Decision Document DD2010-82 Determination of the Safety of Monsanto Canada Inc.'s corn (Zea mays L.) event MON 87460

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Issued: 2010-12

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 document BIO1994-11, "The Biology of Zea mays L. (Corn/Maize)", and Chapter 2.6 of the Regulatory Guidance: Feed Registration Procedures and Labelling Standards, entitled "Guidelines for the Assessment of Novel Feeds: Plant Sources".

The Canadian Food Inspection Agency (CFIA), specifically the Plant Biosafety Office of the Plant Health and Biosecurity Directorate, the Plant and Biotechnology Risk Assessment Unit of the Plant Health Science Directorate and the Animal Feed Division of the Animal Health Directorate, has evaluated information submitted by Monsanto Canada Inc. This information is in regard to the drought tolerant corn event MON 87460. The CFIA has determined that this plant with a novel trait (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 87460 is therefore authorized by the Plant Biosafety Office of the Plant Health and Biosecurity Directorate and the Animal Feed Division of the Animal Health Directorate as of December 17, 2010. Any corn lines derived from corn event MON 87460 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 in Canada, in terms of their potential environmental impact and livestock feed safety, and (iv) the novel genes are expressed at a level similar to that of the authorized line.

Corn event MON 87460 is subject to the same phytosanitary import requirements as its unmodified counterpart.

Please note that the livestock feed and environmental safety assessments 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.

Table of Contents

  1. Brief Identification of the Modified Plant
  2. Background Information
  3. Description of the Novel Trait
    1. Development Method
    2. Drought Tolerance
    3. Resistance to Aminoglycoside Antibiotics
    4. Stable Integration into the Plant Genome
  4. Criteria for the Environmental Assessment
    1. Potential of Corn Event MON 87460 to become a Weed of Agriculture or be Invasive of Natural Habitats
    2. Potential for Gene Flow from Corn Event MON 87460 to Wild Relatives whose Hybrid Offspring May Become More Weedy or More Invasive
    3. Altered Plant Pest Potential of Corn Event MON 87460
    4. Potential Impact of Corn Event MON 87460 on Non-Target Organisms
    5. Potential Impact of Corn Event MON 87460 on Biodiversity
  5. Criteria for the Livestock Feed Assessment
    1. Potential Impact of Corn Event MON 87460 on Livestock Nutrition
    2. Potential Impact of Corn Event MON 87460 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 87460, OECD Unique Identifier MON-87460-4
Applicant:
Monsanto Canada Inc.
Plant Species:
Corn (Zea mays (L.)
Novel Traits:
Drought tolerance (reduced yield loss under water-limited conditions)
Trait Introduction Method:
Agrobacterium-mediated transformation
Intended 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. has developed a drought tolerant corn event. This corn event, designated MON 87460, was developed to reduce yield loss under water-limited conditions. Under well-watered conditions corn event MON 87460 does not perform differently than unmodified corn. Corn event MON 87460 is still subject to some yield loss under drought stress.

The drought tolerance trait was introduced by Agrobacterium-mediated transformation, resulting in the introduction of a gene for a cold shock protein (CSPB) cloned from Bacillus subtilis. Cold shock proteins moderate stress responses in plants and bacteria, primarily by stabilizing RNA. Corn event MON 87460 also contains a neomycin phosphotransferase II (nptII) gene. The NPTII protein confers resistance to the antibiotic kanamycin. Kanamycin was used as a selectable marker during the development of corn event MON 87460 and this trait has no agronomic or environmental significance.

Monsanto Canada Inc. has provided data on the identity of corn event MON 87460, 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 87460 was field tested in the United States and Chile in 2006 and 2007. Some of the locations (Illinois, Iowa, Indiana, Nebraska, Ohio and Pennsylvania) of these trials share similar environmental and agronomic conditions to corn production areas in Southern Ontario and Quebec and were considered representative of major Canadian corn growing regions.

