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Decision Document 98-26:
Determination of the Safety of Monsanto
Canada Inc.'s Yieldgard^TM Insect Resistant
Corn (Zea mays L.) Line MON802

This Decision Document has been prepared to explain the regulatory decision reached under the guidelines Dir94-08 Assessment Criteria for Determining Environmental Safety of Plants with Novel Traits and its companion document Dir94-11 The Biology of Zea mays L. (Corn/Maize) and the guidelines Dir95-03 Guidelines for the Assessment of Livestock Feed from Plants with Novel Traits.

The Canadian Food Inspection Agency (CFIA), specifically the Plant Biotechnology Office and the Feed Section of the Plant Health & Production Division, has evaluated information submitted by Monsanto Canada Inc. This information is in regard to the European Corn Borer (ECB) resistant corn line MON802. CFIA has determined that this plant with novel traits does not present altered environmental interactions or pose concerns for the safety of livestock consuming feed derived from the PNT, when compared to currently commercialized corn varieties in Canada.

Unconfined release into the environment and use as livestock feed of the corn line MON802 is therefore authorized as of March 26, 1997. Any other Zea mays lines and intraspecific hybrids resulting from the same transformation event and all their descendants, may also be released, provided no inter-specific crosses are performed, provided the intended use is similar, provided it is known following thorough characterization that these plants do not display any additional novel traits and are substantially equivalent to currently grown corn, in terms of their potential environmental impact and livestock feed safety and provided that pest resistance management requirements described in the present document are applied.

Please note that, while determining the livestock feed and environmental safety of plants with novel traits are critical steps in the commercialization of these plant types, other requirements still need to be addressed, such as the evaluation of food safety (Health Canada).

Table of Contents

1. Brief Identification of Plant with Novel Traits (PNT)
2. Background Information
3. Description of the Novel Traits
   1. Resistance to the European Corn Borer (ECB)
   2. Glyphosate Tolerance
   3. Development Method
   4. Stable Integration into the Plant's Genome
4. Assessment Criteria for Environmental Safety
   1. Potential of the PNT to Become a Weed of Agriculture or be Invasive of Natural Habitats
   2. Potential for Gene Flow to Wild Relatives Whose Hybrid Offspring May Become More Weedy or More Invasive
   3. Altered Plant Pest Potential
   4. Potential Impact on Non-Target Organisms
   5. Potential Impact on Biodiversity
   6. Potential for Development of ECB Resistance to the PNT
5. Nutritional Assessment Criteria for Use as Livestock Feed
   1. Nutritional Composition of the PNT's
   2. Anti-Nutritional Factors
6. Regulatory Decision

I. Brief Identification of the Plants with Novel Traits (PNT's)

II. Background Information

Monsanto Canada Inc. has developed a corn line resistant to the European Corn Borer (ECB) larvae, a periodic pest of corn in Canada. The Yieldgard^TM corn line, designated as MON802, was developed to provide a method to control yield losses from insect feeding damage caused by the larval stages of ECB, without the use of conventional pesticides and to provide tolerance to the herbicide glyphosate.

MON802 was developed using recombinant DNA technology, resulting in the introduction of bacterial genes conferring ECB resistance and glyphosate herbicide tolerance.

Monsanto Canada Inc. has provided data on the identity of line MON802, a detailed description of the transformation method, data and information on the gene insertion site, copy number and levels of expression in the plant, the role of the inserted genes and regulatory sequences in donor organisms and the full nucleotide sequence. Each novel protein was identified, characterized and compared to the original bacterial protein, including an evaluation of its potential toxicity to livestock and non-target organisms.

These materials have been field tested in Canada under confined conditions in Ontario (1995, 1996) and also in Chile, Argentina, USA, France, Italy, South Africa, and Costa Rica.

Agronomic characteristics of corn hybrids derived from MON802 such as seed dormancy, vegetative vigour, early stand establishment, time to maturity, flowering period, susceptibilities to various Z. mays pests and pathogens, and seed production were compared to those of unmodified Z. mays counterparts.

The Plant Biotechnology Office of the Plant and Health Production Division, CFIA, has reviewed the above information, in light of the assessment criteria for determining environmental safety of plants with novel traits, as described in the regulatory directive Dir94-08:

• potential of the PNT's to become weeds of agriculture or be invasive of natural habitats,
• potential for gene flow to wild relatives whose hybrid offspring may become more weedy or more invasive,
• potential for the PNT's to become plant pests,
• potential impact of the PNT's or their gene products on non-target species, including humans, and
• potential impact on biodiversity.

