Decision document DD2023-143: Determination of the safety of Bayer Crop Science Inc.'s soybean (Glycine max (L.) Merr.) Event MON 94313

Authorized as of October 3, 2023

The Canadian Food Inspection Agency (CFIA) has evaluated information submitted by Bayer CropScience Inc. concerning the herbicide tolerant soybean event MON 94313. The CFIA has determined that soybean event MON 94313 does not present an altered environmental or livestock feed risk when compared to soybean varieties currently grown and permitted to be used in Canada. Soybean event MON 94313 is therefore authorized for these uses as of October 3, 2023, subject to the provisions outlined in this decision document.

This bulletin was created by the CFIA. For further information, please contact the Plant Biosafety Office or the Animal Feed Program by visiting the contact page.

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1. Brief identification of the modified plant

Designation: Soybean event MON 94313, OECD Unique Identifier MON-94313-8

Applicant: Bayer CropScience Inc.

Plant species: Soybean, Glycine max (L.) Merr.

Novel trait: Tolerance to glufosinate-ammonium, dicamba, 2,4-D, and mesotrione herbicides

Trait introduction method: Agrobacterium-mediated transformation

Intended end use: Human consumption and livestock feed uses

Intended area of cultivation: Within the typical production area for soybean in Canada

2. Background information

Bayer CropScience Inc. has developed a soybean event that is tolerant of glufosinate-ammonium, dicamba, 2,4-D, and mesotrione herbicides. Soybean event MON 94313 was developed using recombinant deoxyribonucleic acid (rDNA) technology, resulting in the introduction of a phosphinothricin N-acetyltransferase (pat) gene, a dicamba mono-oxygenase (dmo) gene, a ft_t.1 gene, a triketone dioxygenase (tdo) gene, and associated regulatory elements.

Bayer CropScience Inc. provided:

  • information on the identity of soybean event MON 94313
  • a detailed description of the introduced genetic elements and new proteins encoded by these genetic elements
  • information about how soybean event MON 94313 compares to other soybean varieties in terms of its environmental safety
  • information about how soybean event MON 94313 compares to other soybean varieties in terms of its nutrition and safety as an animal feed

The CFIA has reviewed the above information, in light of the Assessment criteria for determining environmental safety of plants with novel traits (Directive 94-08). The CFIA has considered:

  • the potential for soybean event MON 94313 to become a weed of agriculture or to be invasive of natural habitats
  • the potential for gene flow from soybean event MON 94313 to sexually compatible plants whose hybrid offspring may become more weedy or more invasive
  • the potential for soybean event MON 94313 to become a plant pest
  • the potential impact of soybean event MON 94313 and its gene products on non-target organisms, including humans
  • the potential impact of soybean event MON 94313 on biodiversity

The CFIA has also reviewed the above information with respect to the Guidelines for the Assessment of Novel Feeds: Plant Sources. The CFIA has considered both intended and unintended effects and similarities and differences between soybean event MON 94313 and unmodified soybean varieties relative to the safety and efficacy of feed ingredients derived from soybean event MON 94313 for their intended purpose, including:

  • the potential impact of soybean event MON 94313 on livestock nutrition
  • the potential impact of soybean event MON 94313 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 CFIA has also considered whether feeds derived from soybean event MON 94313 meet the definitions and requirements of feeds as listed in Schedule IV of the Feeds Regulations.

3. Description of the novel traits

3.1 Development method

Soybean event MON 94313 was developed through Agrobacterium-mediated transformation of embryos from soybean variety A3555 using a single plasmid that included 2 separate T-DNAs. The first T-DNA contained 4 gene cassettes for pat, dmo, ft_t.1, and tdo conferring tolerance to glufosinate-ammonium, dicamba, 2,4-D, and mesotrione herbicides, respectively. The second T-DNA contained a selectable marker gene and a gene to improve transformation efficiency. Following transformation, plant breeders used traditional breeding, segregation, selection, and genetic screening to isolate plants containing the T-DNA I sequence but not the T-DNA II.

Soybean event MON 94313 was identified as a candidate for commercial development based on herbicide tolerance and agronomic evaluations.

3.2 Glufosinate-ammonium herbicide tolerance

Glufosinate-ammonium is a Group 10 herbicide which inhibits the plant enzyme glutamine synthetase. Inhibiting glutamine synthetase results in reduced glutamine synthesis, and accumulation of lethal levels of ammonia in susceptible plants. Ammonia is produced by plants as a result of normal metabolic processes, but elevated levels of ammonia can interfere with essential plant processes, like photosynthesis, leading to plant death.

Soybean event MON 94313 was developed to be tolerant of the herbicide glufosinate-ammonium by incorporation of the pat gene. The pat gene encodes the protein phosphinothricin N-acetyltransferase (PAT), which acetylates the primary amino group of glufosinate-ammonium, making the herbicide inactive. Introduction of the pat gene into soybean event MON 94313 confers commercial-level tolerance to the herbicide glufosinate-ammonium. The pat gene was derived from Streptomyces viridochromogenes, a gram-positive soil bacterium, and optimized for expression in soybean. The amino acid sequence of PAT protein produced in soybean event MON 94313 is identical to the amino acid sequence of the native protein.

