DD2008-74: Determination of the Safety of Monsanto Canada Inc.'s Corn (Zea mays L.) Event MON 89034

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Issued: 2008-06

This Decision Document has been prepared to explain the regulatory decision reached under Directive 94-08 (Dir94-08), entitled "Assessment Criteria for Determining Environmental Safety of Plants with Novel Traits", its companion biology document BIO1994-11, "The Biology of Zea mays L. (Corn/Maize)", and Directive 95-03 (Dir95-03), entitled "Guidelines for the Assessment of Novel Feeds: Plant Sources".

The Canadian Food Inspection Agency (CFIA) has evaluated information submitted by Monsanto Canada Inc. This information is in regard to the lepidopteran insect resistant corn event MON 89034. The CFIA has determined that this plant with novel traits (PNT) does not present altered environmental risk nor, as a novel feed, does it present livestock feed safety concerns when compared to currently commercialized corn varieties in Canada.

Taking into account these evaluations, unconfined release into the environment and use as livestock feed of corn event MON 89034 are therefore authorized by Plant Biosafety Office of the Plant Health and Biosecurity Directorate and the Animal Feed Division of the Animal Health Directorate as of June 19, 2008. Any corn lines derived from event MON 89034 may also be released into the environment and used as livestock feed, provided that (i) no inter-specific crosses are performed, (ii) the intended uses are similar, (iii) it is known based on characterization, that these plants do not display any additional novel traits and are substantially equivalent to currently grown corn varieties in Canada, in terms of their potential environmental impact and livestock feed safety, (iv) the novel genes are expressed at a level similar to that of the authorized line and (v) the insect resistance management requirements described in the present document are applied.

Corn event MON 89034 is subject to the same phytosanitary import requirements as its unmodified counterparts.

Please note, that the livestock feed and environmental safety of novel feeds and PNTs are critical steps in the potential commercialization of these plant types. Other requirements, such as the evaluation of food safety by Health Canada, have been addressed separately from this review.

This Decision Document was modified on August 13, 2009 to reflect a change to the insect resistance management plan.

Table of Contents

  1. Brief Identification of the Modified Plant
  2. Background Information
  3. Description of the Novel Traits
    1. Resistance to Lepidopteran Pests of Corn
    2. Development Method
    3. Stable Integration into the Plant Genome
  4. Criteria for the Environmental Assessment
    1. Potential of Corn event MON 89034 to Become a Weed of Agriculture or be Invasive of Natural Habitats
    2. Potential for Gene Flow from Corn event MON 89034 to Wild Relatives Whose Hybrid Offspring May Become More Weedy or More Invasive
    3. Altered Plant Pest Potential of Corn event MON 89034
    4. Potential Impact of Corn event MON 89034 on Non-Target Organisms
    5. Potential Impact of Corn event MON 89034 on Biodiversity
    6. Potential for Development of European Corn Borer Resistance to corn event MON 89034
  5. Criteria for the Livestock Feed Assessment
    1. Potential Impact of Corn event MON 89034 on Livestock Nutrition
    2. Potential Impact of Corn event MON 89034 on Livestock and Workers/Bystanders
  6. New Information Requirements
  7. Regulatory Decision

I. Brief Identification of the Modified Plant

Designation of the Modified Plant: Corn Event MON 89034, OECD Unique Identifier MON-89034-3

Applicant: Monsanto Canada Inc.

Plant Species: Maize (Zea mays L.)

Novel Traits: Resistance to lepidopteran pests of corn, including European Corn Borer (Ostrinia nubilalis), Corn Earworm (Helicoverpa zea), and Fall Armyworm (Spodopera frugiperda).

Trait Introduction Method: Agrobacterium-mediated transformation

Proposed Use of the Modified Plant: Production of corn for human consumption (wet mill products, dry mill products and seed oil) and oil, meal, grain, silage and other byproducts for livestock feed. These plants are not intended to be grown outside the normal production area for corn in Canada.

II. Background Information

Monsanto Canada Inc. developed, through the use of recombinant DNA techniques, a corn event resistant to lepidopteran insect pests of corn in Canada. The corn event, designated as MON 89034, was developed to provide a method to control yield losses from insect feeding damage caused by the European Corn Borer (Ostrinia nubilalis) and other lepidopteran insect pests.