The CFIA has consulted with the Pest Management Regulatory Agency of Health Canada on issues related to potential development of ECB populations resistant to the insecticidal protein produced by the PNT.

The Feed Section of the Animal Health and Production Division, 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 Dir95-03 the following have been considered:

• potential impact to livestock and
• potential impact on livestock nutrition.

III. Description of the Novel Traits

1. Resistance to the European Corn Borer (ECB)

• Bacillus thuringiensis var. kurstaki HD-1(B.t.k.) is a common gram-positive soil- borne bacterium. In its spore forming stage, it produces several insecticidal protein crystals, including the ð-endotoxin Cry1A(b) which is active against certain lepidopteran insects, such as ECB. This protein has been shown to be non-toxic to humans, other vertebrates and beneficial insects. B.t.k. based foliar insecticides have been registered for over 30 years in Canada and have a long history of safe use.
• A synthetic cry1A(b) gene was developed for maximum expression in corn, and introduced into line MON802. The gene codes for a protein similar to the Bacillus thuringiensis var. kurstaki HD-1 insecticidal crystal protein. The protein is insecticidal to lepidopteran larvae after cleavage to a bio-active, trypsin resistant core. 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 results in cell lysis. Specific lepidopteran pests of corn sensitive to the protein are ECB and corn earworm.
• The cry1A(b) gene is linked to a strong constitutive promoter. Average protein expression in micro-grams protein per gram fresh weight tissue evaluated by ELISA in leaf (six locations), grain (six locations) and whole plants (two locations) are as follows:
  - Leaf: 9.55 microgram Bt per gram fresh weight
  - Whole plant: 1.35 micrograms Bt per gram fresh weight
  - Grain: 3.2 micrograms per gram fresh weight
• The Cry1A(b) protein was shown to degrade readily in the environment. The plant expressed protein had DT₅₀ and DT₉₀ values (time to degrade to 50% and 90 % of the original bioactivity) of 1.6 and 15 days respectively.
• Protein allergens are normally resistant to digestion unlike the Cry1A(b) protein which was shown to degrade readily in simulated gastric fluid. Unlike many known allergens the insecticidal protein is not glycosylated. A search for amino acid sequence similarity between the Cry1A(b) protein and known allergens, using a database assembled from the public domain databases GenBank, EMBL, Pir and SwissProt, revealed no significant amino acid sequence homologies. A search of a similarly constructed database of known toxins indicated no amino acid sequence homologies between known toxins and the Cry1A(b) protein, with the exception of homologies to other Bt insecticidal proteins.
• Corn products are an important alternative to wheat flour for individuals afflicted with coeliac disease, an immune mediated food intolerance for which wheat gliadins have been implicated as the causal agent. In light of the importance of corn products to these individuals, a sequence similarity search was conducted and no amino acid sequence homologies between the Cry1A(b) protein and gliadins were detected.
• The full nucleotide sequence and corresponding amino acid sequence were provided.
• The native HD-1 protein has a molecular weight of 131 kD. The inserted, plant expressed cryIA(b) gene codes for a full length protein of 1156 amino acids which is processed by proteases into the 600 amino acid tryptic core fragment which is insecticidally active. After activation by trypsin to the insecticidal form, the resulting proteolytic fragments were compared to the bacterial proteins and shown to be of similar molecular weight, immunological reactivity and trypsin resistance. The protein was not glycosylated and showed similar bioactivity and host range specificity to the native protein.

