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Decision Document 97-19:
Determination of the Safety of Monsanto
Canada Inc.'s Yieldgard^TM Insect Resistant
Corn (Zea mays L.) Line MON810

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 Biosafety Office of the Plant Health and Production Division and the Feed Section of the Animal Health and Production Division, has evaluated information submitted by Monsanto Canada Inc. This information is in regard to the European Corn Borer (ECB) resistant corn line MON810. 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 MON810 is therefore authorized. 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) and Variety Registration (CFIA).

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
   2. Anti-Nutritional Factors
6. Regulatory Decision

I. Brief Identification of Plant with Novel Traits (PNT)

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 YieldgardTM corn line, designated as MON810, 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.

MON810 was developed using recombinant DNA technology, resulting in the introduction of bacterial genes conferring ECB resistance. Herbicide tolerance was used to select modified plants, but the genes conferring herbicide tolerance were presumed to be integrated at separate loci from the cryIA(b) gene and were subsequently segregated out during the development of line MON810. Molecular analysis confirmed that the genes conferring tolerance to glyphosate were absent from line MON810.

Monsanto Canada Inc. has provided data on the identity of line MON810, 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 gene and regulatory sequences in donor organisms and the full nucleotide sequence. The 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. Relevant scientific publications were supplied.

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 MON810 such as seed dormancy, seedling vigour, early stand establishment, plant vigour, 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 Biosafety Office of the Plant Health and 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.

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's.

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:

• 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 MON810. 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 evaluated at six locations was 9.35 µg/g (f.w.) in leaves and 0.31 µg/g (f.w.) in seeds. Protein expression evaluated at one site, was 4.15 µg/g (f.w.) in the whole plant and 0.09 µg/g (f.w.) in pollen, as determined from a single sample. Protein expression ranged from 7.93 to 10.34 µg/g (f.w.) in leaves, from 0.19 to 0.39 µg/g (f.w.) in grain and from 3.65 to 4.65 µg/g (f.w.) in the whole plant. Protein expression declined over the growing season as indicated by the Cry1A(b) levels present in leaves assayed over the growing season.
• 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 2 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 B.t. 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 truncated protein with a molecular weight of 91 kD. 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, amino acid sequence, 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 were originally introduced into the original transformant to provide tolerance to glyphosate, the active ingredient in Roundup® herbicide. The tolerance was used to select transformed plants.
• Molecular data was presented which showed that the genes conferring glyphosate tolerance were not present in line MON810. The genes were presumed to have inserted at genetic loci away from the cryIA(b) gene and then segregated out during subsequent crossing leading to line MON810.

3. Development Method

• MON810 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 one integrated DNA segment which included a truncated copy of the cry1A(b) gene, inserted without rearrangement.
• The corn line has been crossed into several diverse corn genotypes for 4 generations and the protection against ECB has been maintained. MON810 was derived from the third generation of backcrossing and therefore the single insert appears to be stably inherited.

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., MON810 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 MON810, and determined that early stand establishment, 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.

The above considerations, together with the fact that the novel traits have no intended effects on weediness or invasiveness, led CFIA to conclude that MON810 has no altered weed or invasiveness potential compared to currently commercialized corn.

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 MON810 to 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 MON810 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 MON810, 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

MON810 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 MON810 will not result in any overall change in the use of chemical insecticides.

CFIA has therefore concluded that the potential impact on biodiversity of MON810 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.

MON810 steadily produces high levels of B.t.k. in leaves 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 MON810 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 B.t. ð-endotoxins, resulting in the loss of other B.t. 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 MON810 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 MON810 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 MON810 with unmodified corn counterparts, the Plant Biosafety Office of the Plant Health and Production Division, CFIA, has concluded that the novel gene and its corresponding traits does 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 Plant Health and Production Division has concluded that the novel trait does not in itself raise any concerns regarding the safety or nutritional composition of MON810. 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. MON810 and ECB resistant corn hybrids derived from it have been assessed and found to be substantially equivalent to traditional corn varieties. MON810 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 MON810 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.

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

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