Corn event MON 89034 was developed using recombinant DNA technology, resulting in the introduction of the synthetic cry1A.105 gene and the cry2Ab2 gene. The cry1A.105 gene is derived from the cry1Ab, cry1Ac and cry1F genes from Bacillus thuringiensis and encodes a synthetic δ-endotoxin Cry1A.105 protein. The cry2Ab2 gene is also derived from Bacillus thuringiensis and encodes a variant of the wild-type δ-endotoxin Cry2Ab2 protein. The combination of the Cry1A.105 and Cry2Ab2 insecticidal proteins in a single plant provides insect control against a range of lepidopteran insect pests. In addition, a selectable marker gene nptII was used in the initial transformation and early event selection process but was removed from the final event selected. The nptII gene from Escherichia coli encodes the neomycin phosphotransferase II protein conferring resistance to the antibiotic kanamycin. This trait is of no agronomical interest but was used to select transformed from non-transformed plants during the early development phase of corn event MON 89084. Once the transgenic plants were identified, the selectable marker gene nptII was no longer needed. Traditional breeding was used to isolate plants that contain the cry1A.105 and cry2Ab2 genes but do not contain the nptII gene, therefore producing marker-free corn event MON 89034.

Monsanto Canada Inc. has provided data on the identity of corn event MON 89034, a detailed description of the transformation method, data and information on the insertion site, gene copy number and levels of gene expression in the plant and the role of the inserted genes and regulatory sequences. The novel proteins were identified and characterized. Data was provided for the evaluation of the potential toxicity of the novel proteins to livestock and non-target organisms and potential allergenicity of the novel proteins to humans and to livestock.

Corn event MON 89034 has been field tested in the United States and Canada and data for trial years 2004 and 2005 were submitted.

Agronomic characteristics of corn event MON 89034 such as seedling emergence, plant height, time to reproduction, lodging, susceptibilities to various corn pests, pathogens and abiotic stressors and yield parameters were compared to those of unmodified corn counterparts.

Nutritional components of corn event MON 89034, such as proximates, amino acids and fatty acids were compared with those of unmodified corn counterparts.

The Biotechnology Environmental Release Assessment (BERA) Unit of the Science Strategies Directorate, CFIA, has reviewed the above information, in light of the assessment criteria for determining environmental safety of PNTs, as described in the Directive 94-08 (Dir94-08), entitled "Assessment Criteria for Determining Environmental Safety of Plants With Novel Traits". The BERA Unit has considered:

  • potential of corn event MON 89034 to become a weed of agriculture or be invasive of natural habitats;
  • potential for gene flow from corn event MON 89034 to wild relatives whose hybrid offspring may become more weedy or more invasive;
  • potential for corn event MON 89034 to become a plant pest;
  • potential impact of corn event MON 89034 or its gene products on non-target species, including humans; and
  • potential impact of corn event MON 89034 on biodiversity.

The Animal Feed Division of the Animal Health Directorate, CFIA, has also reviewed the above information with respect to the assessment criteria for determining the safety and efficacy of livestock feed, as described in Directive 95-03 (Dir95-03), entitled "Guidelines for the Assessment of Novel Feeds: Plant Sources". The Animal Feed Division has considered:

  • potential impact of corn event MON 89034 on livestock nutrition; and
  • potential impact of corn event MON 89034 on livestock and workers/bystanders

Monsanto Canada Inc. has provided the CFIA with a method for the detection and identification of corn products containing the corn event MON 89034

III. Description of the Novel Traits

1. Resistance to Lepidopteran Pests of Corn

Bacillus thuringiensis is a common gram-positive soil-borne bacterium. In the spore forming stage, it produces several insecticidal protein crystals, including the δ-endotoxin Cry1Ab, Cry1Ac, Cry2Ab and Cry1F proteins which are active against certain lepidopteran insects, such as the European Corn Borer. These proteins are insecticidal to susceptible lepidopteran insects after cleavage by proteases in the insect's gut, forming a protease-resistant active fragment (core toxin) that is the bio-active form of the protein. Insecticidal activity is believed to depend on the binding of the active fragment to specific receptors present in susceptible insects on midgut epithelial cells, forming pores which disrupt osmotic balance and eventually result in cell lysis and insect death. Corn event MON 89034 produces two Bt proteins, namely Cry1A.105 and Cry2Ab2. The Cry1A.105 protein is a chimeric protein that consists of different domains from Cry1Ab, Cry1Ac and Cry1F. It was designed by Monsanto using domain exchange strategy to achieve a high level of activity against target lepidopteran pests. The overall amino acid sequence identity of Cry1A.105 to Cry1Ac, Cry1Ab and Cry1F is 93.6%, 90.0% and 76.7%, respectively. The other Bt protein produced in MON 89304, Cry2Ab2, is identical to Cry2Ab2 protein expressed in Lepidopteran-resistant cotton Bollgard II which has been authorized by the CFIA for livestock feed use. This protein has been shown to be non-toxic to humans and other vertebrates. Except for one amino-acid, the amino acid sequence of the Cry2Ab2 protein produced in Bollgard II cotton and MON 89034 corn is identical to that of the wild-type Bt protein. The cry1A.105 and cry2Ab2 coding sequences were optimized to accommodate the preferred codon usage for maize.