2. Glyphosate Tolerance

• Two genes have been introduced into the corn line MON802, which in combination provide field level tolerance to glyphosate, the active ingredient in Roundup® herbicide.
• A plant-derived coding sequence expressing a chloroplast transit peptide was co- introduced with each of the Roundup Ready^TM genes. This peptide facilitates the import of the newly translated Roundup Ready^TM enzymes into the chloroplasts, the site of both the shikimate pathway and glyphosate mode-of-action.
• Both genes associated with their transit peptide coding sequence are linked to the same constitutive promoter.
• The first gene codes for a bacterial version of a plant enzyme 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS), involved in the shikimate biochemical pathway for the production of the aromatic amino acids. The plant version of this enzyme, ubiquitous in nature, is sensitive to glyphosate - the herbicide disrupts this essential pathway, leading to growth suppression or death of the plant. However, the bacterial derived version of this enzyme is highly insensitive to glyphosate and fulfils the aromatic amino acid needs of the plant. The product of this gene was quantified and averaged:
  - Leaf tissue - 26.99 micrograms protein/ gram (fresh weight)
  - Whole plant - 1.85 micrograms protein/ gram (fresh weight)
  - Grain - 2.27 micrograms protein/ gram (fresh weight)
• The second gene codes for glyphosate oxidase, also a bacterial enzyme from a species ubiquitous in nature. Glyphosate oxidase degrades glyphosate, thereby deactivating the herbicidal effect. The average levels of glyphosate oxidase were determined:
  - Leaf Tissue - 10.18 micrograms protein / gram fresh weight
  - Whole plant - 1.68 micrograms protein / gram fresh weight
  - Grain - not detectable
• The Roundup Ready^TM proteins showed no significant homology with any known toxins or allergens. EPSPS is an enzyme that is ubiquitous in nature, and therefore would not be expected to be toxic or allergenic. For the second novel enzyme, glyphosate oxidase, Monsanto describes experiments with a variety of substrates that show that it has a narrow substrate specificity, and appears not to affect any plant specific pathways. Both proteins are inactivated by heat, and by proteases in simulated mammalian gastric and intestinal fluids.

3. Development Method

• MON802 was co-transformed with two vectors, one carrying a synthetic cry1A(b) gene and the second bearing the two herbicide resistance genes. The two plasmid vectors were introduced by microprojectile bombardment into cultured plant cells. Glyphosate tolerant transformed cells were selected, then cultured in tissue culture medium for plant regeneration.

4. Stable Integration into the Plant's Genome

Southern analysis indicated that there are two sites of integration of the introduced DNA. Backcross data is consistant with that of a single locus behaving in a Mendelian fashion, this suggests that the two insert sites are closely linked.

The traits have remained stable for six generations as shown by assays for Cry1A(b), and herbicide tolerance.

IV. Assessment Criteria for Environmental Safety

1. Potential of the PNT to Become a Weed of Agriculture or be Invasive of Natural Habitats

The biology of corn (Zea mays), described in Dir94-11, shows 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 traits such as lack of seed dormancy, the non-shattering aspect of corn cobs, and poor competitive ability of seedlings. According to the information provided by Monsanto Canada Inc., MON802 and derived corn hybrids were determined not to be different from their counterparts in this respect.

CFIA evaluated data submitted by Monsanto Canada Inc. on the reproductive and survival biology of corn hybrids derived from MON802, and determined that flowering period, vegetative vigour, time to maturity and seed production were within the normal range of expression of these traits currently displayed by commercial corn hybrids. No genes were inserted for cold tolerance or winter survival.

No competitive advantage was conferred to these plants, other than that conferred by resistance to European Corn Borer. Resistance to ECB will not, in itself, render corn weedy or invasive of natural habitats since none of the reproductive or growth characteristics were modified. Glyphosate is commonly used for chemical fallow production and resistant corn volunteer plants will not be controlled. Still, they can be managed by growers using alternative herbicides with different modes of action.

The above considerations, together with the fact that the novel traits have no intended effects on weediness or invasiveness, led CFIA to conclude that MON802 has no altered weed or invasiveness potential compared to currently commercialized corn.

A longer term concern, if there is general adoption of several different crop and specific herbicide weed management systems, is the potential development of crop volunteers with a combination of novel resistances to different herbicides. This could result in the loss of the use of these herbicides and any of their potential benefits. Therefore, agricultural extension personnel, in both the private and public sectors, should promote careful management practices for growers who use these herbicide-tolerant crops, to minimize the development of multiple resistance.

2. Potential for Gene Flow to Wild Relatives Whose Hybrid Offspring May Become More Weedy or More Invasive

The biology of corn, as described in Dir94-11, indicates that there are no wild relatives in Canada that can hybridize with Zea mays.

CFIA therefore concludes that gene flow from MON802 to wild corn relatives is not possible in Canada.

3. Altered Plant Pest Potential

The intended effects of both novel traits are unrelated to plant pest potential, and corn is not a plant pest in Canada (Dir94-11). In addition, agronomic characteristics of the modified corn hybrids were shown to be within the range of values displayed by currently commercialized corn hybrids, and indicate that the growing habit of corn was not inadvertently altered. Field observations did not indicate modifications of disease and pest susceptibilities, other than to ECB.

CFIA has therefore determined that MON802 does not display any altered pest potential.

4. Potential Impact on Non-Target Organisms

The history of use and literature suggest that the bacterial B.t.k. ð-endotoxin is not toxic to humans, other vertebrates, and beneficial insects and the B.t.k. protein produced in corn was shown to be equivalent to the original microbial protein. This protein is active only against specific lepidopteran insects; no lepidopteran species are listed as threatened or endangered species in Canada.