The pat gene in soybean event MON 94313 is linked to a constitutive promoter. Samples of soybean event MON 94313 tissues at various plant growth stages were collected from 5 field trial sites in the USA. The range of PAT protein expression levels in micrograms protein per gram dry weight tissue (µg/g dwt), as evaluated by validated multiplexed immunoassay were as follows: 5.3 to 33 µg/g dwt in leaf, 1.7 to 5.4 µg/g dwt in root, 6.7 to 18 µg/g dwt in forage, and 2.6 to 4.6 µg/g dwt in grain.

The identity and integrity of the PAT protein produced in soybean event MON 94313 was confirmed by molecular weight, immunoreactivity, tryptic peptide mass signatures, and N-terminal sequences. The PAT protein has been expressed in many events in different crop species that are grown in Canada and elsewhere. The PAT protein therefore has a history of safe use.

The lack of potential toxicity and allergenicity of PAT protein has previously been establishedFootnote 1. Updated bioinformatics evaluations of the PAT amino acid sequence were submitted in the current application and confirmed the lack of relevant similarities to known toxins and allergens.

For a more detailed discussion of the potential allergenicity and toxicity of the PAT protein, see Section 5.2: Potential impact of soybean event MON 94313 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.

3.3 Dicamba herbicide tolerance

Dicamba is a Group 4 herbicide that mimics indole-3-acetic acid (IAA), a natural plant hormone of the auxin class. Dicamba application causes rapid and uncontrolled growth of the stems, petioles and leaves of sensitive plants, leading to the destruction of vascular tissue and eventually plant death. Dicamba is used for broadleaf weed control on grain crops, pastures and non-crop areas.

Soybean event MON 94313 was developed to be tolerant of dicamba by incorporation of the dmo gene. The dmo gene encodes the protein dicamba mono-oxygenase (DMO), which converts dicamba to non-herbicidal 3,6-dichlorosalicylic acid. Introduction of the dmo gene into soybean event MON 94313 confers commercial-level tolerance of dicamba. The dmo gene was derived from Stenotrophomonas maltophilia, a gram-negative bacterium commonly present in soil, plants, and aquatic environments, and optimized for expression in soybean. The DMO protein in soybean event MON 94313 is identical to the native DMO protein except for insertion of a single amino acid at position 2 from the N-terminus.

The dmo gene in soybean event MON 94313 is linked to constitutive promoter. Samples of soybean event MON 94313 tissues at various plant growth stages were collected from 5 field trial sites in the USA. The range of DMO protein expression levels, as evaluated by validated multiplexed immunoassay were as follows: 32 to 550 µg/g dwt in leaf, 10 to 27 µg/g dwt in root, 100 to 230 µg/g dwt in forage, and 32 to 49 µg/g dwt in grain.

The identity and integrity of the DMO protein produced in soybean event MON 94313 was confirmed by molecular weight, immunoreactivity, tryptic peptide mass signatures, N-terminal sequences, and functional activity.

The potential allergenicity and toxicity of the DMO protein were evaluated. The weight of evidence indicates that this protein is unlikely to be allergenic, based on the following information:

  • the source of the dmo gene, S. maltophilia, is not commonly associated with allergenicity
  • bioinformatics evaluations of the DMO amino acid sequence confirmed the lack of relevant similarities to known allergens
  • the DMO protein has previously been shown to be rapidly degraded in simulated gastric fluid, and to be inactivated by heat (DD2014-104); and
  • unlike many allergenic proteins, the DMO protein in soybean event MON 94313 was shown to be unglycosylated

The CFIA concluded that the DMO protein is unlikely to be toxic to livestock and non-target organisms because:

  • the DMO protein lacks a mode of action to suggest that it is intrinsically toxic to livestock or non-target organisms; and
  • bioinformatics evaluations of the DMO amino acid sequence confirmed the lack of relevant similarities to known toxins

For a more detailed discussion of the potential allergenicity and toxicity of the DMO protein, see Section 5.2.

3.4 2,4-D herbicide tolerance

The Group 4 herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) is a synthetic auxin. Application of 2,4-D to susceptible plants disrupts new cell growth and results in plant death.

Soybean event MON 94313 was developed to be tolerant of the herbicide 2,4-D by incorporation of the ft_t.1 gene. The ft_t.1 gene is a modified version of the R-2,4-dichlorophenoxypropionate dioxygenase (RdpA) gene from Sphingobium herbicidovorans, a common soil bacterium. The ft_t.1 gene was modified from RdpA to improve the enzymatic activity towards herbicide substrates, including 2,4-D, and to improve the stability of the protein at higher temperatures. The ft_t.1 gene encodes a dioxygenase protein (FT_T.1) which converts 2,4-D into 2,4-dichlorophenol, making the herbicide inactive. Introduction of the ft_t.1 gene into soybean event MON 94313 confers commercial-level tolerance to the herbicide 2,4-D.