The cry1A.105 and cry2Ab2 genes are expressed in event MON 89034 using promoters which confer constitutive expression of proteins. Tissue samples were collected at various growth stages from a MON 89034 hybrid grown at five representative US field trial sites. Levels of Cry1A.105 and Cry2Ab2 proteins were evaluated by enzyme-linked immunosorbent assays. Average Cry1A.105 levels across all stages and all sites, expressed in micro-grams Cry1A.105 protein per gram dry weight tissue (µg/g dwt), ranged from ca. 75-520 in leaf, 11-79 in root and 42-380 in whole plant. The levels of Cry1A.105 protein tended to decline over the growing season. The mean Cry1A.105 protein levels across all sites were 5.9 µg/g dwt in grain and 12 µg/g dwt in pollen.

Average Cry2Ab2 levels across all stages and all sites, expressed in micro-grams Cry2Ab2 protein per gram dry weight tissue (µg/g dwt), ranged from ca. 130-180 in leaf, 21-58 in root and 38-130 in whole plant. The levels of Cry2Ab2 protein tended to decline over the growing season. The mean Cry2Ab2 protein levels across all sites were 1.3 µg/g dwt in grain and 0.64 µg/g dwt in pollen.

The Cry1A.105 and Cry2Ab2 proteins were shown to degrade readily in the soil. Soil degradation experiments used three different soil types representative of corn growing regions. Depending on the soil type, the time to degrade 50% and 90% of the initial spiked concentration of Cry1A.105 protein ranged from 2 to 7 days, and 7 to 19 days, respectively, and the time to degrade 50% and 90% of the initial spiked concentration of Cry2Ab2 protein ranged from 0.5 to 3 days, and 3 to 13 days, respectively.

The Cry1A.105 and Cry2Ab2 proteins were purified from the grain of corn event MON 89034 and characterized. The identity of the purified proteins was confirmed by Western immunoblot analysis, tryptic peptide mass mapping, N-terminal characterization and insecticidal activity.

The levels of Cry1A.105 and Cry2Ab2 proteins in corn MON 89034 tissues were too low to extract sufficient amounts for evaluation of environmental and feed safety. To obtain sufficient quantities of the proteins for safety studies, it was necessary to express the cry1A.105 and cry2Ab2 genes in an E. coli production system. The equivalency of the plant-produced proteins to the E. coli - produced proteins was evaluated by comparing their molecular weight, immunological reactivity, tryptic peptide mass map, insecticidal activity and glycosylation status. Based on the results, Cry1A.105 and Cry2Ab2 proteins produced in corn MON 89034 were found to be equivalent to their respective E. coli - produced counterparts.

The potential mammalian toxicity and allergenicity of the Cry1A.105 and Cry2Ab2 proteins were evaluated. Both proteins lack sequence similarity to known allergens and protein toxins which have adverse effects to mammals. No adverse effects were observed when the Cry1A.105 protein was ingested by mice at a dose of 2,072 mg/kg body weight, or the Cry2Ab2 protein at a dose of 2,198 mg/kg body weight, or the Cry2Ab2 protein at a dose of 2,198 mg/kg body weight, or the Cry2Ab2 protein at a dose of 2,198 mg/kg body weight. In vitro digestive fate studies have shown that both Cry1A.105 and Cry2Ab2 proteins are rapidly degraded in simulated gastric fluid, unlike protein allergens which are normally resistant to digestion. The Cry1A.105 and Cry2Ab2 proteins expressed in event MON 89034 are not glycosylated, unlike many known allergens, providing additional evidence that Cry1A.105 and Cry2Ab2 proteins do not have the properties of known allergens.

2. Development Method

Corn event MON 89034 was developed through Agrobacterium-mediated transformation of immature embryos derived from corn line LH172 using a transformation plasmid vector that includes two separate T-DNAs. The first T-DNA, designated as T-DNA-I contains the cry1A.105 and the cry2Ab2 expression cassettes. The second T-DNA, designated as T-DNA II, contains the nptII expression cassette. Following cultivation with Agrobacterium, the embryos were transferred to a selection medium containing paromomycin to eliminate cells that were not transformed and select cells containing T-DNA-II or T-DNA I + T-DNA II. During subsequent breeding the unlinked insertions of T-DNA I and T-DNA II were segregated. The plants containing only the cry1A.105 and the cry2Ab2 expression cassettes were selected using molecular analysis while the plants containing the nptII expression cassette were eliminated from subsequent breeding. Event MON 89034 was identified as a successful transformant and was chosen for further development.