In addition, Monsanto Canada Inc. has submitted data from dietary toxicity studies on the effect of the microbial B.t.k. protein on non-target insects, including pollinators (honeybees), predaceous insects (green lacewing larvae, ladybird beetles) and parasitic Hymenoptera. There was no discernible effect at approximately 10 times the usual LC₅₀ dose for a target insect.

Based on the above, CFIA has determined that the unconfined release of MON802, when compared with currently commercialized corn, will not result in altered impacts on interacting organisms, including humans, with the exception of specific lepidopteran insect species.

5. Potential Impact on Biodiversity

MON802 has no novel phenotypic characteristics which would extend its use beyond the current geographic range of corn production in Canada. Since corn does not outcross to wild relatives in Canada, there will be no transfer of novel traits to unmanaged environments.

At present, the use of chemical insecticides to control ECB is not recommended for grain corn produced in Canada, as they are only effective when the larvae first emerge and before these begin to move to the whorl of the plant and into the stalk. It is not considered cost-effective to use insecticides to control ECB in grain corn, but treatment is recommended in sweet corn given the higher cash value of the crop and lower consumer tolerance for insect-damaged cobs. Current recommendations by provincial extension services for the control of ECB in grain corn are: to grow corn hybrids which have been traditionally bred for natural resistance to ECB, to avoid planting at densities that are too high for the chosen hybrid, to plant early to reduce infestations by the second generation of ECB, to harvest early in order to decrease losses from dropped ears and broken stalks, and to practice crop rotation. Despite the promotion of no-till practices in corn for soil conservation, it is still recommended to plow-down corn residues in the fall, in order to kill over-wintering larvae. Thus, the cultivation of MON802 will not result in any overall change in the use of chemical insecticides on grain corn.

CFIA has therefore concluded that the potential impact on biodiversity of MON802 will not be altered.

6. Potential for Development of ECB Resistance to the PNT

The potential for lepidopteran insects to develop resistance to conventional chemical insecticides is well documented. A B.t.k. foliar insecticide is currently registered for control of ECB on hybrid seed corn production. Another B.t.k. insecticide is registered to control cabbage looper (but not ECB) on pepper, a crop also visited and attacked by ECB. Resistance may develop as a result of increased use of these B.t.k. foliar sprays; resistance to the B.t.k. proteins could also develop following continued exposure to ECB-resistant hybrid corn.

The development of such a resistance would result in the loss of valuable B.t.k. tools for the control of ECB infestations in corn, and to a lesser extent, pepper.

MON802 steadily produces high levels of B.t.k. in leaves relative to grain resulting in mortality of ECB feeding on this plant. Target insects will thus be exposed to significantly higher levels of B.t.k. than through the current foliar spray treatments, leading to high selection pressures for resistant ECB individuals. It is currently accepted that ECB has one or more generations a year in Canada. The number of ECB generations that will develop in any one season will be influenced by the environmental conditions in a given area, particularly temperature and day length. The potential for development of resistant ECB populations may also increase in areas with multi- generations.

A component of a possible resistance management strategy linked to the use of MON802 is the presence of non-selecting refugia (unmodified corn) in close proximity, where susceptible insect populations are maintained. Should resistant insects occur, they would then be able to mate with susceptible insects to produce heterozygotes, which are expected to be susceptible to the ECB-resistant corn hybrids. The behaviour of ECB during mating is such that individuals migrate to grassy areas adjacent to corn fields to mate, hence increasing the likelihood that any resistant ECB individuals mate with susceptible ones. Initially there will exist sufficient unstructured non-Bt corn refugia and this may delay the development of resistance. Should the acreage of ECB-resistant corn become greater than the non-Bt hybrids, careful management resulting in the maintenance of non-Bt corn (structured refugia) may be necessary to provide the required non-Bt refugia. Even though the majority of the scientific community agrees that this approach sounds effective in theory, it is very difficult at this point to predict the extent and rapidity of resistance development without field validation of the proposed strategy. These corn plants should therefore be responsibly managed and ECB populations monitored for development of resistant individuals in a regular and consistent manner.

Consideration must be given to the possibility that ECB populations developing resistance to the corn produced B.t.k. protein could also develop cross-resistance to other Bt ð-endotoxins, resulting in the loss of other Bt protein types that may be used for the control of ECB infestations.