The FT_T.1 protein is also active on FOP herbicides. However soybean is naturally tolerant of FOP herbicides and the addition of the ft_t.1 gene does not increase the level of tolerance to FOP herbicides in soybean event MON 94313, compared to non-modified soybean.

The ft_t.1 gene in soybean event MON 94313 is linked to a constitutive promoter. Samples of soybean event MON 94313 tissues at various plant growth stages were collected from 5 field trial sites in the USA. The range of FT_T.1 protein expression levels, as evaluated by validated multiplexed immunoassay were as follows: 14 to 36 µg/g dwt in leaf, 2.3 to 6.0 µg/g dwt in root, 7.5 to 15 µg/g dwt in forage, and 4.7 to 7.2 µg/g dwt in grain.

To obtain sufficient quantities of FT_T.1 protein for evaluation of environmental and feed safety, it was necessary to express the ft_t.1 gene in a microbial production system. The equivalency of the FT_T.1 protein produced in soybean event MON 94313 and the FT_T.1 protein produced in themicrobial production system was demonstrated by determining their molecular weights, immunoreactivity, N-terminal sequence analysis, peptide mapping by mass spectrometry, and functional activity. Based on the results, the two proteins were found to be equivalent.

The potential allergenicity and toxicity of the FT_T.1 protein were evaluated. The weight of evidence indicates that this protein is unlikely to be allergenic, based on the following information:

  • the source of the ft_t.1 gene, S. herbicidovorans, is not commonly associated with allergenicity
  • bioinformatics evaluations of the FT_T.1 amino acid sequence confirmed the lack of relevant similarities to known allergens
  • the FT_T protein has previously been shown to be rapidly degraded in simulated gastric fluid, and to be inactivated by heat (DD2020-128); and
  • unlike many allergenenic proteins, the FT_T.1 protein in soybean event MON 94313 was shown to be unglycosylated

It was also concluded that the FT_T.1 protein is unlikely to be toxic to livestock and non-target organisms because:

  • the FT_T.1 protein lacks a mode of action to suggest that it is intrinsically toxic to livestock or non-target organisms
  • bioinformatics evaluations of the FT_T.1 amino acid sequence confirmed the lack of relevant similarities to known toxins; and
  • the microbe-produced FT_T protein has previously been shown to not cause any adverse effects in mice following a single oral dose of 2000 mg FT_T protein/kg body weight (DD2020-128)

For a more detailed discussion of the potential allergenicity and toxicity of the FT_T.1 protein, see Section 5.2

3.5 Mesotrione herbicide tolerance

The p-hydroxyphenylpyruvate dioxygenase (HPPD) enzyme of soybean and other plants catalyzes the formation of homogentisic acid, which is the aromatic precursor of plastoquinone and vitamin E. The Group 27 herbicide mesotrione inhibits the HPPD enzyme in many plants, resulting in depletion of plant plastoquinone and vitamin E levels, leading to bleaching and plant death.

Soybean event MON 94313 was developed to be tolerant to the herbicide mesotrione by incorporation of the tdo gene. The tdo gene encodes the protein triketone dioxygenase (TDO), which oxidizes mesotrione to 5' hydroxy-mesotrione and oxy-mesotrione, making the herbicide inactive. Introduction of the tdo gene into soybean event MON 94313 confers commercial-level tolerance to the herbicide mesotrione. The tdo gene was derived from rice, Oryza sativa, and optimized for expression in soybean. The amino acid sequence of TDO protein produced in soybean event MON 94313 is identical to the amino acid sequence of the native protein.

The tdo gene in soybean event MON 94313 is linked to a constitutive promoter. Samples of soybean event MON 94313 tissues at various plant growth stages were collected from 5 field trial sites in the USA. The range of TDO protein expression levels, as evaluated by validated enzyme-linked immunosorbent assay were as follows: 13 to 54 µg/g dwt in leaf, 8.8 to 17 µg/g dwt in forage, and 2.8 to 8.1 µg/g dwt in grain. TDO protein levels in root tissue ranged from below the limit of quantification (0.50 µg/g dwt) to 1.3 µg/g dwt.

To obtain sufficient quantities of TDO protein for evaluation of environmental and feed safety, it was necessary to express the tdo gene in a microbial production system. The equivalency of the TDO protein produced in soybean event MON 94313 and the TDO protein produced in themicrobial production system was demonstrated by determining their molecular weights, immunoreactivity, N-terminal sequence analysis, peptide mapping by mass spectrometry, and functional activity. Based on the results, the 2 proteins were found to be equivalent.