MON 87460 corn's agronomic characteristics, nutritional components, and physiological responses to drought were observed in the field studies. Responses to biotic stresses were also observed (e.g., insect damage, disease damage).

Agronomic characteristics observed included seedling vigour, early stand count, days to 50% pollen shed, days to 50% silking, plant height, ear height, pollen morphology, pollen viability, final stand count, stay green, stalk lodged plants, root lodged plants, dropped ears, grain moisture, test weight, yield, kernels per ear, dormancy/germination, volunteer potential, and potential to survive in unmanaged ecosystems.

Physiological responses included leaf extension rate, photosynthetic rate, carbon fixation, stomatal conductance and relative water content. Monsanto Canada Inc. also provided data to address the potential for corn event MON 87460 to have altered tolerance to abiotic stresses other than drought (e.g., salt, heat, cold).

Nutritional components focussed on those described in the OECD "Consensus Document on Compositional Considerations for new Varieties of Maize (Zea mays): Key Food and Feed Nutrients, Anti-Nutrients and Secondary Plant Metabolites". Forage and grain samples were measured for moisture, ash, protein, fat, carbohydrate, fibre composition, minerals, amino acids, fatty acids, vitamins, and anti-nutrients.

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

  • the potential of corn event MON 87460 to become a weed of agriculture or to be invasive of natural habitats;
  • the potential for gene-flow from corn event MON 87460 to wild relatives whose hybrid offspring may become more weedy or more invasive;
  • potential for soybean event MON 87701 to become a plant pest;
  • the potential for corn event MON 87460 to become a plant pest;
  • the potential impact of corn event MON 87460 and its gene products on non-target species, including humans; and
  • the potential impact of corn event MON 87460 on biodiversity.

The Animal Feed 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 Chapter 2.6 of the Regulatory Guidance: Feed Registration Procedures and Labelling Standards, entitled "Guidelines for the Assessment of Novel Feeds: Plant Sources". The Animal Feed Division has considered:

  • the potential impact of corn event MON 87460 on livestock nutrition; and
  • the potential impact of corn event MON 87460 on livestock and workers/bystanders.

Monsanto Canada Inc. has provided the CFIA with a method for the detection and identification of corn event MON 87460.

III. Description of the Novel Trait

1. Development Method

Corn event MON 87460 was developed through Agrobacterium-mediated transformation of immature corn embryos with a T-DNA plasmid containing the cspB and nptII genes and their regulatory elements. Corn event MON 87460 was selected based on the presence of CSPB protein, tolerance to kanamycin, its reduced yield loss under water-limited conditions, its phenotypic characteristics and its molecular profile.

2. Drought Tolerance

Corn event MON 87460 has reduced yield loss under water-limited conditions compared to conventional corn. Corn event MON 87460 may still experience some yield loss under water-limited conditions, particularly during drought-sensitive growth stages such as flowering and grain fill. Under well-watered conditions, corn event MON 87460 does not perform differently than conventional corn.

The gene cspB in corn event MON 87460 encodes the cold shock protein B (CSPB) protein from B. subtilis. The CSPB protein belongs to the family of CSPs, which participate in the bacterial cold shock response. CSPs contain highly conserved RNA-binding sequences, which are known as cold shock domains (CSDs). In bacteria, environmental stresses can result in the production of RNA secondary structures, which leads to a reduction in protein synthesis and contributes to the disruption of normal cell physiology. Proteins that contain CSDs, such as CSPs, can bind RNA, helping to sustain translation, maintain RNA levels, and improve cellular function. In corn event MON 87460, the CSPB protein is similarly expected to help maintain cellular function during stress by stabilizing RNA.

The cspB gene in corn event MON 87460 is identical to that in B. subtilis with the exception of a single amino acid change at the second position from leucine to valine that was necessary for cloning purposes.