3. Stable Integration into the Plant Genome

Molecular characterization by Southern blot analysis demonstrated that corn event MON 89034 contains one copy of the cry1A.105 and cry2Ab2 gene cassettes inserted at a single site in the corn genome. No additional elements, including intact or partial DNA fragments of the cry1A.105 or cry2Ab2 cassette, T-DNA II sequences or backbone sequences from the plasmid vector, linked or unlinked to the intact insert, were detected in corn event MON 89034. Sequencing of the introduced DNA confirmed the organization and sequence of the genetic elements with two exceptions. Some elements of the promoter that regulates the expression of the cry1A.105 gene have been deleted and the right border sequence present in the transformation vector has been replaced by a left border in MON 89034 corn. These modifications have no effect on the functionality of the DNA insert.

The stability of the inserted DNA was demonstrated by Southern blot analysis across seven generations in the breeding history of MON 89034 corn. Analysis of the inheritance pattern of the cry1A.105 and cry2Ab2 genes and the Cry1A.105 and Cry2Ab2 proteins across five generations of event MON 89034 confirmed the stability of the inserted DNA and the stability of Cry1A.105 and Cry2Ab2 protein expression. The results of the analysis are consistent with the finding of a single site of insertion that segregates according to the Mendelian law of genetics.

IV. Criteria for the Environmental Assessment

1. Potential of Corn event MON 89034 to Become a Weed of Agriculture or be Invasive of Natural Habitats

The biology of corn, described in the CFIA Biology Document BIO1994-11, states that unmodified plants of this species are not invasive of unmanaged habitats in Canada. Corn does not possess the potential to become weedy due to the lack of seed dormancy, the non-shattering nature of corn cobs and the poor competitive ability of seedlings. According to the information provided by Monsanto Canada Inc., corn event MON 89034 was determined to be similar to unmodified corn in this respect. The introduction of novel traits did not alter the dormancy, germination and volunteer potential of the seed of MON 89034 compared to conventional corn seed.

MON 89034 corn hybrids were tested in the United States corn belt at 9 locations in 2004 and 9 locations in 2005, and in Canada at 5 locations in 2005. A total of 14 phenotypic and agronomic traits were evaluated. These agronomic traits covered a broad range of characteristics that encompass the entire life cycle of the maize plant and included data assessing seedling emergence, vegetative vigour, time to reproduction and yield characteristics. For the majority of agronomic traits, no statistically significant differences between MON 89034 corn hybrids and their non-transformed counterpart were observed. Although instances of statistically significant differences between MON 89034 and control hybrids were observed for some traits, there were no consistent trends in the data across locations or years that would indicate that any of these differences were due to the genetic modification.

In addition to the phenotypic and agronomic characteristics, susceptibility of MON 89034 corn to abiotic stressors and various corn pests and pathogens was evaluated. Differences in susceptibility to wind damage or excess moisture were observed between MON 89034 and control corn at a few trial sites in 2004; however, they were not considered biologically meaningful since no consistent trends were observed across sites and years. These observations indicated that MON 89034 corn had no altered susceptibility or tolerance to the biotic and abiotic stressors compared to control hybrids, except for the introduced lepidopteran-resistance trait.

The results showed that no competitive advantage was conferred to corn event MON 89034 by the expression of the Cry1A.105 and Cry2Ab2 proteins, other than that conferred by resistance to lepidopteran insect pests. None of the corn's reproductive or growth characteristics were modified, and MON 89034's tolerance to abiotic and biotic stresses was unchanged except for the intended trait of resistance to lepidopteran insect pests. As feeding damage by lepidopteran larvae is not known to be a major factor restricting the establishment or distribution of corn in Canada, the introduction of this novel trait does not make MON 89034 corn weedy or invasive of natural habitats.

The above considerations led the CFIA to conclude that corn event MON 89034 has no increased weediness or invasiveness potential compared to currently commercialized corn varieties.

2. Potential for Gene Flow from Corn event MON 89034 to Wild Relatives Whose Hybrid Offspring May Become More Weedy or More Invasive

The biology of corn, as described in CFIA Biology Document BIO1994-11, indicates that there are no wild relatives in Canada that can hybridize with corn. None of the data submitted by Monsanto Canada Inc. on corn MON 89034 indicated any change in sexual compatibility as a result of the genetic modification.