The potential allergenicity and toxicity of the TDO protein were evaluated. The weight of evidence indicates that this protein is unlikely to be allergenic, based on the following information:

  • the source of the tdo gene, O. sativa, is not commonly associated with allergenicity
  • bioinformatics evaluations of the TDO amino acid sequence confirmed the lack of relevant similarities to known allergens
  • unlike many allergenic proteins, which tend to resist digestion, the TDO protein is rapidly degraded in simulated gastric fluid, and is not heat-stable; and
  • unlike many allergenenic proteins, the TDO protein in soybean event MON 94313 was shown to be unglycosylated

The CFIA also concluded that the TDO protein is unlikely to be toxic to livestock and non-target organisms because:

  • the source of the tdo gene, O. sativa, has a long history of safe use in agriculture, feed and food
  • the TDO protein lacks a mode of action to suggest that it is intrinsically toxic to livestock or non-target organisms
  • bioinformatics evaluations of the TDO amino acid sequence confirmed the lack of relevant similarities to known toxins; and
  • the microbe-produced TDO protein did not cause any adverse effects in mice following a single oral dose of 2000 mg TDO protein/kg body weight

For a more detailed discussion of the potential allergenicity and toxicity of the TDO protein, see Section 5.2.

3.6 Stable integration into the plant genome

Molecular characterization by junction sequence analyses and directed sequencing demonstrated that soybean event MON 94313 contains 1 intact copy of the T-DNA with the pat, dmo, ft_t.1, and tdo coding sequences, along with the associated regulatory elements. No sequences from the other T-DNA were detected in MON 94313. No additional elements, including intact or partial T-DNA fragments or backbone sequences, linked or unlinked to the intact insert, were detected in soybean event MON 94313.

The stability of the inserted DNA was demonstrated by junction sequence analysis and western blots across 5 generations in the breeding history of soybean event MON 94313. The inheritance pattern of the insert was evaluated by genotypic analyses across 3 segregating generations of soybean event MON 94313. The combined analysis showed that the insert is stably inherited and segregates according to the Mendelian rules of inheritance for a single genetic locus.

4. Criteria for the environmental assessment

The CFIA used information on the biology of soybean and conclusions from previous assessments of plants tolerant to dicamba, glufosinate, glyphosate or metrione to inform the environmental assessment of soybean event MON 94313, as described in the following sections.

4.1 Potential for soybean event MON 94313 to become a weed of agriculture or be invasive of natural habitats

As indicated in BIO2021-01 - The Biology of Glycine max (L.) Merr. (Soybean), soybean does not invade 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 Bayer CropScience Inc., soybean event MON 94313 was determined not to be significantly different from unmodified soybean varieties in this respect.

The CFIA evaluated data submitted by Bayer CropScience Inc. on the reproductive biology and life history traits of soybean event MON 94313. This event was field-tested in the USA at 8 sites in the 2020 growing season. The CFIA determined that the USA locations share similar environmental and agronomic conditions to Canadian soybean production areas and were considered representative of the major Canadian soybean growing regions. During the field trials, soybean event MON 94313 was compared to an unmodified control soybean variety with a similar genetic background. 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, days to flowering, days to maturity, plant height, lodging, seed loss, final stand count, moisture, seed weight, and yield. For the majority of the traits, no statistically significant differences were observed between soybean event MON 94313 and the unmodified control soybean variety. Instances of statistically significant differences were observed between soybean event MON 94313 and the unmodified control soybean variety in the individual-site analyses. There was only one difference in the combined-site analysis related to decreased seed weight. For seed weight, the values for soybean event MON 94313 were within the range established for the reference soybean varieties included in the same field trials. Since the values were within established range, the results were not considered to be biologically meaningful. These results support a conclusion of phenotypic equivalence to currently grown soybean varieties.

Bayer CropScience Inc. provided information on the dormancy and germination of soybean event MON 94313 under 2 different temperature regimes. Seed germination characteristics were evaluated including percent germinated seed (normal or abnormal), percent hard seed, percent dead seed, and percent firm swollen seed. Soybean event MON 94313 was compared to an unmodified control soybean variety with a similar genetic background. As well, 4 reference soybean varieties were included to provide a range of comparative values for each germination characteristic. No statistically significant differences were detected between soybean event MON 94313 and the unmodified control soybean variety with respect to percentages of viable firm swollen seed and viable hard seed (viable hard seed is associated with dormancy). Statistically significant differences were observed between soybean event MON 94313 and the unmodified control soybean variety in both temperature regimes for percent germinated seed and percent dead seed. However, the values were within the reference range established for commercial soybean varieties and not considered to be biologically meaningful. Therefore, the introduction of the novel traits did not have a biologically meaningful impact on the germination of the soybean seed and did not confer dormancy to the soybean seed.

The response of soybean event MON 94313 to abiotic stressors was evaluated in the field at the same locations as the agronomic characteristic studies. The stressors observed included:

  • cold temperatures
  • drought
  • excessive rain
  • hail
  • high temperatures
  • high winds
  • mineral toxicity
  • nutrient deficiency
  • soil compaction, and
  • sun scald

No trend in increased or decreased susceptibility to these abiotic stressors was observed in soybean event MON 94313 compared to the unmodified control soybean variety.

The susceptibility of soybean event MON 94313 to soybean pests and pathogens was evaluated in the field at the same locations as the agronomic characteristic studies (further details provided below in Section 4.3: Potential for soybean event MON 94313 to become a plant pest). No trend in increased or decreased susceptibility to pests or pathogens was observed in soybean event MON 94313 compared to the unmodified control soybean variety.

