid, and p-coumaric acid).

There were two statistically significant differences between the test (MON 89034) and the control (LH198 x LH172) variety in the combined sites analysis observed for 18:0 stearic acid and 20:0 arachidic acid. These differences were small (3.4 - 4.0%), considering the natural variability, and the mean levels and ranges of MON 89034 were within the 99% tolerance intervals established from the conventional reference corn substances. Other statistically significant differences observed between the test and the control substances in the individual sites analyses were not consistently observed across all sites and the means and ranges of all components from MON 89034 showing a statistical difference were within the 99% tolerance intervals of conventional corn and/or within the ranges observed in the International Life Sciences Institute Crop Composition Database.

7. Toxicology

The available evidence suggests that the novel proteins present in MON 89034 corn are unlikely to be toxins. The Cry1A.105 and Cry2Ab2 proteins were not observed to be acutely toxic in mice at doses orders of magnitude greater than the range typically associated with toxic proteins. The Cry1A.105 protein was administered at an acute oral dose of 2072 mg/kg bw and the Cry2Ab2 protein was administered at an oral dose of 2198 mg/kg bw, without any observable acute effects. Neither of the proteins is homologous to any known mammalian toxins, and therefore would not be expected to possess toxic activity associated with these proteins.

The available evidence suggests that the novel proteins present in MON 89034 corn are unlikely to resemble typical food allergens. The amino acid sequence of the Cry1A.105 and Cry2Ab2 proteins are not homologous to those of any known allergenic proteins and therefore should not be expected to possess activity associated with these allergens. Additionally, the Cry1A.105 and Cry2Ab2 proteins are expressed at low levels, are heat labile and rapidly digested in simulated gastric fluid. These observed characteristics indicate that both proteins are not similar to known food allergens.

Conclusion:

Health Canada's review of the information presented in support of the food use of corn lines containing event MON 89034 concluded that the food use of corn lines containing this event does not raise concerns related to safety. Health Canada is of the opinion that MON 89034 is similar to non-transgenic parental strains of corn in terms of being an acceptable food source.

Health Canada's opinion deals only with the human food use of corn lines containing event MON 89034 . Issues related to growing corn lines containing event MON 89034 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

Telephone: (613) 941-5535
Facsimile: (613) 952-6400