DD2009-76: Determination of the Safety of Pioneer Hi-Bred Production Ltd.'s Soybean (Glycine max (L.) Merr.) Event 305423

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Issued: 2009-04

This Decision Document has been prepared to explain the regulatory decision reached under Directive 94-08 (Dir94-08), entitled "Assessment Criteria for Determining Environmental Safety of Plants with Novel Traits", its companion biology document BIO1996-10, "The Biology of Glycine max (L.) Merr. (Soybean)", and Directive 95-03 (Dir95-03), entitled "Guidelines for the Assessment of Novel Feeds: Plant Sources".

The Canadian Food Inspection Agency (CFIA), has evaluated information submitted by Pioneer Hi-Bred Production Ltd. This information is in regard to the high oleic soybean event 305423. The CFIA has determined that this plant with novel traits (PNT) does not present altered environmental risk nor, as a novel feed, does it present livestock feed safety concerns when compared to currently commercialized soybean varieties in Canada.

Taking into account these evaluations, unconfined release into the environment and use as livestock feed of soybean event 305423 is therefore authorized by Plant Biosafety Office of the Plant Health and Biosecurity Directorate and the Animal Feed Division of the Animal Health Directorate as of April 30, 2009. Any soybean lines derived from event 305423 may also be released into the environment and used as livestock feed, provided that (i) no inter-specific crosses are performed, (ii) the intended uses are similar, and (iii) it is known based on characterization, that these plants do not display any additional novel traits and are substantially equivalent to currently grown soybean in Canada, in terms of their potential environmental impact and livestock feed safety.

Soybean event 305423 is subject to the same phytosanitary import requirements as its unmodified counterparts.

Please note, that the assessment of livestock feed and environmental safety of novel feeds and PNTs are critical steps in the potential commercialization of these plant types. Other requirements, such as the evaluation of food safety by Health Canada, have been addressed separately from this review.

Table of Contents

  1. Brief Identification of the Modified Plant
  2. Background Information
  3. Description of the Novel Traits
    1. Production of High Levels of Oleic Acid in the Seed
    2. Tolerance to ALS-Inhibiting Herbicides
    3. Development Method
    4. Stable Integration into the Plant Genome
  4. Criteria for the Environmental Assessment
    1. Potential of Soybean Event 305423 to Become a Weed of Agriculture or be Invasive of Natural Habitats
    2. Potential for Gene Flow from Soybean Event 305423 to Wild Relatives Whose Hybrid Offspring May Become More Weedy or More Invasive
    3. Altered Plant Pest Potential of Soybean Event 305423
    4. Potential Impact of Soybean Event 305423 on Non-Target Organisms
    5. Potential Impact of Soybean Event 305423 on Biodiversity
  5. Criteria for the Livestock Feed Assessment
    1. Potential Impact of Soybean Event 305423 on Livestock Nutrition
    2. Potential Impact of Soybean Event 305423 on Livestock and Workers/Bystanders
  6. New Information Requirements
  7. Regulatory Decision

I. Brief Identification of the Modified Plant

Designation of the Modified Plant: High Oleic Soybean Event 305423, OECD Unique Identifier DP-3Ø5423-1

Applicant: Pioneer Hi-Bred Production Ltd.

Plant Species: Soybean (Glycine max (L.) Merr.)

Novel Traits: Increased levels of oleic acid and decreased levels of linoleic and linolenic acids in seeds, Tolerance to ALS-inhibiting herbicides.

Trait Introduction Method: Microprojectile bombardment (gene gun) mediated transformation

Proposed Use of the Modified Plant: Commercial production of soybean grain for human consumption and for livestock feed. These plants are not intended to be grown outside the normal cultivation area for soybean in Canada.

II. Background Information

Pioneer Hi-Bred Production Ltd. developed, through the use of recombinant DNA techniques, a soybean producing high levels of oleic acid in the seeds. The soybean event, designated as event 305423, was developed to provide the food and industrial oil sectors with a highly stable vegetable oil suitable for frying applications without the need for hydrogenation, and for formulation of industrial fluids, respectively. Soybean event 305423 is also tolerant to ALS-inhibiting herbicides, however Pioneer Hi-Bred Production Ltd. does not plan to promote 305423 soybean varieties as tolerant to ALS-inhibiting herbicides as this trait was only used as a selectable marker during the development of the event.