The development of resistance to the Cry1Ab protein by non-target insect pests, that may then cause further problems in other crops, is another consideration. Armyworm (Lepidoptera: Pseudaletia unipuncta) is a sporadic pest in Canada. It feeds on corn and other crops such as forage grasses (e.g. timothy), wheat, oats and barley. Corn earworm (Lepidoptera: Helicoverpa zea) feeds on the silks and developing ears (pre- dough stage of kernels) of corn. Presently, B.t.k. foliar sprays are not registered for control of these insects in Canada, so even should resistance occur, control of these insects would not be compromised. CFIA has therefore concluded that development of resistance in non-target insect pests is unlikely to have an impact on the conventional control of these pests.

CFIA believes that sound management practices can reduce and delay the development of resistant ECB populations, and that 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 a pest resistance management plan that includes the following key components:

• The early detection of ECB populations resistant to the corn-expressed insecticidal protein 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 enforcement procedures in case of resistance development.
• 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.
• Monsanto Canada Inc. will have 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 and use of appropriate bioassays to evaluate suspected Cry1Ab resistant individuals, and a protocol for immediate action to control resistant individuals.
• Detection of confirmed resistant ECB populations and following action plan will immediately be reported to CFIA.
• Integrated Pest Management practices will be promoted, such as prediction of infestation problems from previous years and crop rotation.
• The strategy for resistance management of ECB when using plants that continually produce high concentrations of a B.t.k. ð-endotoxin and refugia has not been previously tested in the field on a large scale. Continued research in this area using sound science will be conducted.
• The developed plans, information and data from the above are available to CFIA.

CFIA also strongly encourages Monsanto Canada Inc. to develop novel ECB control systems with different modes of action that would offer additional or alternative management practices to growers.

If at any time, Monsanto Canada Inc. becomes aware of any information regarding risk to the environment, including risk to agriculture such as development of ECB resistance, or risk to animal or human health, that could result from release of these materials in Canada, or elsewhere, Monsanto Canada Inc. will immediately provide such information to CFIA. On the basis of such new information, CFIA will re-evaluate the potential impact of the proposed release, and will re-evaluate its decision with respect to the unconfined release of these corn hybrids.

V. Nutritional Assessment Criteria for Use as Livestock Feed

1. Nutritional Composition of the PNT

Comparisons of protein, fat and fibre of corn grain and whole plant material from the PNT line and its parent line were made. In both the grain and the whole plant, there were occasional significant differences between the PNT and the control but there was no consistent pattern of differences from the control for any nutrient for either grain or the whole plant.

Protein, fat and fibre concentration were within the published range for corn, in both the grain and the whole plant in the PNT. The observed variations in nutritional composition were judged to arise from normal variability rather than as a result of the inserted novel traits. CFIA has determined that line MON802 is substantially equivalent to traditional corn varieties.

2. Anti-Nutritional Factors

The parent plant Zea mays is not known for the production of anti-nutritional factors and the transformation event which produced MON802 would not be expected to induce their synthesis.

VI. Regulatory Decision

Based on the review of data and information submitted by Monsanto Canada Inc., and through comparisons of corn hybrids derived from MON802 with unmodified corn counterparts, the Plant Biotechnology Office of the Plant Health and Production Division, CFIA, has concluded that the novel genes and their corresponding traits do not confer to these plants any characteristic that would result in intended or unintended environmental effects following unconfined release. Monsanto Canada Inc. has developed and will implement a resistance management plan.

Based on the review of submitted data and information, the Feed Section of the Animal Health and Production Division has concluded that the novel trait does not in itself raise any concerns regarding the safety or nutritional composition of MON802. 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. MON802 and ECB resistant corn hybrids derived from it have been assessed and found to be substantially equivalent to traditional corn varieties. MON802 and its byproducts are considered to meet present ingredient definitions and are approved for use as livestock feed ingredients in Canada.

Unconfined release into the environment and use as livestock feed of the corn line MON802 is therefore authorized. Any other Zea mays lines and intraspecific hybrids resulting from the same transformation event, and all their descendants, are also approved, provided no inter-specific crosses are performed, provided the intended use is similar, provided it is known following thorough characterization that these plants do not display any additional novel traits and are substantially equivalent to currently grown corn, in terms of their potential environmental impact and livestock feed safety and provided that pest resistance management requirements described in the present document are applied.

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This bulletin is published by the Plant Health and Production Division and the Animal Health and Production Division CFIA. For further information, please contact the Plant Biosafety Office or the Feed Section at: