Decision Document DD2014-107 Determination of the Safety of Monsanto Canada Inc.'s Soybean (Glycine max (L.) Merr.) Event MON 87751

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This Decision Document has been prepared to explain the regulatory decisions reached under Directive 94-08 (Dir94-08) Assessment Criteria for Determining Environmental Safety of Plants with Novel Traits, its companion document BIO1996-10 - The Biology of Glycine max (L.) Merr. (Soybean) and Section 2.6 - Guidelines for the Assessment of Novel Feeds: Plant Sources, of Chapter 2 of the RG-1 Regulatory Guidance: Feed Registration Procedures and Labelling Standards.

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 concerns the insect resistant soybean event MON 87751. 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 or nutrition concerns when compared to soybean varieties currently grown and permitted to be used as livestock feed in Canada.

Taking into account the conclusion of the environmental risk assessment and recognizing that incidental release into the environment could occur when this PNT is used as intended, unconfined release into the environment of soybean event MON 87751 and of any lines derived from it is therefore authorized by the Plant Biosafety Office of the Plant Health and Biosecurity Directorate as of October 31, 2014 on a condition that no sale of seed takes place in Canada. This condition is being applied to ensure that the PNT is used as intended.

Taking into account livestock feed assessment, use as livestock feed of soybean event MON 87751 and of any lines derived from it is authorized by the Animal Feed Division of the Animal Health Directorate as of October 31, 2014.

Authorizations of the soybean lines derived from soybean event MON 87751 are conditional on the following:

  1. no inter-specific crosses are performed,
  2. the intended uses are similar,
  3. it is known based on characterization that these plants do not display any additional novel traits and are substantially equivalent to soybean varieties that are currently grown and permitted to be used as livestock feed in Canada, in terms of their potential environmental impact and livestock feed safety; and
  4. the novel genes are expressed at levels similar to those of the authorized line.

Soybean event MON 87751 is subject to the same phytosanitary import requirements as unmodified soybean varieties. Soybean event MON 87751 is required to meet the requirements of other jurisdictions, including but not limited to, the Food and Drugs Act and the Pest Control Products Act.

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 assessment of food safety by Health Canada, have been addressed separately from this review.

(publié aussi en français)
October 31, 2014

This bulletin is published by the Canadian Food Inspection Agency. For further information, please contact the Plant Biosafety Office or the Animal Feed Division at:

1-800-442-2342
59 Camelot Drive, Ottawa
Ontario K1A 0Y9

Table of Contents

I. Brief Identification of the Modified Plant

Designation of the Modified Plant:
Soybean Event MON 87751, OECD Unique Identifier MON-87751-7
Applicant:
Monsanto Canada Inc.
Plant Species:
Soybean (Glycine max (L.) Merr.)
Novel Traits:
Resistance to lepidopteran pests of soybean
Trait Introduction Method:
Agrobacterium-mediated transformation
Intended Use of the Modified Plant:
Human consumption and livestock feed use. Soybean event MON 87751 is not intended for cultivation in Canada.

II. Background Information

Monsanto Canada Inc. developed, through the use of recombinant DNA techniques, a soybean event resistant to lepidopteran insect pests of soybean. The soybean event, designated as MON 87751, was developed to provide a method to control yield losses from insect feeding damage caused by key lepidopteran insect pests including the corn earworm (Helicoverpa zea) and the velvetbean caterpillar (Anticarsia gemmatalis) as well as secondary pests from the genus Spodoptera (e.g. fall armyworm, S. frugiperda).

Soybean event MON 87751 was developed using recombinant DNA technology, resulting in the introduction of the synthetic cry1A.105 gene and the cry2Ab2 gene, similar to previously authorized corn event MON 89034. As described in DD2008-74: Determination of the Safety of Monsanto Canada Inc.'s Corn (Zea mays L.) Event MON 89034, the cry1A.105 gene is derived from the cry1Ab, cry1Ac and cry1F genes from Bacillus thuringiensis and encodes a synthetic δ-endotoxin Cry1A.105 protein. The cry2Ab2 gene is also derived from B. thuringiensis and encodes a variant of the wild-type δ-endotoxin Cry2Ab2 protein. The combination of the Cry1A.105 and Cry2Ab2 insecticidal proteins in a single plant provides control against a range of lepidopteran insect pests. In addition, selectable marker genes aadA and splA were used in the initial transformation and early event selection process. The aadA gene encodes the aminoglycoside-modifying enzyme conferring resistance to the antibiotics spectinomycin and streptomycin. The splA gene encodes the sucrose phosphylase protein and, when expressed during embryo development, interferes with sucrose metabolism leading to a shrunken appearance. These traits are of no agronomic interest but were used to select transformed plants during development of soybean event MON 87751. Traditional breeding was used to isolate plants that contain the cry1A.105 and cry2Ab2 genes but do not contain the aadA and splA genes, therefore producing marker-free soybean event MON 87751.

Monsanto Canada Inc. has provided data on the identity of soybean event MON 87751, a detailed description of the transformation method, data and information on the gene 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. Information 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.

Soybean event MON 87751 was field tested at 17 sites in the United States (US) in 2012, of which 10 were considered to be representative of major Canadian soybean growing regions. Agronomic characteristics of soybean event MON 87751, such as early stand count, plant vigour, days to 50% flowering, flower colour, plant lodging, pod shattering, plant height, final stand count, grain moisture, 100 seed weight, yield, growth stage and susceptibility to various soybean biotic and abiotic stressors, were compared to those of an unmodified control soybean variety, which shares the same genetic background as soybean event MON 87751 but has not been modified. Several reference soybean varieties were also included in these trials to establish a reference range for typical soybean behaviour.

Nutritional components of soybean event MON 87751 grain and forage, such as protein, fat, moisture, ash, amino acids, fatty acids, vitamins, minerals and anti-nutrients, were compared to those of the unmodified control soybean variety and to the reference range established by the reference soybean varieties.