Expression of cspB is driven by a constitutive promoter in corn event MON 87460. Samples of tissues were collected from corn plants at various growth stages from six field trial sites in the United States. Average CSPB protein expression across all plant stages was evaluated by enzyme-linked immunosorbent assay. Average CSPB protein concentrations expressed in micro-grams CSPB protein per gram dry weight tissue (µg/g dwt), ranged from 0.47-3.1 µg/g dwt in leaves, 0.029-1.4 µg/g dwt in roots and 0.67-2.8 µg/g dwt in whole plants. Average CSPB protein concentrations were 0.072 µg/g dwt in grain, 13 µg/g dwt in pollen, 1.2 µg/g dwt in silk, 0.042 µg/g dwt in stover, and 0.10 µg/g dwt in forage. Consistent with the use of a constitutive promoter, no differences were observed in CSPB protein levels in tissues collected from plants grown under well-watered or water-limited conditions.

The CSPB protein was purified from the grain of corn event MON 87460. The identity of the purified protein was confirmed by western blot analysis, N-terminal sequence analysis, mass spectral analysis of tryptic peptides, and functional activity assay.

The concentration of CSPB protein in corn event MON 87460 was too low to enable extraction of sufficient amounts for evaluation of environmental and feed safety. To obtain sufficient quantities of the protein for safety studies, it was necessary to express the cspB gene in an E. coli production system. The equivalency of the plant produced CSPB protein to the E. coli-produced protein was evaluated by comparing their molecular weights, immunological reactivity, N-terminal sequence, functional activity and glycosylation status. Based on the results, the CSPB protein produced in corn event MON 87460 was found to be equivalent to its E. coli-produced counterpart.

The potential mammalian toxicity and allergenicity of the CSPB protein was evaluated. The CSPB protein expressed in corn event MON 87460 is highly similar to other CSPs present in bacterial species broadly used by the food industry and with CSD-containing proteins in plant species used as food. The CSPB protein lacks sequence similarity to known allergens and protein toxins which have adverse effects on mammals. No adverse effects were observed when CSPB protein was ingested by mice at a dose of approximately 4.7 mg/kg body weight in acute oral toxicity studies. In vitro digestive fate studies have shown that CSPB protein is rapidly degraded in simulated gastric fluid, unlike protein allergens which are normally resistant to digestion. A small, transiently stable CSPB protein fragment was observed but this was quickly degraded during short exposure to simulated intestinal fluid. The CSPB protein expressed in corn event MON 87460 is not glycosylated, unlike many known allergens, providing additional evidence that the CSPB protein does not have the properties of known allergens. Therefore, based on the weight of evidence, the CSPB protein expressed in corn event MON 87460 is unlikely to be toxic or allergenic to mammals.

3. Resistance to Aminoglycoside Antibiotics

Aminoglycoside antibiotics bind to bacterial ribosomes, disrupting normal protein synthesis and killing the bacterial cell. The nptII gene, which originates from the E. coli transposon Tn5, produces an enzyme that phosphorylates aminoglycoside antibiotics, preventing them from binding to ribosomes and thereby rendering the cells resistant. Thus, the NPTII protein confers positive selection of genetically transformed plant cells on media containing aminoglycoside antibiotics.

The nptTII gene in corn event MON 87460 is linked to a constitutive promoter. Samples of tissues were collected from corn plants at various growth stages from six field trial sites in the United States. Average NPTII protein expression across all plant stages was evaluated by enzyme-linked immunosorbent assay. Average NPTII protein concentrations across growth stages, expressed in micro-grams NPTII protein per gram dry weight tissue (µg/g dwt), were 2.6 µg/g dwt in leaves, 0.47 µg/g dwt in roots, and 0.12 µg/g dwt in forage. Levels of NPTII protein in grain were below the limit of quantitation (<0.0047 µg/g fwt).