No competitive advantage was conferred to soybean event MON 94313, other than that conferred by tolerance to the dicamba, glufosinate-ammonium, 2,4-D, and mesotrione herbicides. The reproductive characteristics, growth characteristics, and tolerance to biotic and abiotic stressors of soybean event MON 94313 were comparable to those of the unmodified control soybean variety. Tolerance to the dicamba, glufosinate-ammonium, 2,4-D, and mesotrione herbicides provides a competitive advantage only when these herbicides are used, alone or in combination, and will not, in and of itself, make the herbicide-tolerant plant weedier or more invasive of natural habitats. Soybean event MON 94313 plants growing as volunteers will not be controlled if dicamba, glufosinate-ammonium, 2,4-D, or mesotrione herbicides are used as the only weed control tools. However, control of soybean event MON 94313 as a volunteer weed in subsequent crops or in fallow ground can be achieved by the use of other classes of herbicides or by mechanical means.

The CFIA considers the changes in usual agronomic practices that may arise from volunteer plants with novel herbicide tolerances. To address this issue, a herbicide tolerance management plan, which includes sustainable volunteer management strategies, should be implemented.

The CFIA has therefore concluded that soybean event MON 94313 has no altered weediness potential in Canada compared to currently grown soybean varieties, when a herbicide tolerance management plan is implemented.

4.2 Potential for gene flow from soybean event MON 94313 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 BIO2021-01 - 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.

This information, together with the expectation that the novel traits have no intended effects on soybean reproductive biology, led the CFIA to conclude that there is minimal potential for gene flow from soybean event MON 94313 to related species in Canada.

4.3 Potential for soybean event MON 94313 to become a plant pest

The potential for soybean event MON 94313 to harbor new or increased populations of pathogens or pests was evaluated. Soybean is not considered to be a plant pest in Canada. The dicamba, glufosinate-ammonium, 2,4-D, and mesotrione herbicides tolerance traits introduced into soybean event MON 94313 are unrelated to plant pest potential. The DMO, PAT, FT_T.1, and TDO proteins expressed in soybean event MON 94313 are not intended or expected to impact the plant pest potential of the soybean plant.

As expected, field observations showed no differences in the response of soybean event MON 94313 to pests or pathogens when compared with the unmodified control soybean variety. The pests observed included:

  • aphids
  • armyworms
  • bean leaf beetles
  • grasshoppers
  • European corn borer
  • grasshoppers
  • green cloverworms
  • Japanese beetles
  • loopers
  • Mexican bean beetles
  • potato leaf hoppers
  • spider mites
  • stink bugs
  • thistle caterpillar
  • thrips
  • whiteflies

The pathogens observed include:

  • anthracnose
  • Asian soybean rust
  • bacterial blight
  • bacterial pustule
  • bean pod mottle virus
  • brown spot
  • brown stem rot
  • Cercospora leaf blight
  • Macrophomina phaseolina (charcoal rot)
  • downy mildew
  • frogeye leaf spot
  • Phytophtora root rot
  • Rhizoctonia foliar blight
  • soybean mosaic virus
  • sudden death syndrome
  • white mold

The CFIA has therefore concluded that soybean event MON 94313 does not display any altered plant pest potential compared to currently grown soybean varieties.

4.4 Potential impact of soybean event MON 94313 and its gene products on non-target organisms, including humans

The CFIA evaluated the potential impacts of the novel traits expressed by soybean event MON 94313, as well as the proteins which confer the novel traits, on non-target organisms. The glufosinate-ammonium, dicamba, 2,4-D, and mesotrione herbicide tolerance traits introduced into soybean event MON 94313 are unrelated to potential toxicity to non-target organisms.

The PAT, DMO, and FT_T.1 proteins have been previously evaluated by the CFIA and are not known toxins or allergens (see Section 5.2).

The CFIA evaluated the potential impact of the TDO protein in soybean event MON 94313 on non-target organisms. The CFIA concluded that the TDO protein is unlikely to cause adverse impacts to non-target organisms on the basis of:

  • the TDO protein lacks a mode of action to suggest that it is intrinsically toxic to non-target organisms; and
  • the TDO protein does not display characteristics of a toxin or allergen

For further discussion of the potential allergenicity and toxicity of the TDO protein, see Section 5.2.

Field evaluations of soybean event MON 94313 did not show any increased resistance to arthropod pests or pathogens compared to the unmodified control soybean (see Section 4.3).

Based on the above, the CFIA has determined that the unconfined release of soybean event MON 94313 in Canada will not result in altered impacts on non-target organisms, including humans, when compared to soybean varieties that are currently grown in Canada.

4.5 Potential impact of soybean event MON 94313 on biodiversity

Soybean event MON 94313 expresses no novel phenotypic characteristics that would extend its range beyond the current geographic range of soybean production in Canada. The only known sexually compatible wild relative of soybean, G. soja, does not occur in unmanaged habitats in Canada, and the possibility of soybean outcrossing to G. soja is very low.Soybean event MON 94313 is unlikely to cause adverse effects on non-target organisms and does not display increased invasiveness or plant pest potential.