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) - Assessment Criteria for Determining Environmental Safety of Plants with Novel Traits.

The PBRA Unit has considered:

  • the potential for soybean event MON 87751 to become a weed of agriculture or to be invasive of natural habitats;
  • the potential for gene flow from soybean event MON 87751 to sexually compatible plants whose hybrid offspring may become more weedy or more invasive;
  • the potential for soybean event MON 87751 to become a plant pest;
  • the potential impact of soybean event MON 87751 and its gene products on non-target organisms, including humans; and
  • the potential impact of soybean event MON 87751 on biodiversity.

The Animal Feed Division (AFD) of the 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 Section 2.6 - Guidelines for the Assessment of Novel Feeds: Plant Sources, of Chapter 2 of the RG-1 Regulatory Guidance: Feed Registration Procedures and Labelling Standards.

The AFD has considered both intended and unintended effects and similarities and differences between soybean event MON 87751 and unmodified soybean varieties relative to the safety and efficacy of feed ingredients derived from soybean event MON 87751 for their intended purpose, including:

  • the potential impact of soybean event MON 87751 on livestock nutrition; and
  • the potential impact of soybean event MON 87751 on animal health and human safety, as it relates to the potential transfer of residues into foods of animal origin and worker/bystander exposure to the feed.

The AFD has also considered whether feeds derived from soybean event MON 87751 meet the definitions and requirements of feeds as listed in Schedule IV of the Feeds Regulations.

Monsanto Canada Inc. has provided the CFIA with a method for the detection and identification of soybean event MON 87751.

III. Description of the Novel Traits

1. Development Method

Soybean event MON 87751 was developed through Agrobacterium-mediated transformation of meristem tissue excised from embryos of germinated conventional seed, using a transformation plasmid vector that includes two separate T-DNAs. The first T-DNA, designated as T-DNA I, contains the cry1A.105 and the cry2Ab2 expression cassettes. The second T-DNA, designated as T-DNA II, contains the aadA and splA expression cassettes. Following cultivation with Agrobacterium, the embryos were transferred to a selection medium containing spectinomycin to eliminate cells that were not transformed and thereby select cells containing T-DNA-II or T-DNA I + T-DNA II. Agrobacterium was removed by placing the tissue on a selection medium containing carbenicillin disodium salt, cefotaxime sodium salt and ticarcillin disodium salt/potassium clavulanate mixture. During subsequent breeding the unlinked insertions of T-DNA I and T-DNA II were segregated. The plants containing only the cry1A.105 and the cry2Ab2 expression cassettes were selected, while the plants containing the aadA and splA expression cassettes were eliminated from subsequent breeding. Soybean event MON 87751 was identified as a successful transformant and was chosen for further development.

2. Resistance to Lepidopteran Pests of Soybean

B. thuringiensis is a common gram-positive soil-borne bacterium. In the spore forming stage, it produces several insecticidal protein crystals, including the δ-endotoxin Cry1Ab, Cry1Ac, Cry2Ab and Cry1F proteins which are active against certain lepidopteran insects. These proteins are insecticidal to susceptible lepidopteran insects after cleavage by proteases in the insect's gut, forming a protease-resistant active fragment (core toxin) that is the bio-active form of the protein. Insecticidal activity is believed to depend on the binding of the active fragment to specific receptors present in susceptible insects on midgut epithelial cells, forming pores which disrupt osmotic balance and eventually result in cell lysis and insect death. Soybean event MON 87751 produces two Bt proteins, namely Cry1A.105 and Cry2Ab2.

The Cry1A.105 protein is a chimeric protein that consists of different domains from Cry1Ab, Cry1Ac and Cry1F. It was designed by Monsanto Canada Inc. using a domain exchange strategy to achieve a high level of activity against target lepidopteran pests. With the exception of four additional amino acids at its N-terminus from a chloroplast transit peptide, the Cry1A.105 protein expressed in soybean event MON 87751 shares 100% amino acid identity with the Cry1A.105 protein present in corn event MON 89034. No effects on the functionality or safety of the Cry1A.105 protein were observed as a result of this addition.

The Cry2Ab2 protein in soybean event MON 87751, with the exception of 18 fewer amino acids at its N-terminus, shares 100% amino acid identity with the Cry2Ab2 protein present in corn event MON 89034 and cotton event Bollgard II™. No effects on the functionality or safety of the Cry2Ab2 proteins were observed as a result of this deletion.

Monsanto Canada Inc. has submitted information to demonstrate that the protease-resistant core domains of the Cry1A.105 and Cry2Ab2 proteins expressed in soybean event MON 87751, which are responsible for insecticidal activity and specificity, share 100% amino acid identity to the proteins expressed in corn event MON 89034 (for both Cry1A.105 and Cry2Ab2) and cotton event Bollgard II™ (for Cry2Ab2 only). Corn event MON 89034 was previously authorized for unconfined environmental release and livestock feed use in Canada (DD2008-74: Determination of the Safety of Monsanto Canada Inc.'s Corn (Zea mays L.) Event MON 89034). Cotton event Bollgard II™ was previously authorized for livestock feed use in Canada (DD2003-45: Determination of the Safety of Monsanto's Insect Resistant Bollgard II™ Cotton (Gossypium hirsutum L)).

The cry1A.105 and cry2Ab2 genes are expressed in soybean event MON 87751 using promoters which confer constitutive expression of proteins. Tissue samples were collected at various growth stages from a soybean event MON 87751 hybrid grown at five representative US field trial sites. Levels of Cry1A.105 and Cry2Ab2 proteins were evaluated by enzyme-linked immunosorbent assays (ELISA).

The average Cry1A.105 protein expression levels in micrograms of protein per gram of dry weight tissue (μg/g dwt) from plants, as evaluated by ELISA, were as follows: 580 μg/g dwt in over season leaf (OSL) at the V3-4 stage, 590 μg/g dwt in leaf at the V5-7 stage, 400 μg/g dwt in OSL at the R2-3 stage, 790 μg/g dwt in OSL at the R6 stage, below the limit of detection in root at the R6 stage, 230 μg/g dwt in forage at the R6 stage and 2.4 μg/g dwt in seed at the R8 stage. For pollen/anther the Cry1A.105 protein expression levels were reported in micrograms of protein per gram of fresh weight tissue (μg/g fwt) at 11 μg/g fwt.