NPTII protein was purified from leaves of corn event MON 87460. The identity of the purified protein was confirmed by western blot analysis. The equivalency of the NPTII protein produced in corn event MON 87460 to a previously characterized E. coli-produced protein was evaluated by comparing their molecular weight and immunological reactivity. Previous assessments have shown that the NPTII protein lacks sequence similarity to known allergens and protein toxins that have adverse effects on mammals. No adverse effects were observed when NPTII protein was ingested by mice at a dose of approximately 5,000 mg/kg body weight in acute oral toxicity studies. The NPTII protein is ubiquitous in the environment, and is present in several previously authorized biotechnology-derived crops in Canada.

4. Stable Integration into the Plant Genome

Molecular characterization by Southern blot analysis demonstrated that corn event MON 87460 contains one intact copy of the cspB and nptII gene cassettes inserted at a single site in the corn genome. No additional elements, including intact or partial DNA fragments of the cspB or nptII cassette or backbone sequences from the plasmid vector, linked or unlinked to the intact insert, were detected in corn event MON 87460. Sequencing of the introduced DNA confirmed the organization of the genetic elements and revealed that the right and left border regions were truncated. The truncation in the right border region included part of the promoter driving expression of cspB. Sequencing of the flanking genomic DNA revealed a 22 bp deletion at the DNA-insert junction. The truncations and deletion had no effect on the functionality of the DNA insert or the plant and such phenomena have been previously observed with Agrobacterium transformation.

The stability of the insert within corn event MON 87460 was verified by Southern blot analysis over seven generations. The inheritance pattern of the cspB gene across three segregating backcross generation of corn event MON 87460 showed that the cspB gene segregates according to Mendelian rules of inheritance for a single genetic locus.

IV. Criteria for the Environmental Assessment

1. Potential of Corn Event MON 87460 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. Monsanto Canada Inc. provided data on phenotypic and agronomic traits to establish that corn event MON 87460 is not weedier or more invasive than commercially available Canadian corn hybrids.

Corn event MON 87460 was compared to an unmodified counterpart that had a similar genetic background but was not transformed with the cspB gene. Commercially available hybrids were also grown in these trials to provide a reference range for typical corn behaviour. Field trials were conducted under imposed well-watered and water-limited conditions, as well as conditions where water availability was not managed (i.e., naturally-occurring conditions). For the sites where water-limited conditions were imposed, the only sites used for statistical analysis for the effect of water treatment were those in which the reference hybrids had a 15% yield penalty in the water-limited plots (in comparison to the well-watered plots).

Corn event MON 87460 hybrids were field tested in the United States and Chile in 2006 and 2007. Some of the locations (Illinois, Iowa, Indiana, Nebraska, Ohio and Pennsylvania) of these trials share similar environmental and agronomic conditions to corn production areas in Southern Ontario and Quebec and were considered representative of major Canadian corn growing regions, of which this product is intended for cultivation. Phenotypic and agronomic traits were evaluated, covering a broad range of characteristics that encompass the entire life cycle of the corn plant. The traits included seedling vigour, early stand count, days to 50% pollen shed, days to 50% silking, plant height, ear height, pollen morphology, pollen viability, final stand count, stay green, stalk lodged plants, root lodged plants, dropped ears, grain moisture, test weight, yield, and kernels per ear.

For the majority of agronomic traits, no statistically significant or biologically significant differences between corn event MON 87460 and its unmodified counterpart were observed. Although instances of statistically significant differences were observed between corn event MON 87460 and control hybrids for some traits, there were few consistent trends in the data across locations or years that would indicate that any differences that could be associated with weediness were due to the genetic modification. A few traits (e.g., stalk lodging, root lodging) indicated that there was a trend occurring in corn event MON 87460 that was not occurring in the comparator, but these values were within the reference range established as typical corn behaviour in the same field trials.

Monsanto Canada Inc. provided information on the dormancy and germination of corn event MON 87460 seed under several different temperature regimes. No biologically significant differences were detected with respect to percentages of dead seed, normal germinated seed, abnormal germinated seed, total germinated seed, viable firm swollen seed, and viable hard seed.