Soybean event MON 94313 has tolerance to the herbicides dicamba, 2,4-D, glufosinate-ammonium, and mesotrione. The use of these herbicides in cropping systems has the intended effect of reducing local weed populations within agro-ecosystems. This may result in a reduction in local weed species biodiversity, and may have effects on other trophic levels that utilize these weed species. It must be noted, however, that the goal of reducing weed biodiversity in agricultural fields is not unique to the use of PNTs, soybean event MON 94313 or the cultivation of soybean. It is therefore unlikely that soybean event MON 94313 will have any indirect effects on biodiversity, in comparison to the effects that would be expected from cultivation of currently grown soybean varieties.

The CFIA also considers the potential that continued application of the same herbicide in subsequent rotations may lead to increased selection pressure for herbicide-tolerant weed populations. To address these issues, a herbicide tolerance management plan that includes integrated weed management strategies should be implemented. This plan may include a recommendation to rotate or combine weed management products with alternate modes of action and to employ other weed management practices.

The CFIA has therefore concluded that the potential impact on biodiversity of soybean event MON 94313 is unlikely to be different from that of the soybean varieties that are currently grown in Canada, when a herbicide tolerance management plan is implemented.

5. Criteria for the livestock feed assessment

The CFIA considered the safety and efficacy of feed ingredients derived from soybean event MON 94313, including nutrient and anti-nutrient profiles; 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. The CFIA also assessed whether feeds derived from soybean event MON 94313 meet the definitions and requirements of feeds as listed in Schedule IV of the Feeds Regulations.

5.1. Potential impact of soybean event MON 94313 on livestock nutrition

Based on the data provided by Bayer Crop Science, the nutritional composition of soybean event MON 94313 is similar to that of the unmodified control and conventional soybean.

A field trial comparing MON 94313 test line with the A3555 check line (which has a genetic background similar to MON 94313) was conducted in 2020 at five field sites in the United States representative of commercial growing conditions (Iowa, Illinois, Missouri, Nebraska, Wisconsin). The compositional analysis of MON 94313 seed (grain) included proximates, fibre, minerals, vitamins, amino acids, fatty acids, anti-nutrients (phytic acid, raffinose, soybean lectin, stachyose and trypsin inhibitor) and isoflavones (daidzein, genistein and glycitein).

Statistically significant (P < 0.05) differences were observed between MON 94313 and the unmodified control for seven components (ADF (acid-detergent fibre), vitamin K, cystine, tyrosine, palmitic acid, linolenic acid, and glycitein). The biological relevance of these differences was assessed by comparing them to the natural variation in soybean varieties reported in the published literature. All nutrient and anti-nutrient levels were within the published range for conventional soybean and were therefore not considered biologically relevant. These differences in nutrient composition are expected to have little impact on the total diet in normal livestock feeding practises and minimal effect on the composition of co-products such as soybean meal, soy protein concentrate and soy isolate.

Conclusion

Based on the data provided by Bayer CropScience Inc., the nutritional composition of soybean event MON 94313 is similar to that of conventional soybean varieties. Soybean seeds and co-products derived from soybean event MON 94313 are considered to meet present ingredient definitions for soybean in Schedule IV of the Feeds Regulations.

5.2 Potential impact of soybean event MON 94313 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

A weight-of-evidence approach was used to evaluate the risk to livestock consuming feed ingredients derived from soybean event MON 94313, to humans consuming foods of animal origin derived from those livestock, and to workers/bystanders exposed to the feed ingredients from this soybean event. The following was specifically considered:

  • TDO protein
  • FT_T.1 protein
  • DMO protein
  • PAT protein

TDO protein

To obtain sufficient quantities of the TDO protein for safety evaluation, it was necessary to express the TDO protein in a microbial production system. Equivalency was demonstrated between the TDO protein expressed in soybean event MON 94313 and the TDO protein produced in the Escherichia coli (E. coli) production system. Equivalency was established by comparing their molecular weight, immunoreactivity, peptide identity and N-terminal amino acid sequence by LC-MS/MS, functional activity, and glycosylation. Based on the results, the proteins were found to be equivalent. This demonstration of equivalency allows studies with the E. coli-produced TDO protein to support the safety of soybean event MON 94313.

The potential allergenicity and toxicity of the TDO protein were evaluated. Regarding its potential allergenicity, the host organism, soybean, has a history of safe use in feed and food, although it is a known allergen. The source of the TDO gene, Oryza sativa, is not commonly associated with allergenicity. The amino acid sequence of TDO in soybean event MON 94313 is identical to that of the naturally occurring rice protein. A bioinformatics evaluation of the TDO protein amino acid sequence confirmed the lack of relevant similarities between this protein and known allergens. Unlike many allergens, the TDO protein produced in the E. coli production system was experimentally shown to be rapidly degraded in simulated gastric fluid and simulated intestinal fluid. This E. coli-derived TDO protein was also experimentally shown to lose functional activity when exposed to heat. The weight of evidence thus indicates that the TDO protein is unlikely to be allergenic.

In terms of potential toxicity, the TDO protein lacks a mode of action suggesting it is intrinsically toxic. The source of the TDO gene, Oryza sativa, is not commonly associated with toxicity. A bioinformatics evaluation of its amino acid sequence confirmed the lack of relevant similarities between the TDO protein and known toxins. Livestock exposure to the TDO protein is expected to be very low, as this protein is expressed at low levels in soybean event MON 94313 grain (maximum 8.1 µg/g dry weight) and forage (maximum 17 µg/g dry weight). Additionally, the E. coli-derived TDO protein was shown experimentally to be rapidly degraded under conditions simulating the mammalian digestive tract and to lose functional activity under heating conditions expected during the processing of some soybean products.

Furthermore, no test substance-related adverse effects were observed when the E. coli-produced TDO protein was administered by oral gavage to male and female CD-1 mice at a single dose of  mg/kg body weight. A repeat-dose oral gavage of the E. coli-produced TDO protein in mice at dose levels of 10, 100, and 1000 mg/kg/day for 28 days demonstrated no test substance-related effects. The weight of evidence thus indicates that the TDO protein is unlikely to be toxic.

Therefore, the TDO protein in soybean event MON 94313 is unlikely to pose a risk to livestock, humans, or workers/bystanders.

FT_T.1 protein

The potential mammalian allergenicity and toxicity of the FT_T.1 protein were evaluated. Regarding its potential allergenicity, the source of the ft_t.1 gene, Sphingobium herbicidovorans, is not commonly associated with allergenicity. The amino acid sequence of the FT_T.1 protein in soybean event MON 94313 is nearly identical to the previously assessed and authorized FT_T protein in corn event MON 87429 (DD2020-128). A bioinformatics evaluation of the FT_T.1 protein amino acid sequence confirmed the lack of relevant similarities between this protein and known allergens. Bridging data submitted in support of soybean event MON 94313 protein safety demonstrated that this protein is rapidly degraded under conditions simulating the mammalian digestive tract. The weight of evidence thus indicates that the FT_T.1 protein is unlikely to be allergenic.

In terms of potential toxicity, the FT_T.1 protein lacks a mode of action suggesting it is intrinsically toxic. The source of the ft_t.1 gene, Sphingobium herbicidovorans, is not commonly associated with toxicity. The bioinformatics evaluation of the FT_T.1 protein amino acid sequence confirmed the lack of relevant similarities between the FT_T.1 protein and known toxins. Additionally, livestock exposure to the FT_T.1 protein is expected to be very low, as this protein is expressed at low levels in soybean event MON 94313 grain (maximum 7.7 µg/g dry weight) and forage (maximum 15 µg/g dry weight). Bridging data submitted in support of the FT_T.1 protein's safety demonstrated that this protein is rapidly degraded under conditions simulating the mammalian digestive tract. The weight of evidence thus indicates that the FT_T.1 protein is unlikely to be toxic.

Therefore, the FT_T.1 protein in soybean event MON 94313 is unlikely to pose a risk to livestock, humans, or workers/bystanders.

DMO protein

The potential mammalian allergenicity and toxicity of the DMO protein were evaluated. Regarding its potential allergenicity, the source of the dmo gene, Stenotrophomonas maltophilia, is not commonly associated with allergenicity. The amino acid sequence of the DMO protein in soybean event MON 94313 is identical to the previously assessed and authorized DMO protein in corn event MON 87429 (DD2020-128) and is identical to the wild-type DMO, except for a leucine insertion at position 2 in soybean event MON 94313. A bioinformatics evaluation of the DMO protein amino acid sequence confirmed the lack of relevant similarities between this protein and known allergens. Bridging data submitted in support of the DMO protein safety in soybean event MON 94313 demonstrated that this protein is rapidly degraded under conditions simulating the mammalian digestive tract. The weight of evidence thus indicates that the DMO protein is unlikely to be allergenic.

In terms of potential toxicity, the DMO protein lacks a mode of action suggesting it is intrinsically toxic. The source of the dmo gene, Stenotrophomonas maltophilia, is not commonly associated with toxicity. A bioinformatics evaluation of the DMO protein amino acid sequence confirmed the lack of relevant similarities between the DMO protein and known toxins. Additionally, livestock exposure to the DMO protein is expected to be low, as this protein is expressed at levels of 49 µg/g dry weight in soybean event MON 94313 grain and 230 µg/g dry weight in forage.

Bridging data submitted in support of the DMO protein's safety demonstrated that this protein is rapidly degraded under conditions simulating the mammalian digestive tract. The weight of evidence thus indicates that the DMO protein is unlikely to be toxic.

The weight of evidence thus indicates that the DMO protein is unlikely to pose a risk to livestock, humans, or workers/bystanders.