The CFIA has therefore concluded that gene flow from corn event MON 89034 to wild relatives is not possible in Canada.

3. Altered Plant Pest Potential of Corn event MON 89034

The expression of the Cry1A.105 and Cry2Ab2 proteins is unrelated to plant pest potential, and corn is not considered a plant pest in Canada. Field evaluation of MON 89034 corn did not show any increase or decrease in susceptibility to disease or insect stressors, other than to some lepidopteran insects, compared to unmodified corn counterparts. Feeding damage by lepidopteran larvae is not known to be a major factor restricting the establishment or distribution of corn in Canada.

The CFIA has, therefore, determined that the corn event MON 89034 does not display any altered pest potential compared to currently commercialized corn varieties.

4. Potential Impact of Corn event MON 89034 on Non-Target Organisms

The history of use and available literature indicate that Bacillus thuringiensis δ-endotoxins are active only against specific insect groups, and are not toxic to other organisms including humans and other vertebrates. The Cry1A.105 expressed in MON 89034 corn is a chimeric protein derived from the well characterized Cry1Ab, Cry1Ac and Cry1F proteins. The spectra of activity of these three proteins are restricted to some lepidopteran insects. The Cry2Ab2 protein expressed in MON 89034 corn is identical to the Cry2Ab2 protein expressed in Bollgard II cotton and has been shown to be specific to certain lepidopteran species (see DD2003-45).

Monsanto Canada Inc. provided studies on the insecticidal activities of the Cry1A.105 and Cry2Ab2 proteins on a range of insects from three taxa. Insect species tested included four lepidopterans (European corn borer, corn earworm, fall armyworm and black cutworm), two coleopterans (boll weevil, southern corn rootworm) and two hemipterans (western tarnished plant bug and green peach aphid). The results confirmed that Cry1A.105 and Cry2Ab2 proteins have activity against all four lepidopteran species but no activity against the coleopteran or hemipteran species.

Consequently, the endangered species assessment focused on larvae of the order Lepidoptera. The habitat of the Karner blue butterfly (Lycaeides melissa samuelis) has the potential to occur in proximity of corn fields; however, the Karner blue butterfly is listed by the Committee on the Status of Endangered Wildlife as extirpated in Canada (Please refer to http://www.cosewic.gc.ca/index.htm for more information). Even should Karner blue butterfly populations be recovered in Canada, Karner blue larvae would have low likelihood of exposure to corn pollen because corn pollen shed typically occurs after Karner blue larvae have finished feeding. The lepidopteran species currently listed by the Committee on the Status of Endangered Wildlife in Canada as being a threatened or endangered species are usually not found in proximity of corn fields. Therefore the potential exposure of threatened or endangered lepidopteran species in Canada to Cry1A.105 or Cry2Ab2 protein expressed in MON 89034 corn will be minimal.

Among the butterfly species of special concern in Canada, the monarch butterfly (Danaus plexippus) is the only species that may be exposed to significant amounts of the Cry1A.105 and Cry2Ab2 proteins expressed in MON 89034. As monarch butterfly larvae feeding on milkweed plants may be exposed to corn pollen drifting from adjacent MON 89034 corn plants, a risk assessment was conducted for quantification of the potential impact of corn pollen from MON 89034 corn on populations of monarch butterflies in the North American corn belt. Data from an extended dietary assay conducted on newly-hatched monarch larvae indicate that pollen from MON 89034 corn has no adverse effect on survival and development of monarch larvae to adult at pollen densities lower or equal to 147 grains/cm2 on milkweed leaves. An estimation of monarch exposure to MON 89034 corn pollen was conducted using published procedures and parameters described in Sears et al. (2001), Pleasants et al. (2001) and Dively et al. (2004). These assessment results indicate that MON 89034 corn would affect approximately 0.6% of the monarch population across the North American corn belt, which includes Ontario. Similar levels are anticipated for the other Canadian corn growing areas, including Quebec. Such small population effects are unlikely to have any significant impact on the sustainability of monarch butterfly populations in Canada.

An assessment of the potential for interaction between Cry1A.105 and Cry2Ab2 proteins using two sensitive lepidopteran species demonstrated that the proteins only showed additive insecticidal activity and no synergistic or antagonistic activities. The lack of interaction between Cry1A.105 and Cry2Ab2 proteins allows for each protein to be tested independently.