Volunteer potential was assessed at three locations in the United States. Corn event MON 87460 seeds were seeded in the fall of 2006. No volunteers were detected the following spring.

The potential for corn event MON 87460 to survive in unmanaged ecosystems was also measured at four locations. Seeds from an F2 generation carrying the corn event MON 87460 trait were planted in unmanaged environments. These seeds were genetically typical of those that would be produced in a corn event MON 87460 grain field. The F2 plants set seed at only one of the four locations. There were 39 corn event MON 87460 F2 plants per plot in that location at reproductive maturity, yet only 8 seeds were produced per plot. This indicates that any population of corn event MON 87460 that establishes outside of cultivation will decrease over time and eventually disappear.

Therefore, the introduction of novel traits did not alter the dormancy, germination and volunteer potential of the seed of corn event MON 87460 compared to conventional corn seed.

Monsanto Canada Inc. also provided information from studies to establish that corn event MON 87460 has no increased tolerance to other abiotic stresses (e.g., salt, heat, cold). These studies were conducted in greenhouses and growth chambers because these stresses are very difficult to reliably establish in the field. Corn event MON 87460 behaved similarly to other commercially available corn hybrids in these and in field tests and demonstrated no additional stress tolerances beyond drought tolerance.

The susceptibility of corn event MON 87460 to various corn pests and pathogens and additional abiotic stresses was evaluated in the field. Across 31 sites, 21 arthropods, 19 diseases, and 15 abiotic stressors were observed. They were: cold, compaction, frost, hail, heat, nitrogen deficiency, nutrient deficiency, flood/water logging, wind damage, drought, green snap, mineral toxicity, heavy thunderstorm, anthracnose, crazy top, ear rot, ear and kernel rot, eye spot, Fusarium, gray leaf spot, gray mold, leaf blight, maize dwarf mosaic virus, northern corn leaf blight, northern leaf spot, Pythium, root rot, rust, seedling blight, southern corn leaf blight, smut, stalk rot, Stewarts wilt, yellow leaf blight, aphid, armyworm, billbugs, corn earworms, corn rootworm beetles, cutworms, European corn borers, flea beetles, grape colaspis, grasshoppers, Japanese beetles, leafhoppers, leafrollers, mites, northern corn rootworms, seed corn maggot, southwestern corn borers, spider mites, thrip, western bean cutworms, white grubs, and wireworm. No qualitative differences were observed for 386 of 388 observations, and the other two observations (one difference for grasshoppers and the other for European corn borer) were not consistent trends.

These observations indicated that corn event MON 87460 had no altered susceptibility or tolerance to the biotic and abiotic stressors compared to control hybrids, except for the introduced drought tolerance trait.

The results showed that no competitive advantage was conferred to corn event MON 87460 by the expression of the drought tolerance trait, other than that conferred by reduced yield loss under water-limited conditions. None of the reproductive or growth characteristics of corn event MON 87460 were modified, and tolerance to abiotic and biotic stresses was unchanged. Although it can have a significant impact on yield, water availability is not considered to be a major factor restricting the establishment or distribution of corn in Canada. The introduction of this novel trait does not make corn event MON 87460 weedy or invasive of natural habitats.

The above considerations led the CFIA to conclude that corn event MON 87460 has no increased weediness or invasiveness potential compared to currently commercialized corn varieties.

2. Potential for Gene Flow from Corn Event MON 87460 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. The drought tolerance trait is unrelated to sexual compatibility and the information submitted by Monsanto Canada Inc. on corn event MON 87460 indicates that it behaves similarly to commercially available corn in terms of reproduction.

The CFIA has therefore concluded that gene flow from corn event MON 87460 to wild relatives is not possible in Canada.

3. Altered Plant Pest Potential of Corn Event MON 87460

The drought tolerance trait is unrelated to plant pest potential, and corn is not considered to be a plant pest in Canada. Field evaluation of corn event MON 87460 did not show any increase or decrease in susceptibility to disease or insect stressors compared to its unmodified counterpart.

The CFIA has therefore determined that corn event MON 87460 does not display any altered pest potential compared to currently commercialized corn varieties.

4. Potential Impact of Corn Event MON 87460 on Non-Target Organisms

The gene cspB in corn event MON 87460 encodes the cold shock protein B (CSPB) protein from B. subtilis. The B. subtilis bacterium is common in soil and has no known toxic or allergenic properties. It is not pathogenic and it is present in some fermented foods with a history of safe consumption. The United States Food and Drug Adminstration has designated enzyme preparations from this bacterium as Generally Recognized As Safe (GRAS).

Cold shock proteins also have no known toxicity or allergenicity. Proteins with cold shock domains are ubiquitous in nature, including many plants and some bacterial species which are part of the gastrointestinal flora.

The CSPB protein shows no sequence similarity with known allergens, gliadins, glutenins, or protein toxins. The full-length protein is 99% rapidly digested in simulated gastric fluid (SGF) within 30 seconds. A small transiently stable fragment which was not degraded in SGF was rapidly degraded in simulated intestinal fluid (SIF), indicating that it is unlikely that CSPB protein would reach intestinal mucosa and thereby be absorbed into the human bloodstream. Proteins which are rapidly degraded do not tend to be allergens.

Although there is no indication of toxicity associated with CSPB protein, acute oral toxicity studies were performed on mice. There were no adverse effects at 4.7 mg/kg body weight. This is 1000 to 10,000 times greater than the conservative estimates for expected human exposures to CSPB protein from corn event MON 87460. CSPB protein makes up no more than 0.000007% of total protein in grain of corn event MON 87460.

Since there are no allergenic or toxic properties associated with CSPB protein, there is no indication that any endangered species associated with corn fields will be put at any additional risk by the cultivation of corn event MON 87460. For example, there will be no adverse effects posed by the cultivation of corn event MON 87460 on either the Karner blue butterfly (Lycaeides melissa samuelis) (listed by the Committee on the Status of Endangered Wildlife as extirpated in Canada) or the monarch butterfly (Danaus plexippus).

Monsanto Canada Inc. also provided information on an abundance of pest and beneficial insects in corn event MON 87460 field plots. Pest insects included aphids, corn flea beetles, delphacid planthoppers, grasshoppers, leafhoppers, northern corn rootworms, sap beetles, southern corn rootworms, and western corn rootworms. Beneficial insects included brown lacewings, green lacewings, ladybird beetles, macro-parasitic hymenopterans, micro-parasitic hymenopterans, nabids, Orius (minute pirate bugs), spiders, and big-eyed bugs (Geocoris). Quantitative assessments at seven sites over two years identified eight differences out of 326 comparisons. Of the eight differences, there were no consistent effects or trends except for a few transient decreases in micro-parasitic hymenoptera which each recovered by the final observation point. This demonstrates that corn event MON 87460 does not have any impact on the abundance of tested organisms.

Composition analyses showed that the levels of key nutrients and anti-nutrients in corn event MON 87460 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 87460 will not result in altered impacts on non-target organisms, including humans.

5. Potential Impact of Corn Event MON 87460 on Biodiversity

Corn event MON 87460 has no novel phenotypic characteristics that would extend its range beyond the current geographic range of corn production in Canada. Cold temperatures are a much larger factor than water availability in determining the geographic range in which corn can survive to reproductive maturity. Monsanto Canada Inc. has provided information to demonstrate that corn event MON 87460 is no more tolerant to cold temperature stress than commercially available corn hybrids.

Monsanto Canada Inc. has also provided information to demonstrate that the drought tolerance trait in corn event MON 87460 will not spread into or persist in unmanaged environments. Corn has no wild relatives with which it can outcross in Canada, so there will be no transfer of the novel trait to other species in unmanaged environments. Corn event MON 87460 does not pose an increased risk to interacting non-target organisms.

The CFIA has therefore concluded that the potential impact on biodiversity of corn event MON 87460 is equivalent to that of currently commercialized corn varieties.

V. Criteria for the Livestock Feed Assessment

1. Potential Impact of corn event MON 87460 on Livestock Nutrition

Nutritional Composition

The compositional equivalence of corn event MON 87460 to its isogenic unmodified counterpart and commercial corn varieties was assessed from six field sites in the United States under typical corn production conditions in 2006 and three sites in Chile under well-watered and water-limited growing conditions in 2006/2007. In both the US and Chile trials, forage and grain samples were collected from replicated plots at each site, and analyzed for protein, moisture, fat, ash, acid detergent fibre (ADF), neutral detergent fibre (NDF), total dietary fibre (TDF), amino acids, fatty acids, vitamins and minerals. No statistically significant differences were observed between forage from corn event MON 87460 and its unmodified counterpart for moisture, protein, fat, ash, ADF, NDF, calcium and phosphorus in the United States and Chile trial (well-watered). Except for fat, which was significantly higher in corn event MON 87460 than in its unmodified counterpart, all other forage nutrients in corn event MON 87460 and its unmodified counterpart were similar under the water-limited conditions. Statistically significant differences were observed between grain from corn event MON 87460 and the unmodified counterpart for ash (trial in the United States), fat and magnesium (well-watered); however the means for both corn event MON 87460 and its unmodified counterpart were within the normal variation of commercial corn and the range of literature values. No statistically significant differences were observed between grain from corn event MON 87460 and its unmodified counterpart for all other proximate, fibres, mineral and vitamins analyzed under all three production conditions. Statistically significant differences were observed between grain from corn event MON 87460 and its unmodified counterpart for stearic (trial in the United States) and eicosenoic acids (well-watered and water-limited conditions). The means of stearic and eicosenoic acids in both corn event MON 87460 and its unmodified counterpart were within the normal variation of commercial corn varieties and the range of literature values. No statistically significant differences were observed between grain from corn event MON 87460 and its unmodified counterpart for amino acids measured under all three production conditions.

Anti-Nutrients and Secondary Metabolites

Phytic acid, raffinose, ferulic acid and p-coumaric acid were analyzed in corn event MON 87460 grain and compared to its unmodified counterpart under the three production conditions described above. Phytic acid, raffinose, ferulic acid and p-coumaric acid in corn event MON 87460 were not statistically significantly different from the unmodified counterpart under all production conditions. All means were within the commercial ranges and literature values. Since corn event MON 87460 is expected to be grown in regions subject to frequent drought stress, additional secondary metabolites associated with stress responses and osmoprotectants were measured (salicylic acid, abscisic acid, sucrose, glucose, fructose, glycerol, free proline, glycine, betaine and choline) in forage and grain samples collected in the 2006/2007 Chile trial. No statistically significant differences were observed between forage from corn event MON 87460 and its unmodified counterpart for all metabolites under water-limited conditions. Under well-watered conditions, abscisic acid was statistically significantly higher in corn event MON 87460 compared to its unmodified counterpart, and was outside the range for commercial corn. The applicant provided evidence to show that the difference between corn event MON 87460 and its unmodified counterpart, which was small, was due to high abscisic acid levels which occurred at only one site and was not reproducible across sites. Abscisic acid is a non-toxic, natural plant hormone and further evidence showed that higher levels are not expected to impact the safety of livestock that would consume corn event MON 87460 forage. Apart from sucrose in corn grain under the water-limited conditions, no statistically significant differences were observed between grain from corn event MON 87460 and its unmodified counterpart for all other additional metabolites under either well-watered or water-limited conditions. All means were within the normal variation for conventional corn.

Broiler performance on corn event MON 87460 diets

A 42 day broiler study was performed to evaluate the effects of transgenic corn event MON 87460 diets on the growth performance of 1100 birds on diets formulated using corn from corn event MON 87460, unmodified control (DM1718) and six other commercial corn varieties. Diets were formulated to be isocaloric and contain the same amount of corn: 59% for starters (0-21 days) and 63% for grower/finishers (22-42 days). Broiler mortality ranged from 1-5% and was not related to any dietary treatment. No statistically significant differences were observed between birds fed on corn event MON 87460 diets and other controls for body weight gain, feed intake and efficiency throughout the study. A statistically significant difference was observed between birds on corn event MON 87460 corn diets and unmodified control diets for adjusted feed:gain ratio. However, further comparison of corn event MON 87460 fed birds to birds fed the unmodified control and commercial corn varieties (as a group), showed no significant differences for growth performance. No statistically significant differences were observed among diets for percent moisture, protein and fat in thigh and breast meat of birds. Carcass measurements of chill weight, fat pad, breast, wing, drum and thigh weights, breast and thigh moisture, protein and fat were similar for birds fed corn event MON 87460, unmodified control and commercial corn. No adverse health effects were observed among birds over the course of the trial.

The evidence provided by Monsanto supports the conclusion that the nutritional composition and efficacy of corn event MON 87460 are substantially equivalent to that of commercial corn varieties.

2. Potential Impact of corn event MON 87460 on Livestock and Workers/Bystanders

Cold shock protein genes are found in a wide variety of bacteria. The CSPB protein of B. subtilis is not a known toxin or allergen and does not have sequence similarity with any known allergens or toxins. The CSPB protein was not glycosylated, was shown to be heat labile and was rapidly degraded under conditions similar to those encountered in the gastrointestinal tract. This information suggests that the CSPB protein is unlikely to be a toxin or allergen.

The NPTII protein is also commonly found in bacteria and is present in previously authorized crops in Canada. The NPTII protein is not a known toxin or allergen and does not have sequence similarity with any known allergens or toxins. The NPTII protein was shown to be rapidly degraded under conditions similar to those encountered in the gastrointestinal tract. This information suggests that the NPTII protein is unlikely to be a toxin or allergen.

No adverse effects from the CSPB and NPTII proteins were observed in acute oral toxicity studies in male and female mice using approximately 330 and 45,000 times the highest predicted livestock dose level per kg body weight. No adverse effects on nutrition or health were observed in a broiler feeding trial comparing corn event MON 87460 grain to several conventional varieties of corn grain in the diet.

The evidence provided by Monsanto Canada Inc. indicates there is no potential impact of corn event MON 87460 on livestock and workers/bystanders when compared to 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 87460 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 corn event MON 87460 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 87460.

VII. Regulatory Decision

Based on the review of the data and information submitted by Monsanto Canada Inc., and through comparisons of corn event MON 87460 with its unmodified counterpart, the Plant and Biotechnology Risk Assessment Unit of the Plant Health Science Directorate, CFIA, has concluded that the novel drought tolerance trait does not confer to corn event MON 87460 any characteristic that would result in unintended environmental effects following unconfined release.

Based on the review of submitted data and information by Monsanto Canada Inc., including comparisons of corn event MON 87460 with its unmodified corn counterpart, the Animal Feed Division of the Animal Health Directorate, CFIA, has concluded that the novel drought tolerance trait will not confer to corn event MON 87460 any characteristic that would raise any concerns regarding the safety or nutritional composition of corn event MON 87460. 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 87460 has been assessed and found to be as safe as and as nutritious as traditional corn varieties. Corn event MON 87460 and its products 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 corn event MON 87460 is therefore authorized by the Plant Biosafety Office of the Plant Health and Biosecurity Directorate and the Animal Feed Division of the Animal Health Directorate as of December 17, 2010. Any Zea mays lines derived from corn event MON 87460 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, and the novel genes are expressed at levels similar to those in the authorized line.

Corn event MON 87460 is subject to the same phytosanitary import requirements as its unmodified counterpart.

Please refer to Health Canada's Decisions on Novel Foods for a description of the food safety assessment of corn event MON 87460. The food safety decisions are available at the following Health Canada web site.

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