PAT protein

The potential allergenicity and toxicity of the PAT protein were evaluated. Regarding its potential allergenicity, the source of the pat gene, Streptomyces viridochromogenes, is not commonly associated with allergenicity. The amino acid sequence of the PAT protein produced in soybean event MON 94313 is identical to those of the PAT protein in previously assessed and authorized events currently in commercial use. A bioinformatics evaluation of the PAT protein's amino acid sequences confirmed the lack of relevant similarities between this protein and known allergens. Bridging data submitted in support of the PAT protein's safety demonstrated that this protein is rapidly degraded under conditions simulating the mammalian digestive tract. The weight of evidence thus indicates that the PAT protein is unlikely to be allergenic.

In terms of potential toxicity, the PAT protein lacks a mode of action to suggest that it is intrinsically toxic. The source of the pat gene, Streptomyces viridochromogenes, is not commonly associated with toxicity. The amino acid sequence of the PAT protein produced in soybean event MON 94313 is identical to that of the PAT protein in previously assessed and authorized events. A bioinformatics evaluation of the PAT protein's amino acid sequence confirmed the lack of relevant similarities between this protein and known toxins. Additionally, livestock exposure to the PAT protein is expected to be negligible, as this protein is expressed at very low levels in soybean event MON 94313 grain (maximum 4.6 µg/g dry weight) and forage (maximum 18 µg/g dry weight). Bridging data submitted in support of the PAT protein's safety demonstrated that this protein is rapidly degraded under conditions simulating the mammalian digestive tract. The weight of evidence thus indicates that the PAT protein is unlikely to be toxic.

Therefore, the PAT protein in soybean event MON 94313 is unlikely to pose a risk to livestock, humans, or workers/bystanders.

Conclusion

It was concluded, based on the evidence provided by Bayer CropScience Canada Inc., that the novel TDO protein, FT_T.1 protein, DMO protein, and PAT protein trait will not confer to soybean event MON 94313 any characteristic that would raise concerns regarding the safety of soybean event MON 94313. Thus, soybean event MON 94313 is unlikely to pose a risk to livestock consuming feed ingredients derived from soybean event MON 94313, to humans consuming foods of animal origin derived from those livestock, and to workers/bystanders exposed to the feed ingredients from this event.

6. New information requirements

If at any time, Bayer CropScience Inc. becomes aware of any new information regarding risk to the environment, livestock, or human health, which could result from the unconfined environmental release or livestock feed use of soybean event MON 94313 or lines derived therefrom, Bayer CropScience 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 94313 on the environment, livestock and human health, and may re-evaluate its decision with respect to the livestock feed use and unconfined environmental release authorizations of soybean event MON 94313.

7. Regulatory decision

Environmental release

Based on the review of the data and information submitted by Bayer CropScience Inc. and input from other relevant scientific sources, the CFIA has concluded that the unconfined environmental release of soybean event MON 94313 does not present altered environmental risk when compared to soybean varieties that are currently grown in Canada when a herbicide tolerant management plan is implemented.

According to Bayer CropScience Inc., soybean event MON 94313 is not intended to be commercialized as an individual event in Canada. Therefore, a herbicide tolerance management plan specific to this product is not required at this time. Before soybean event MON 94313 is commercialized in Canada as an individual event or in combination with other soybean events to stack novel traits, Bayer CropScience Inc. must submit a herbicide tolerance management plan to the CFIA.

Unconfined release into the environment of soybean event MON 94313 is therefore authorized by the CFIA as of October 3, 2023.

Livestock feed use

Based on the review of the data and information submitted by Bayer CropScience Inc. and input from other relevant scientific sources, the CFIA has concluded that the herbicide tolerance traits will not confer any characteristics to soybean event MON 94313 that would raise concerns regarding the safety or nutritional value. Livestock feeds derived from soybean are currently listed in Schedule IV of the Feeds Regulations. Soybean event MON 94313 has been determined to be as safe as and as nutritious as currently and historically grown soybean varieties. Soybean event MON 94313 and its products are considered to meet present ingredient definitions.

Use as livestock feed of soybean event MON 94313 is therefore authorized by the CFIA as of October 3, 2023.

Lines derived from the authorized line

Any soybean lines derived from soybean event MON 94313 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 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 nutrition
  • the novel genes are expressed at a level similar to that of the authorized line

Cultivation of plants derived from soybean event MON 94313 is subject to herbicide tolerance management requirements.

Other regulatory requirements

Soybean event MON 94313 is subject to the same phytosanitary import requirements as unmodified soybean varieties. Soybean event MON 94313 must meet the requirements of other applicable Canadian legislation, including but not limited to, the Food and Drugs Act and the Pest Control Products Act.

Other requirements, such as the assessment of novel foods by Health Canada, have been addressed separately from this assessment. Refer to Health Canada's Decisions on Novel Foods for a description of the food safety assessment of soybean event MON 94313.

8. Contact us

For more information on this decision, please contact the CFIA's Plant Biosafety Office at PBO@inspection.gc.ca or Animal Feed Program at cfia.afp-paa.acia@inspection.gc.ca.

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