Monsanto Canada Inc. has submitted data from dietary toxicity studies on the effect of Cry1A.105 or Cry2Ab2 protein on non-target invertebrates, including the honeybee larvae and adult (Apis mellifera), minute pirate bug (Orius insidiosus), ladybird beetle (Coleomegilla maculata), a parasitic wasp (Ichneumon promissorius), and earthworm (Eisenia foetida). Collembola (Folsomia candida) were fed an artificial diet containing 50% of MON 89034 leaf tissue. In all cases, the Cry1A.105 and Cry2Ab2 proteins were demonstrated to be safe to these indicator species at doses equal to or exceeding 14 times the estimated environmental concentration of Cry1A.105 and Cry2Ab2 proteins in the diet of non-target invertebrates feeding on MON 89034 tissues or exposed to MON 89034 corn via their preys. In addition, no adverse effects were observed when the aquatic invertebrate Daphnia magna was exposed to MON 89034 corn pollen at a concentration of 100 mg/L, which indicates that no hazard is anticipated to aquatic invertebrates from exposure to MON 89034 corn pollen.

Data was also submitted on non-target vertebrates including the mouse, the bobwhite quail and broiler chicken. No adverse effects were detected when mice were exposed to a single oral dose of 2,072 mg Cry1A.105 protein/kg body weight or 2,198 mg Cry2Ab2 protein /kg body weight. These doses represent several thousand times the worst-case daily dose of Cry1A.105 and Cry2Ab2 proteins to humans or livestock feeding on MON 89034 grain. No adverse effects were detected when bobwhite quail or broiler chicken were fed a diet containing 50% MON 89034 corn grain for 8 days and 42 days, respectively.

Composition analyses showed that the levels of key nutrients and anti-nutrients in corn MON 89034 grain and forage are comparable to those in commercial corn varieties.

Based on the above, the CFIA has determined that, compared to current commercial corn varieties, the unconfined release of corn event MON 89034 will not result in altered impacts on non-target organisms, including humans.

5. Potential Impact of Corn event MON 89034 on Biodiversity

Corn event MON 89034 has no novel phenotypic characteristics that would extend its range beyond the current geographic range of corn production in Canada. Since corn has no wild relatives with which it can outcross in Canada, there will be no transfer of the novel traits to other species in unmanaged environments. In addition the novel traits were determined to pose minimal risks to non-target organisms.

At present, the use of chemical insecticides and Bt corn hybrids to control corn pests is common practice in Canada. Therefore, the reduction in local pest species as a result of the release of MON 89034 corn does not present a significant change from existing agricultural practices.

The CFIA has therefore concluded that the potential impact on biodiversity of corn event MON 89034 is equivalent to that of currently commercialized corn varieties.

6. Potential for Development of European Corn Borer Resistance to Corn event MON 89034

In order to significantly minimize the likelihood of the development of insect pest resistance to modified plants expressing novel insect resistance, the CFIA requires that an insect resistance management (IRM) plan be implemented for these products. Lepidopteran insects have a significant ability to develop resistance to conventional chemical insecticides. Therefore, it is reasonable to expect that resistance to the insecticidal properties of corn event MON 89034 may develop. The following IRM design is intended to reduce or delay European corn borer (ECB) resistance to the Cry1A.105 and Cry2Ab2 proteins. A component of the IRM strategy that will be used with MON 89034 corn is the establishment of a refuge of ECB-susceptible corn within or near the MON 89034 corn field. Should resistant insects occur, they would then be able to mate with susceptible insects from the refuge to keep the frequency of resistance genes diluted in the insect population.

As Cry1A.105 and Cry2Ab2 proteins are highly active against ECB larvae, each protein individually achieves 95% or greater control of ECB at the concentrations produced in MON 89034 corn leaves. In addition, as Cry1A.105 and Cry2Ab2 proteins have different primary structures, share only 14% of amino acid sequence identity and bind differently to distinct proteins in the midgut of ECB , the likelihood of cross-resistance between the two proteins is very low. In view of the dual effective dose delivered by MON 89034 corn and the lack of cross-resistance between the two proteins, the likelihood of ECB resistance development to MON 89034 corn is significantly reduced compared to corn events expressing a single Bt protein. Therefore, MON 89034 corn cultivation is expected to be sustainable using a reduced refuge. The current refuge size requirement for single Bt protein corn products is 20% of total corn acres. Based on conservative mathematical models, a 5% refuge will preserve the durability of MON 89034 corn.

CFIA believes that sound management practices and IRM strategies can significantly reduce and delay the development of ECB populations resistant to Cry1A.105 protein and/or Cry2Ab2 protein. However, the ECB populations must be monitored for the development of resistance in a regular and consistent manner.