The average Cry2Ab2 protein expression levels, as evaluated by enzyme-linked immunosorbent assay (ELISA), were as follows: 24 μg/g dwt in over season leaf (OSL) at the V3-4 stage, 26 μg/g dwt in leaf at the V5-7stage, 32 μg/g dwt in OSL at the R2-3 stage, 24 μg/g dwt in OSL at the R6 stage, 15 μg/g dwt in root at the R6 stage, 14 μg/g dwt in forage at the R6 stage and 4 μg/g dwt in seed at the R8 stage. For pollen/anther the Cry2Ab2 protein expression levels were reported in micrograms of protein per gram of fresh weight tissue at 7.7 μg/g fwt.

The Cry1A.105 and Cry2Ab2 proteins were purified from the seed of soybean event MON 87751 and characterized. The identity of the purified proteins was confirmed by western immunoblot analysis, tryptic peptide mass mapping, N-terminal characterization and insecticidal activity.

The levels of Cry1A.105 and Cry2Ab2 proteins in soybean event MON 87751 tissues were too low to extract sufficient amounts for assessment of environmental and livestock feed safety. To obtain sufficient quantities of the proteins for safety studies, it was necessary to express the cry1A.105 and cry2Ab2 genes in an E. coli production system. The equivalency of the plant-produced proteins to the E. coli - produced proteins was evaluated by comparing their molecular weight, immunological reactivity, tryptic peptide mass map, insecticidal activity and glycosylation status. Based on the results, Cry1A.105 and Cry2Ab2 proteins produced in soybean event MON 87751 were found to be equivalent to their respective E. coli-produced counterparts.

The potential toxicity and allergenicity of the Cry1A.105 and Cry2Ab2 proteins to livestock and non-target organisms were evaluated. Both proteins lack sequence similarity to known allergens and protein toxins which have adverse effects to livestock and non-target organisms. No adverse effects were observed when the Cry1A.105 protein was ingested by mice at a dose of 2072 mg/kg body weight, or the Cry2Ab2 protein at a dose of 2198 mg/kg body weight. In vitro digestive fate studies have shown that both Cry1A.105 and Cry2Ab2 proteins are rapidly degraded in simulated gastric fluid, unlike protein allergens which are normally resistant to digestion. The Cry1A.105 and Cry2Ab2 proteins expressed in soybean event MON 87751 are not glycosylated, unlike many known allergens, providing additional information to support that Cry1A.105 and Cry2Ab2 proteins do not have the properties of known allergens.

3. Stable Integration into the Plant Genome

Molecular characterization by DNA sequence analysis and Southern blot analysis demonstrated that soybean event MON 87751 contains one copy of the cry1A.105 and cry2Ab2 gene cassettes inserted at a single site in the soybean genome. No additional elements, including intact or partial DNA fragments of the cry1A.105 or cry2Ab2 cassette, T-DNA II sequences or backbone sequences from the plasmid vector, linked or unlinked to the intact insert, were detected in soybean event MON 87751. Sequencing of the introduced DNA and the flanking genomic DNA confirmed the organization of the genetic elements and revealed that there was a 1 base pair (bp) insertion and a 7 bp deletion at the insertion site, and also a 16 bp deletion in the flanking genomic DNA, 5' to the insertion site. Such phenomena are commonly observed with Agrobacterium transformation. The deletions and insertion had no observable effect on the functionality of the DNA insert or the plant, as evidenced by the phenotypic and agronomic equivalence of soybean event MON 87751 to currently grown soybean varieties (further detail provided below in Section IV, part 1: Potential of Soybean Event MON 87751 to Become a Weed of Agriculture or be Invasive of Natural Habitats).

The stability of the inserted DNA was demonstrated by DNA sequence analysis and western blot analysis across five generations in the breeding history of soybean event MON 87751. Analysis of the inheritance pattern of the cry1A.105 and cry2Ab2 genes and the Cry1A.105 and Cry2Ab2 proteins across five generations of soybean event MON 87751 confirmed the stability of the inserted DNA and the stability of Cry1A.105 and Cry2Ab2 protein expression. The results of the analysis are consistent with the finding of a single site of insertion that segregates according to the Mendelian law of genetics.

IV. Criteria for the Environmental Assessment

1. Potential for Soybean Event MON 87751 to Become a Weed of Agriculture or be Invasive of Natural Habitats

The biology of soybean, as described in the CFIA biology document BIO1996-10 - The Biology of Glycine max (L.) Merr. (Soybean), is such that unmodified plants of this species are not invasive of unmanaged habitats in Canada. Soybean does not possess an intrinsic potential to become weedy in Canada due to traits such as the lack of seed dormancy and the poor competitive ability of seedlings. According to the information provided by Monsanto Canada Inc., soybean event MON 87751 was determined not to be significantly different from unmodified soybean varieties in this respect.

The CFIA evaluated data submitted by Monsanto Canada Inc. on the reproductive biology and life history traits of soybean event MON 87751. As previously mentioned, this event was field-tested in the US at 17 locations in the 2012 growing season, of which 10 were considered to be representative of major Canadian soybean growing regions. During the field trials, soybean event MON 87751 was compared to the unmodified control soybean variety. Reference soybean varieties were also included in these trials to establish ranges of comparative values that are representative of currently grown soybean varieties. Phenotypic and agronomic traits were evaluated, covering a broad range of characteristics that encompass the entire life cycle of the soybean plant. The traits included early stand count, plant vigour, days to 50% flowering, flower colour, plant lodging, pod shattering, plant height, final stand count, grain moisture, 100 seed weight, yield and growth stage. Although instances of statistically significant differences were observed between soybean event MON 87751 and the unmodified control soybean variety for some traits in the individual-site analyses, there was no consistent trend in the data across locations that would indicate the differences were due to the genetic modification, and the values for soybean event MON 87751 were within the reference range established for the reference soybean varieties included in the same field trials. Therefore, the statistical analysis of these observations showed no biologically meaningful differences between soybean event MON 87751 and the unmodified control soybean variety, and supports a conclusion of phenotypic and agronomic equivalence to currently grown soybean varieties.

Monsanto Canada Inc. evaluated the germination of soybean event MON 87751 seed under six temperature regimes. The following seed germination characteristics were evaluated: percent germinated seed (normal and abnormal), percent hard seed (viable and non-viable), percent dead seed and percent swollen seed (viable and non-viable). Soybean event MON 87751 was compared to the unmodified control soybean variety. Twelve reference soybean varieties were also included to provide a range of comparative values for each germination characteristic. Seed lots were produced at three sites in the US. Although instances of statistically significant differences were observed between soybean event MON 87751 and the unmodified control soybean variety in the individual-site analyses for percent viable hard seed, percent dead seed and percent viable firm swollen seed for three temperature regimes, these differences were not consistently detected across locations, and the values were within the range of reference established for conventional soybean varieties. Statistically significant differences were observed between soybean event MON 87751 and the unmodified control soybean variety for one temperature regime for percent normal germinated seed and percent abnormal germinated seed in the individual-site and combined-site analyses. However, the values were within the range established for reference soybean varieties, and were therefore not considered to be biologically meaningful.

Soybean event MON 87751 was exposed to drought, frost, hail injury, heat, mineral toxicity, nutrient deficiency, soil compaction, sun scald, wet soil and wind. No trend in increased or decreased susceptibility to these abiotic stressors was observed in soybean event MON 87751 compared to the unmodified control soybean variety.

The susceptibility of soybean event MON 87751 to soybean pests and pathogens was evaluated in the field at the same locations as the agronomic characteristic studies (further detail provided below in Section IV, part 3: Potential for Soybean Event MON 87751 to Become a Plant Pest). No trend in increased or decreased susceptibility to pests or pathogens was observed in soybean event MON 87751 compared to the unmodified control soybean variety.

Soybean event MON 87751 is not intended to be cultivated in Canada, and seed is not authorized for sale to ensure this intended use. Therefore, environmental exposure to soybean event MON 87751 plants will be minimal.

Based on the above information, the CFIA has concluded that soybean event MON 87751 is unlikely to become a weed of agriculture or invasive of natural habitats as a result of incidental environmental release.

2. Potential for Gene Flow from Soybean Event MON 87751 to Sexually Compatible Plants Whose Hybrid Offspring May Become More Weedy or More Invasive

Natural hybridization between cultivated soybean and the wild annual species Glycine soja can occur. However, G. soja is not naturalized in North America, and although this species is occasionally grown in research plots, there are no reports of its escape to unmanaged habitats nor of it becoming a weed in Canadian agro-ecosystems. The biology of soybean, as described in the CFIA biology document BIO1996-10 - The Biology of Glycine max (L.) Merr. (Soybean), shows that soybeans exhibit a high degree of self-fertilization. Cross pollination is usually less than one percent, suggesting that any pollen flow from cultivated soybeans to related species is minimal.

Monsanto Canada Inc. provided information on the viability, diameter and morphology of pollen of soybean event MON 87751. No statistically significant differences were detected between soybean event MON 87751 and the unmodified control soybean variety for percent viable pollen or pollen diameter. Furthermore, no visual differences in general morphology were observed between soybean event MON 87751 and the unmodified control soybean variety. Therefore the introduction of the novel trait did not impact the viability and morphology of soybean pollen.

Soybean event MON 87751 is not intended to be cultivated in Canada, and seed is not authorized for sale to ensure this intended use. Therefore, environmental exposure to soybean event MON 87751 plants will be minimal.

Based on the above information, the CFIA has concluded that the potential for gene transfer from soybean event MON 87751 to soybean relatives in managed ecosystems is negligible, and that there is no potential for gene transfer to sexually compatible species in Canada as a result of incidental environmental release.

3. Potential for Soybean Event MON 87751 to Become a Plant Pest

Soybean is not considered a plant pest in Canada, and the lepidopteran resistance traits introduced into soybean event MON 87751 are unrelated to plant pest potential (e.g. the potential for the plant to harbour new or increased populations of pathogens or pests).

The susceptibility of soybean event MON 87751 to various soybean arthropods and pathogens was evaluated in the field at the same locations as the agronomic characteristic studies. The stressors observed included aphids (Aphididae), bean leaf beetles (Cerotoma trifurcate), blister beetles (Meloidae), corn rootworm beetles (Diabrotica sp.), grape colaspis (Colaspis brunnea), grasshoppers (Acrididae), Japanese beetles (Popillia japonica), kudzu bugs (Megacopta cribraria), Mexican bean beetles (Epilachna varivestis), soybean stem borers (Dectes texanus), spider mites (Tetranychus sp.), stink bugs (Pentatomidae), striped flea beetles (Phyllotreta sp.), three-cornered alfalfa hoppers (Spissistilus festinus), thrips (Thysanoptera), whiteflies (Aleyrodidae), Alternaria leaf spot, Asian rust, bacterial blight, bacterial leaf spot, bean pod mottle, soybean brown spot, brown stem rot, Cercospora leaf disease, charcoal rot, damping-off, downy mildew, frogeye leaf spot, leaf bacterial pustule, Phytophthora root rot, pod and stem blight, powdery mildew, Pythium sp., Rhizoctonia sp., root knot nematode, soybean rust, Septoria sp., soybean mosaic virus, soybean stem cankers, sudden death syndrome and white mould. No qualitative differences were observed between soybean event MON 87751 and the unmodified control soybean variety for any of the 154 observations for plant damage caused by arthropods and for any of the 191 observations for plant damage cause by diseases.

Monsanto Canada Inc. quantitatively assessed bean leaf beetle (C.  trifurcate) and stink bug (Pentatimidae) damage in the field at the same locations as the agronomic characteristic studies. There were no statistically significant differences detected between soybean event MON 87751 and the unmodified control soybean variety for plant damage caused by the bean leaf beetle or stink bug.

Monsanto Canada Inc. quantitatively assessed the arthropod abundance in the field at the same locations as the agronomic characteristic studies at five collection times. The pest and beneficial arthropods observed included aphids (Aphididae), bean leaf beetles, corn rootworm beetles (Diabrotica sp.), Japanese beetles (P. japonica), kudzu bugs (M. cribraria), minute brown scavenger beetles (Latridiidae), leaf beetles (Chrysomelidae), leafhoppers (Cicadellidae), tarnished plant bugs (Lygus spp.), plant bugs (Miridae), spider mites (Tetranychus sp.), stink bugs, thrips (Thysanoptera), treehoppers (Membracidae), whiteflies (Aleyrodidae), ant-like flower beetles (Anthicidae), spiders (Araneae), assassin bugs (Reduviidae), big-eyed bugs (Geocoridae), brown lacewings (Hemerobiidae), green lacewings (Chrysopidae), damsel bugs (Nabis spp.), ladybird beetles (Coccinellidae), micro-parasitic Hymenoptera, minute pirate bugs (Orius sp.) and predatory mites (Phytoseiidae). Although instances of statistically significant differences were detected between soybean event MON 87751 and the unmodified control soybean variety for some of the pest and beneficial arthropods in the individual-site analyses, there was no trend observed across collections or sites. Therefore, the differences were not considered biologically meaningful in terms of plant pest potential.

Since soybean event MON 87751 is not intended to be cultivated in Canada, an insect resistance management plan specific to this product is not required.

Soybean event MON 87751 is not intended to be cultivated in Canada, and seed is not authorized for sale to ensure this intended use. Therefore, environmental exposure to soybean event MON 87751 plants will be minimal.

Based on the above information, the CFIA has concluded that soybean event MON 87751 would not be expected to display altered plant pest potential as a result of incidental environmental release.

4. Potential Impact of Soybean Event MON 87751 and Its Gene Products on Non-Target Organisms, Including Humans

The Cry1A.105 and Cry2Ab2 proteins were introduced into soybean event MON 87751 as protection against feeding by lepidopteran soybean pests. The history of use and available literature indicate that the insecticidal Cry proteins derived from B. thuringiensis are only active against specific insect groups and are not toxic to other organisms, including humans. In addition, foliar insecticides based on Cry proteins have been registered for over 30 years in Canada and have a long history of safe use.

The Cry1A.105 and Cry2Ab2 proteins produced in soybean event MON 87751 have been demonstrated to be active only against certain lepidopteran insects. With the exception of four additional amino acids at its N-terminus from a chloroplast transit peptide, the Cry1A.105 protein expressed in soybean event MON 87751 shares 100% amino acid identity with the Cry1A.105 protein present in corn event MON 89034. Similarly, the Cry2Ab2 protein in soybean event MON 87751, with the exception of 18 fewer amino acids at its N-terminus, shares 100% amino acid identity with the Cry2Ab2 protein present in corn event MON 89034 and cotton event Bollgard II™. No effects on the functionality or safety of the proteins were observed as a result of these changes. The safety of the Cry1A.105 and Cry2Ab2 proteins for non-target organisms, including humans, has been previously established (DD2008-74: Determination of the Safety of Monsanto Canada Inc.'s Corn (Zea mays L.) Event MON 89034, DD2003-45: Determination of the Safety of Monsanto's Insect Resistant Bollgard II™ Cotton (Gossypium hirsutum L)). Furthermore, Monsanto Canada Inc. has submitted information to demonstrate that the protease-resistant core domains of the Cry1A.105 and Cry2Ab2 proteins expressed in soybean event MON 87751, which are responsible for insecticidal activity and specificity, share 100% amino acid identity to the proteins expressed in corn event MON 89034 (for both Cry1A.105 and Cry2Ab2) and cotton event Bollgard II™ (for Cry2Ab2 only).

Monsanto Canada Inc. has previously submitted an evaluation of the potential toxicity of the Cry1A.105 and Cry2Ab2 proteins to selected non-target invertebrates in support of the safety of corn event MON 89034. The detailed hazard assessment included dietary toxicity studies using honeybee larvae and adults (Apis mellifera), minute pirate bugs (Orius insidiosus), ladybird beetles (Coleomegilla maculata), parasitic wasps (Ichneumon promissorius), earthworms (Eisenia foetida) and Collembola (Folsomia candida). In all cases, the Cry1A.105 and Cry2Ab2 proteins were demonstrated to be safe at doses equal to or exceeding 14 times the estimated environmental concentration of Cry1A.105 and Cry2Ab2 proteins in the diet of non-target invertebrates feeding on plant tissues or exposed via their prey. In addition, no adverse effects were observed when the aquatic invertebrate Daphnia magna was exposed to pollen at a concentration of 100 mg/L, which indicates that no hazard to aquatic invertebrates is anticipated through exposure to pollen.

Data were also previously submitted on non-target vertebrates including mice, bobwhite quails and broiler chickens in support of the safety of corn event MON 89034. No significant adverse effects were detected when mice were exposed to Cry1A.105 protein or Cry2Ab2 protein at a single oral dose of approximately 2000 mg/kg body weight, which represents several thousand times the maximum daily dose of Cry1A.105 and Cry2Ab2 proteins to humans or livestock. No significant adverse effects were detected when bobwhite quail or broiler chicken were fed a diet containing 50% grain from corn event MON 89034 for 8 days and 42 days, respectively.

Furthermore, a 42-day feeding study with broiler chickens comparing the nutritional value of diets containing soybean meal produced from soybean event MON 87751 and the unmodified control soybean variety demonstrated that there were no biologically relevant differences (see Section V, part 1: Potential Impact of Soybean Event MON 87751 on Livestock Nutrition).