CFIA understands that Monsanto Canada Inc. has developed, and will implement, an IRM plan that includes the following key components:

  1. The use of structured refugia to provide a population of European corn borers that have not been exposed to the Cry1A.105 and Cry2Ab2 proteins and are available to reproduce with potentially resistant ECB individuals which may emerge from the MON 89034 crop.
  2. The early detection of ECB populations resistant to MON 89034 corn-expressed insecticidal proteins is extremely important. Close monitoring for the presence of such populations, in ECB-resistant corn fields and surrounding areas, is therefore warranted. Monitoring includes the development of appropriate detection tools such as visual field observations and laboratory bioassays, education of growers, reporting schedules, and mitigation procedures in case of resistance development.
  3. Education tools will be developed and provided to all growers, district managers and field managers. These will include information on product performance, resistance management, monitoring procedures and timetables, detection protocols for resistant ECB individuals, instructions to contact Monsanto Canada Inc. and strategies to be followed if unexpected levels of ECB damage occur.
  4. Monsanto Canada Inc. will have documented procedures in place for responding to these reported instances of unexpected ECB damage. These procedures will include, where warranted, the collection of plant tissue and ECB individuals and use of appropriate bioassays to evaluate suspected resistant individuals, and a protocol for immediate action to control resistant individuals.
  5. Detection of confirmed resistant ECB populations and mitigation measures will be immediately reported to CFIA.
  6. Integrated Pest Management practices will be promoted, such as prediction of infestation problems from field histories.

Note: The Plant Biosafety Office periodically audits compliance with the IRM requirements.

V. Criteria for the Livestock Feed Assessment

1. Potential Impact of Corn event MON89034 on Livestock Nutrition

Nutritional Composition

The compositional equivalence of MON 89034 to its isogenic non-transgenic control (LH198 x LH172) was assessed from five sites in the US during the 2004 growing season. Forage and grain samples were collected from replicated plots and analyzed for proximate, acid detergent fibre (ADF), neutral detergent fibre (NDF), total detergent fibre (TDF), minerals, amino acids, fatty acids, vitamins and secondary metabolites. There were no statistically significant differences between MON 89034 and control across locations for crude protein, total fat, ash, moisture, carbohydrates, ADF and NDF, TDF in forage and grain. Crude protein and NDF were higher in test than control at one site, but means were within the normal variation of commercial corn. Phosphorus in forages was significantly higher in MON 89034 than control; however, the means were within the tolerance interval for commercial reference corn and literature values. No statistically significant differences were observed between MON 89034 and control grain for all minerals across sites. Significant differences between test and control were observed for Cu and Fe at two sites and Ca and Mn at one site, but all means were within literature ranges. Except for stearic and arachidic acids, all other fatty acids in grain were similar between MON 89034 and control across locations. Stearic and arachidic acid levels were within the commercial tolerance interval and literature values. Linolenic, palmitoleic, oleic, linoleic and eicosenoic acids were significantly different between test and control at one site, but all means were within the tolerance interval for commercial corn. No statistically significant differences were observed between test and control for amino acids across locations, while significant differences were observed at one site; however, no consistent trend was observed. All vitamin levels were statistically similar between test and control. Ferulic and p-coumaric acid in MON 89034 were not statistically significantly different from the control in the combined analyses. All means were within the commercial tolerance interval and literature values.

Anti-nutritional factors

Phytic acid was analyzed in MON 89034 grain and compared to its non transgenic control (LH198 x LH172). No statistically significant differences were observed between MON 89034 and control for phytic acid. All means were within the conventional tolerance interval and literature values.

Nutrient Bioavailability

A 42-d broiler study was performed to evaluate the effects of transgenic MON 89034 on broiler performance of 600 birds on six diets (including diets of MON 89034; near isogenic control and four reference commercial corn hybrids). Broiler mortality averaged 4% and was not related to any dietary treatment. Body weights, total feed intake, feed conversion, carcass yield, fat pad, breast, wing, drum and thigh weight, percent moisture, protein and fat in thigh and breast meat were similar across treatments. Adjusted feed conversion for birds on MON 89034 diet was significantly lower than the control diet, but similar to three of the commercial corn reference varieties. Further comparison of MON 89034 to control and commercial corn varieties (as a group) showed no statistically significant differences between the two groups. No adverse health effects were observed in the course of the trial.

The evidence provided by Monsanto supports the conclusion that the nutritional composition of MON 89034 corn is substantially equivalent to its non transgenic control varieties.

2. Potential Impact of Corn event MON 89034 on Livestock and Workers/Bystanders

Acute oral toxicity studies in mice demonstrated a non-observed-effect-level (NOEL) value of 2072 mg/kg-bw/day for Cry1A.105, which is much higher than the predicted worst case exposure values of 19.38, 15.96, and 11.88 mg/kg-bw/day predicted for Cry1A.105 in broilers, swine, and cattle, respectively. Acute oral toxicity studies in mice demonstrated a non-observed-effect-level (NOEL) value of 2198 mg/kg-bw/day for Cry2Ab2, which is much higher than the predicted worst case exposure values of 6.63, 5.46, and 4.06 mg/kg-bw/day predicted for Cry2Ab2 in broilers, swine, and cattle, respectively. A feeding trial in broiler chickens using realistic levels of exposure of the two proteins in MON 89034 compared with conventional diets showed no effects on performance or health. The proteins Cry1A.105 and Cry2Ab2 have been demonstrated to be heat labile and rapidly digested under conditions present in the gastrointestinal tract. These proteins have shown no sequence similarities with known allergens or toxins which would indicate potential allergenicity or toxicity issues. Maximum exposures to the proteins are mitigated by their digestibility, processing, as well as the blending of the MON 89034 corn with other varieties.

These findings indicate that MON 89034, which produces Cry1A.105 and Cry2Ab2 proteins, is not expected to result in adverse toxicological effects in livestock fed MON 89034 corn or in humans exposed to the proteins. Based on the large disparity between predicted exposure levels and no effect levels, and the lack of inherent allergenic properties demonstrated by the proteins, no significant risk to livestock and workers/bystanders is expected from exposure to the Cry1A.105 and Cry2Ab2 proteins produced by the MON 89034 corn.

Based on the detailed characterization provided for MON 89034 (nutritional composition and agronomic data of the modified plant compared to the unmodified comparator), it is unlikely that the modification has had any unintended effects on the modified plant.

The evidence provided by Monsanto Canada Inc. supports the conclusion that the potential impact on livestock and workers/by-standers of corn event MON 89034 is equivalent to that of currently commercialized corn lines.

VI. New Information Requirements

If at any time Monsanto Canada Inc. becomes aware of any information regarding risk to the environment, including risk to human or animal health, which could result from release of corn event MON 89034 materials in Canada or elsewhere, Monsanto Canada Inc. will immediately provide such information to the CFIA. On the basis of such new information, the CFIA will re-evaluate the potential impact of event MON 89034 on the environment, livestock and human health, and may re-evaluate its decision with respect to the livestock feed use and environmental release authorizations of corn event MON 89034.

VII. Regulatory Decision

Based on the review of the data and information submitted by Monsanto Canada Inc., and through comparisons of corn event MON 89034 with unmodified corn counterparts, the Biotechnology Environmental Release Assessment Unit, CFIA, has concluded that the novel genes and their corresponding traits do not confer to corn event MON 89034 any characteristic that would result in unintended environmental effects following unconfined release.

Based on the review of submitted data and information by Monsanto Inc., including comparisons of corn event MON 89034 with its unmodified corn counterparts, the Animal Feed Division of the Animal Health Directorate has concluded that the introduced genes and their corresponding traits will not confer to corn event MON 89034 any characteristic that would raise any concerns regarding the safety or nutritional composition of corn event MON 89034. Grain corn, its byproducts and corn oil are currently listed in Schedule IV of the Feeds Regulations and are, therefore, approved for use in livestock feeds in Canada. Corn event MON 89034 has been assessed and found to be as safe as and as nutritious as traditional corn varieties. Corn event MON 89034 and its products are considered to meet present ingredient definitions and are approved for use as livestock feed ingredients in Canada.

Taking into account these evaluations, unconfined release into the environment and use as livestock feed of corn event MON 89034 are therefore authorized by Plant Biosafety Office of the Plant Health and Biosecurity Directorate and the Animal Feed Division of the Animal Health Directorate as of June 19, 2008. Any corn lines derived from event MON 89034 may also be released into the environment and used as livestock feed, provided that no inter-specific crosses are performed; the intended uses are similar; it is known based on characterization, that these plants do not display any additional novel traits and are substantially equivalent to currently grown corn varieties in Canada, in terms of their potential environmental impact and livestock feed safety; the novel genes are expressed at levels similar to that of the authorized line; and the insect resistance management requirements described in the present document are applied.

Corn event MON 89034 is subject to the same phytosanitary import requirements as its unmodified counterparts.

Please refer to Health Canada's Decisions on Novel Foods for a description of the food safety assessment of corn event MON 89034. The food safety decisions are available at the following Health Canada web site: http://www.hc-sc.gc.ca/fn-an/gmf-agm/appro/index-eng.php

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