Field evaluations of soybean event MON 87751 did not show any increased resistance to non-target pests or pathogens compared to the unmodified control soybean variety (see Section IV, part 3: Potential for Soybean Event MON 87751 to Become a Plant Pest).

Composition analyses showed that the levels of key nutrients and anti-nutrients in soybean event MON 87751 grain and forage are comparable to those in commercial soybean varieties (see Section V, part 1: Potential Impact of Soybean Event MON 87751 on Livestock Nutrition). Therefore, it is very unlikely that the introduction of the novel traits may have caused unintended changes to the composition of soybean event MON 87751 tissues that would negatively impact organisms interacting with soybean event MON 87751.

Detailed characterization of the Cry1A.105 and Cry2Ab2 proteins expressed in soybean event MON 87751 led to the conclusion that neither of these proteins displays any characteristic of a potential toxin or allergen (see Section V, part 2: Potential Impact of Soybean Event MON 87751 on Animal Health and Human Safety as it Relates to the Potential Transfer of Residues into Foods of Animal Origin and Worker/Bystander Exposure to the Feed) to non-lepidopteran species. Therefore, no negative impacts resulting from exposure of organisms to the Cry1A.105 and Cry2Ab2 proteins expressed in soybean event MON 87751 are expected.

Soybeans interact with a symbiotic plant-associated nitrogen-fixing bacterium (Bradyrhizobium japonicum), which results in the fixation of atmospheric nitrogen to a plant-available from inside root nodules. Soybean event MON 87751 was compared to an unmodified control soybean variety with respect to nitrogen fixation and nodulation. In addition, reference ranges were established for six reference soybean varieties. Nodule number and dry weight, shoot dry weight, root dry weight, shoot total nitrogen in percentage and shoot total nitrogen in grams were observed in a greenhouse study. No biologically meaningful differences between soybean event MON 87751 and the unmodified control soybean variety were observed for any of these characteristics, indicating that nodulation and nitrogen fixation by B. japonicum are not affected by the lepidopteran resistance trait.

Soybean event MON 87751 is not intended to be cultivated in Canada, and seed is not authorized for sale to ensure this intended use. Therefore, environmental exposure to soybean event MON 87751 plants will be minimal.

Based on the above information, the CFIA has concluded that that soybean event MON 87751 will not result in altered impacts on non-target organisms, including humans, as a result of incidental environmental release.

5. Potential Impact of Soybean Event MON 87751 on Biodiversity

Soybean event MON 87751 expresses no novel phenotypic characteristics that would extend its range beyond the current geographic range of soybean production in Canada. The only sexually compatible wild relative of soybean in Canada, G. soja, does not occur in unmanaged habitats, and the possibility of soybean outcrossing to G. soja is very low. Soybean event MON 87751 is unlikely to cause adverse effects on non-target organisms and does not display increased weediness, invasiveness, or plant pest potential. It is therefore unlikely that soybean event MON 87751 will have any direct effects on biodiversity, in comparison to the effects that would be expected from the cultivation of soybean varieties that are currently grown in Canada.

As summarized above, soybean event MON 87751 does not pose a risk to non-target organisms. In addition, Monsanto Canada Inc. provided data showing that the trait conferring resistance to targeted lepidopteran pests does not alter the symbiotic relationship between the bacterium B. japonicum and soybean event MON 87751 compared to the unmodified control soybean variety.

Soybean event MON 87751 is not intended to be cultivated in Canada, and seed is not authorized for sale to ensure this intended use. Therefore, environmental exposure to soybean event MON 87751 plants will be minimal.

Based on the above information, the CFIA has concluded that the potential impact on biodiversity of soybean event MON 87751 would be minimal as a result of incidental environmental release.

V. Criteria for the Livestock Feed Assessment

The AFD considered nutrient and anti-nutrient profiles; the safety of feed ingredients derived from soybean event MON 87751, including the presence of gene products, residues and metabolites in terms of animal health and human safety as it relates to the potential transfer of residues into foods of animal origin and worker/bystander exposure to the feed; and whether feeds derived from soybean event MON 87751 meet the definitions and requirements of feeds as listed in Schedule IV of the Feeds Regulations.

1. Potential Impact of Soybean Event MON 87751 on Livestock Nutrition

Nutrient and Anti-Nutrient Composition

The nutritional equivalence of soybean event MON 87751 to the unmodified control soybean variety and 19 reference soybean varieties was determined from 8 replicated field trials in the US during the 2012 growing season. Forage and harvested soybeans were collected from soybean event MON 87751, the unmodified control soybean variety and reference soybean varieties from each plot and analysed for moisture, ash, protein, crude fat, carbohydrates (by calculation), acid detergent fibre (ADF) and neutral detergent fibre (NDF). Harvested soybeans were further analysed for amino acids, fatty acids, vitamins, minerals, isoflavones (diadzein, genistein and glycitein) and anti-nutrients (lectin, trypsin inhibitors, phytic acid, raffinose and stachyose) as recommended by the OECD consensus document for new varieties of soybeans (OECD, 2012). Composition data were analysed statistically using a mixed model analysis of variance, and statistical differences among the soybean types were identified and assessed (P<0.05). The composition data from the reference soybean varieties were combined across all field sites to calculate a 99% tolerance interval for each component to estimate the natural variability of each component in soybean. The biological relevance of any significant difference among soybean varieties was assessed by comparing the observed values to the range of the values observed in the unmodified control soybean variety and reference soybean varieties grown in the trials, and in the published scientific literature (ILSI, 2011).

No statistically significant differences were observed between the unmodified control soybean variety and soybean event MON 87751 forage for protein, ash, carbohydrates and ADF. Statistically significant effects were found between soybean event MON 87751 and the unmodified control soybean variety forage for fat and NDF. However, the means were within the range of the values observed in the reference soybean varieties grown within the trials and in the published literature (ILSI, 2011). No statistically significant differences were observed between the unmodified control soybean variety and soybean event MON 87751 for fat, ash, carbohydrates, NDF and ADF. A statistically significant difference was found between soybean MON 87751 event and the unmodified control soybean variety for protein. However, all means were within the range of the values observed in the reference soybean varieties grown in the trials and in the published literature (ILSI, 2011). No statistically significant difference was observed between the unmodified control soybean variety and soybean event MON 87751 for calcium. A statistically significant difference was found between soybean event MON 87751 and the unmodified control soybean variety for phosphorus. However, this difference was not compositionally meaningful from a feed safety perspective. Except for glycine and proline, no statistically significant differences were found between soybean event MON 87751 and the unmodified control soybean variety for amino acids. Mean levels of glycine and proline were within the range of the values observed in the reference soybean varieties grown in the trials and the published scientific literature (ILSI, 2011). No statistically significant differences were observed in the fatty acids between the unmodified control soybean variety and soybean event MON 87751. No statistically significant differences were observed in the unmodified control soybean variety and soybean event MON 87751 for vitamin K. A statistically significant difference was found between soybean event MON 87751 and the unmodified control soybean variety for vitamin E. However, the mean differences were negligible and not biologically meaningful as the means were within the range of the values observed in the reference soybean varieties grown in the trials and in the published scientific literature (ILSI, 2011). No statistically significant differences were observed between soybean event MON 87751 and the unmodified control soybean variety for isoflavones (diadzein, genistein and glycitein). There was a statistically significant difference between soybean event MON 87751 and the unmodified control soybean variety for raffinose. However, all means were within the range of the values observed in the reference soybean varieties grown in the trials and in the published scientific literature (ILSI, 2011), therefore the difference was not considered biologically meaningful.

Broiler Performance

Eight hundred male and female broilers were used in a trial to evaluate the effects of diets containing soybean meal from soybean event MON 87751 and the unmodified control soybean variety and soybean reference varieties in a 42-day trial. The treatments were comprised of soybean meal from soybean event MON 87751, soybean meal from the unmodified control soybean variety, and soybean meal from reference soybean varieties. There was no significant difference in mortality or growth performance between the birds fed soybean meal from soybean event MON 87751 and that from the unmodified control soybean meal and the soybean reference varieties. Carcass yield measurements were not different for broilers fed diets containing soybean meal produced from soybean event MON 87751 compared to those fed diets containing soybean meal produced from the unmodified control soybean variety, with the exception of breast meat weight, expressed as percent of chilled carcass weight, and thigh weight, expressed as percent of chilled carcass weight. However, the mean differences were negligible and not biologically meaningful. No unexpected effects on broiler performance and health were found when broilers were fed diets containing soybean meal from soybean event MON 87751.

Conclusion

It was concluded, based on the evidence provided by Monsanto Canada Inc., that the nutritional composition of soybean event MON 87751 is similar to that of the reference soybean varieties grown in the trials and to that reported for other soybeans in the published scientific literature. Feed ingredients derived from soybean event MON 87551 are considered to meet present ingredient definitions for soybeans in Schedule IV of the Feeds Regulations.

2. Potential Impact of Soybean Event MON 87751 on Animal Health and Human Safety as it Relates to the Potential Transfer of Residues into Foods of Animal Origin and Worker/Bystander Exposure to the Feed

Soybean event MON 87751 is resistant to lepidopterans due to production of the Cry1A.105 and Cry2Ab2 proteins. The assessment of soybean event MON 87751 evaluated the impact of the following potential hazards relative to the safety of feed ingredients derived from this event:

  • The presence of the novel proteins: Cry1A.105 and Cry2Ab2

Novel Cry1A.105 Protein

To obtain sufficient quantities of Cry1A.105 proteins for assessment of environmental and feed safety, it was necessary to express the cry1A.105 gene in a microbial production system. Equivalency was demonstrated between soybean event MON 87751-produced Cry1A.105 protein and E. coli-produced Cry1A.105 protein by comparing their molecular weights, immunoreactivity, glycosylation and functional activity. Based on the results, the proteins were found to be equivalent.

Demonstration of equivalence between the Cry1A.105 protein produced in E. coli and the Cry1A.105 protein produced in soybean event MON 87751 allows the Cry1A.105 protein produced in E. coli to be used in studies to confirm the safety of the Cry1A.105 protein produced in soybean event MON 87751.

The potential allergenicity and toxicity of the Cry1A.105 protein to livestock were evaluated. With respect to its potential allergenicity, no single experimental method yields decisive evidence, thus a weight-of-evidence approach was taken, taking into account information obtained with various test methods. The source of the cry1A.105 gene, B. thuringiensis, is not known to produce allergens and a bioinformatics evaluation of the Cry1A.105 amino acid sequence confirmed the lack of relevant similarities between the Cry1A.105 protein and known allergens. E. coli-produced Cry1A.105 protein safety studies indicated that, unlike many allergens, this protein is rapidly degraded in simulated gastric fluid, is not heat stable and is not glycosylated. The weight of evidence thus indicates that the Cry1A.105 protein is unlikely to be allergenic.

In terms of its potential toxicity to livestock, the Cry1A.105 protein lacks a mode of action to suggest that it is intrinsically toxic to livestock and a bioinformatics evaluation of the Cry1A.105 amino acid sequence confirmed the lack of relevant similarities between the Cry1A.105 protein and known toxins. In addition, no significant adverse effects were observed when E. coli-produced Cry1A.105 protein was ingested by mice at a single dose of approximately 2000 mg/kg body weight. A 42-day feeding study with broilers demonstrated that there were no biologically relevant differences in broiler performance, carcass yield, and mortality between broilers fed diets containing soybean event MON 87751 and those fed diets containing the unmodified control soybean varieties. The weight of evidence thus indicates that the Cry1A.105 protein is unlikely to be toxic to livestock.

The livestock exposure to the Cry1A.105 protein is expected to be negligible as the protein is expressed at very low levels in soybean event MON 87751, is rapidly degraded under conditions which simulate the mammalian digestive tract and is unstable under heating conditions expected to be encountered during processing of some soybean products.

Novel Cry2Ab2 Protein

To obtain sufficient quantities of Cry2Ab2 protein for assessment of environmental and feed safety, it was necessary to express the cry2Ab2 gene in a microbial production system. Equivalency was demonstrated between soybean event MON 87751-produced Cry2Ab2 protein and E. coli-produced Cry2Ab2 protein by comparing their molecular weights, immunoreactivity, glycosylation and functional activity. Based on the results, the proteins were found to be equivalent.

Demonstration of equivalence between the Cry2Ab2 protein produced in E. coli and the Cry2Ab2 protein produced in soybean event MON 87751 allows the Cry2Ab2 protein produced in E. coli to be used in studies to confirm the safety of the Cry2Ab2 protein produced in soybean event MON 87751.

The potential allergenicity and toxicity of the Cry2Ab2 protein to livestock were evaluated. With respect to its potential allergenicity, no single experimental method yields decisive evidence, thus a weight-of-evidence approach was taken, taking into account information obtained with various test methods. The source of the cry2Ab2 gene, B. thuringiensis, is not known to produce allergens and a bioinformatics evaluation of the Cry2Ab2 amino acid sequence confirmed the lack of relevant similarities between the Cry2Ab2 protein and known allergens. E. coli-produced Cry2Ab2 protein safety studies indicated that, unlike many allergens, this protein is rapidly degraded in simulated gastric fluid, is not heat stable and is not glycosylated. The weight of evidence thus indicates that the Cry2Ab2 protein is unlikely to be allergenic.

In terms of its potential toxicity to livestock, the Cry2Ab2 protein lacks a mode of action to suggest that it is intrinsically toxic to livestock and a bioinformatics evaluation of the Cry2Ab2 amino acid sequence confirmed the lack of relevant similarities between the Cry2Ab2 protein and known toxins. In addition, no significant adverse effects were observed when E. coli-produced Cry2Ab2 protein was ingested by mice at a total dose of approximately 2000 mg/kg body weight. A 42-day feeding study in broilers demonstrated that there were no biologically relevant differences in broiler performance, carcass yield, and mortality between broilers fed diets containing soybean event MON 87751 and those fed diets containing the unmodified control soybean varieties. The weight of evidence thus indicates that the Cry2Ab2 protein is unlikely to be toxic to livestock.

The livestock exposure to the Cry2Ab2 protein is expected to be negligible as the protein is expressed at very low levels in soybean event MON 87751, is rapidly degraded under conditions which simulate the mammalian digestive tract, and is unstable under heating conditions expected to be encountered during processing of some soybean products.

Conclusion

It was concluded, based on the evidence provided by Monsanto Canada Inc., that the novel Cry1A.105 and Cry2Ab2 protein-based insect resistance traits will not confer to soybean event MON 87751 any characteristic that would raise concerns regarding the safety of soybean event MON 87751. Feed ingredients derived from soybean event MON 87751 are considered to meet present ingredient definitions for soybean.

VI. New Information Requirements

If at any time, Monsanto Canada Inc. becomes aware of any new information regarding risk to the environment, livestock or human health, which could result from release or livestock feed use of soybean event MON 87751 or lines derived from it, Monsanto Canada Inc. is required to immediately provide such information to the CFIA. On the basis of such new information, the CFIA will re-evaluate the potential impact of soybean event MON 87751 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 soybean event MON 87751.

VII. Regulatory Decision

Based on the review of the data and information submitted by Monsanto Canada Inc. and input from other relevant scientific sources, the Plant and Biotechnology Risk Assessment Unit of the Plant Health Science Directorate, CFIA, has concluded that the unconfined environmental release of soybean event MON 87751 does not present altered environmental risk when compared to soybean varieties that are currently grown in Canada.

Based on the review of the data and information submitted by Monsanto Canada Inc. and input from other relevant scientific sources, the Animal Feed Division of the Animal Health Directorate, CFIA, has concluded that the novel genes and their corresponding traits will not confer to soybean event MON 87751 any characteristic that would raise concerns regarding the safety or nutritional composition of soybean event MON 87751. Livestock feeds derived from soybeans are currently listed in Schedule IV of the Feeds Regulations and are, therefore approved for use in livestock feeds in Canada. Soybean event MON 87751 has been found to be as safe as and as nutritious as currently and historically grown soybean varieties. Soybean event MON 87751 and its products are considered to meet present ingredient definitions and are approved for use as livestock feed ingredients in Canada.

Taking into account the conclusion of the environmental risk assessment and recognizing that incidental release into the environment could occur when this PNT is used as intended, unconfined release into the environment of soybean event MON 87751 and of any lines derived from it is therefore authorized by the Plant Biosafety Office of the Plant Health and Biosecurity Directorate as of October 31, 2014 on a condition that no sale of seed takes place in Canada. This condition is being applied to ensure that the PNT is used as intended.

Taking into account livestock feed assessment, use as livestock feed of soybean event MON 87751 and of any lines derived from it is authorized by the Animal Feed Division of the Animal Health Directorate as of October 31, 2014.

Authorizations of the soybean lines derived from soybean event MON 87751 are conditional on the following:

  1. no inter-specific crosses are performed,
  2. the intended uses are similar,
  3. it is known based on characterization that these plants do not display any additional novel traits and are substantially equivalent to soybean varieties that are currently grown and permitted to be used as livestock feed in Canada, in terms of their potential environmental impact and livestock feed safety; and
  4. the novel genes are expressed at levels similar to those of the authorized line.

Soybean event MON 87751 is subject to the same phytosanitary import requirements as unmodified soybean varieties. Soybean event MON 87751 is required to meet the requirements of other jurisdictions, including but not limited to, the Food and Drugs Act and the Pest Control Products Act.

Please refer to Health Canada's Decisions on Novel Foods for a description of the food safety assessment of soybean event MON 87751.

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