Soybean event 305423 was developed using recombinant DNA technology, resulting in the introduction of the gm-fad2-1 gene fragment and the gm-hra gene. The gm-fad2-1 gene fragment is derived from the endogenous soybean FAD2-1 gene that encodes an omega-6 desaturase that converts oleic acid to linoleic acid. The gm-fad2-1 gene fragment does not encode a functional protein, instead its transcription acts to silence expression of the endogenous FAD2-1 gene, resulting in an increased level of oleic acid and decreased levels of linoleic and linolenic acids in 305423 soybean seeds. The gm-hra gene encodes a modified acetolactate synthase (ALS) enzyme that was altered by site-directed mutagenesis to contain two amino acid differences from the wild type enzyme. These differences render the enzyme, and thus 305423 soybean, tolerant to ALS-inhibiting herbicides while retaining its ability to function as a key enzyme in the synthesis of branched chain-amino acids.

Pioneer Hi-Bred Production Ltd. has provided data on the identity of soybean event 305423, 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 protein was identified and characterized. Data were provided for the evaluation of the potential toxicity of the novel protein to livestock and non-target organisms and potential allergenicity of the novel protein to humans and to livestock.

Soybean event 305423 has been field tested at 7 locations in the United States and at 2 locations in Canada in 2005, and at 4 locations in the United States in 2006. The United States locations of these trials share similar environmental and agronomic conditions to southwestern Ontario and were considered representative of major Canadian soybean growing regions.

Agronomic characteristics of soybean event 305423 such as germination and seed dormancy, vegetative vigour, time to maturity, susceptibilities to various soybean pests and pathogens, and seed production were compared to those of unmodified soybean counterparts.

Nutritional components of soybean event 305423, such as proximates, amino acids and fatty acids were compared with those of unmodified soybean counterparts.

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

  • the potential of soybean event 305423 to become a weed of agriculture or be invasive of natural habitats;
  • the potential for gene flow from soybean event 305423 to wild relatives whose hybrid offspring may become more weedy or more invasive;
  • the potential for soybean event 305423 to become a plant pest;
  • the potential impact of soybean event 305423 or its gene products on non-target species, including humans; and
  • the potential impact of soybean event 305423 on biodiversity.

The Animal Feed Division of the Animal Health Directorate, 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 Directive 95-03 (Dir95-03), entitled "Guidelines for the Assessment of Novel Feeds: Plant Sources". The Animal Feed Division has considered:

  • the potential impact of soybean event 305423 on livestock nutrition; and
  • the potential impact of soybean event 305423 on livestock and workers/bystanders.

Pioneer Hi-Bred Production Ltd. has provided the CFIA with a method for the detection and identification of soybean containing the soybean event 305423.

III. Description of the Novel Traits

1. Production of High Levels of Oleic Acid in the Seed

The synthesis of polyunsaturated fatty (linoleic and linolenic) acids (e.g. in developing soybean seed) is catalyzed by two fatty acid desaturases that sequentially add a second and third double bond to oleic acid. The second double bond is added by an omega-6 desaturase encoded by the FAD2-1 gene and the third double bond is added by an omega-3 desaturase encoded by the FAD3 gene. One of the approaches to decrease the content of polyunsaturated fatty acids and elevate the level of oleic acid in soybean seeds is to down-regulate the expression of the FAD2-1 gene. Decrease in the level of the omega-6 desaturase encoded by the FAD2-1 gene inhibits the conversion of oleic acid into linoleic acid, thus elevating the level of oleic acid and decreasing the levels of linoleic and linolenic acids in soybean seed.

Silencing of the endogenous FAD2-1 gene in seeds of soybean event 305423 was achieved through the introduction of a FAD2-1 gene fragment (namely gm-fad2-1) driven by a seed-specific promoter. Transcription of the gm-fad2-1 gene fragment in the seed acts to silence transcription of the endogenous FAD2-1 gene. The resulting modified fatty acid profile of 305423 soybean seeds is characterized by elevated levels of oleic acid, which represents approximately 75% of the total fatty acids in 305423 soybean seeds, compared to approximately 20% in seeds from conventional soybeans, and decreased levels of linoleic and linolenic acids.

Introduction of the gm-fad2-1 gene fragment also caused slight reduction of transcription of the closely related FAD2-2 gene in developing seeds, however the expression of the FAD3 gene was not affected.

Silencing of the endogenous KTi3 gene encoding the Kunitz trypsin inhibitor was observed in 305423 soybean seed. This was likely caused by the use of the promoter from the KTi3 gene to drive expression of the gm-fad2-1 gene fragment.

The gm-fad2-1 gene fragment does not code for a functional protein, therefore no novel protein is produced from the gm-fad2-1 gene cassette.

2. Tolerance to ALS-Inhibiting Herbicides

The gm-hra gene, which was introduced into soybean event 305423, imparts tolerance to ALS-inhibiting herbicides. These herbicides target the ALS enzyme, a key enzyme in the synthesis of the essential branched-chain amino acids isoleucine, leucine, and valine. Herbicide-induced ALS inhibition results in a lethal decrease in protein synthesis. Unmodified soybean has an inherent level of tolerance to some ALS-inhibiting herbicides. Expression of the GM-HRA protein increases this inherent level and provides tolerance to a broader range of ALS-inhibiting herbicides.

The gm-hra gene was engineered by introducing two point mutations into the soybean gm-als gene, which codes for the soybean ALS enzyme, resulting in two amino acid changes in the mature protein. The two amino acid substitutions in the modified GM-HRA protein alter the binding site for ALS-inhibiting herbicides, resulting in the herbicide-tolerant phenotype of 305423 soybean. The gm-hra gene was used as a selectable marker gene in the process of development of soybean event 305423 and does not confer commercial levels of tolerance to ALS-inhibiting herbicides.

GM-HRA expression in 305423 soybeans is driven by a constitutive promoter. Samples of soybean tissues were collected from plants from six field trial sites across Canada and the United States. Average GM-HRA protein expression in micro-grams protein per gram dry weight tissue (µg/g dwt) as evaluated by enzyme-linked immunosorbent assay (ELISA) are as follows: 4 µg/g dwt in leaf, 5.7 µg/g dwt in forage, 0.18 µg/g dwt in root, and 2.5 µg/g dwt in grain.

To obtain sufficient quantities of GM-HRA protein for evaluation of environmental and feed safety, it was necessary to express the gm-hra gene in an E. coli production system. The equivalency of the GM-HRA protein produced in soybean event 305423 to the E. coli-produced GM-HRA protein was evaluated by comparing their molecular weights, immunoreactivity, glycosylation status, N-terminal sequence analysis, and MALDI-TOF mass spectrometry. Based on the results of these tests, the proteins were found to be equivalent. In addition, it was demonstrated that the E. coli -produced GM-HRA protein has equivalent ALS activity both in the presence and absence of an ALS-inhibiting herbicide. Demonstration of protein equivalence between E. coli and soybean 305423-produced GM-HRA proteins allows utilization of the GM-HRA protein produced in E. coli to be used in studies to confirm the safety of the GM-HRA protein produced in 305423 soybean.

Pioneer Hi-Bred Production Ltd. provided an updated bioinformatics evaluation of the GM-HRA amino-acid sequence, which confirmed the lack of relevant similarities between the GM-HRA protein and known toxins. Furthermore, the E. coli GM-HRA protein did not cause any adverse effects in mice following a single oral dose of 582 mg GM-HRA protein /kg body weight. These data support the conclusion that the GM-HRA protein is unlikely to be a toxin.

ALS proteins are ubiquitous proteins present in many species, including bacteria, fungi, algae and plants. ALS proteins have never been associated with allergic reactions. The ALS protein from soybean is not considered a soy allergen. Pioneer Hi-Bred Production Ltd. provided an updated bioinformatics evaluation of the GM-HRA amino-acid sequence, which confirmed the lack of relevant similarities between the GM-HRA protein sequence and sequences of known allergens. In vitro digestive fate studies showed that the GM-HRA protein is rapidly degraded in simulated gastric and intestinal fluids unlike protein allergens, which are normally resistant to digestion. The GM-HRA protein produced in 305423 soybean is not glycosylated, unlike many known allergens, providing additional evidence that GM-HRA protein produced in 305423 soybean does not have the properties of known allergens.

3. Development Method

Soybean event 305423 was developed through microprojectile bombardment-mediated transformation of secondary somatic embryos derived from explants of immature soybean seeds of the soybean ‘Jack' variety. The DNA used in the microprojectile bombardment contained two separate cassettes, namely the gm-hra gene cassette and the gm-fad2-1 gene cassette. Transformants were selected based on tolerance to chlorsulfuron, an ALS-inhibiting herbicide. The presence of gm-fad2-1 and gm-hra gene cassettes in the transformants was later confirmed by Southern blot analysis. Event 305423 was identified as a successful transformant and was chosen for further development.

4. Stable Integration into the Plant Genome

Based on Southern blot analysis and sequence data, it was determined that a single intact copy of the gm-hra gene cassette and multiple intact and truncated copies of the gm-fad2-1 gene cassette have been inserted into soybean event 305423 genome. A small, non-functional fragment of the plasmid backbone DNA has also been inserted. Elements of the gm-fad2-1 gene cassettes present in 305423 soybean genome include 8 copies of the KTi3 promoter, 7 copies of the gm-fad2-1 fragment and 5 copies of the KTi3 terminator. The inserted DNA comprises 4 insertions, which have been fully characterized. The DNA sequences flanking the 4 insertions were sequenced and any novel junctions between endogenous soybean DNA and inserted DNA were analyzed to identify potential open reading frames. The analysis revealed that no novel protein could be produced from DNA junction sequences.

Southern blot analysis was conducted on individual plants from 3 generations of self-and cross-pollinated 305423 soybean plants to study the stability of the inserted DNA during 305423 soybean breeding. A single recombination event was observed among 1,100 segregating individuals. Consistent hybridization patterns were observed for all other segregating and non-segregating individuals. Segregating 305423 soybean plants were examined by PCR and it was confirmed that no other recombinant individuals were present in the population. The occurrence of recombinant individuals at very low frequency does not raise concerns to the stability of the inserted DNA in transformed plants, therefore the inserted DNA was considered stable across generations of soybean event 305423. This analysis also confirmed that the 4 insertions are genetically linked at a single genetic locus.

The inheritance pattern of the inserted DNA and high oleic acid phenotype has been studied in 3 generations of 305423 soybeans. The results of the analysis are consistent with the finding of a single locus of insertion that segregates according to the Mendelian laws of genetics.

IV. Criteria for the Environmental Assessment

1. Potential of Soybean Event 305423 to Become a Weed of Agriculture or be Invasive of Natural Habitats

The biology of soybean, described in the CFIA Biology Document BIO1996-10, shows that unmodified plants of this species are not invasive of unmanaged habitats in Canada. Soybean does not possess the potential to become weedy due to the lack of seed dormancy and the poor competitive ability of seedlings.

Soybean event 305423 was tested in four locations in the US and in two locations in Canada. The US locations share similar environmental and agronomic conditions to southwestern Ontario and were considered to be representative of major Canadian soybean regions. A total of 8 phenotypic characteristics were evaluated: early population, seedling vigour, days to maturity, plant height, lodging, pod shattering, final population and yield. The phenotypic characteristic data showed no biologically meaningful differences between 305423 soybean and the unmodified isoline Jack, and support a conclusion of phenotypic equivalence to currently commercialized soybean lines. The seed dormancy and germination of soybean event 305423 were compared with the unmodified control variety isoline Jack. No significant differences were detected in percent germinated seed, percent dead seed and percent viable firm, swollen seed and viable hard seed.

Data on the susceptibility to a range of insect pest and disease, and response to abiotic stressors were also recorded at each of the field trial sites. No increase or decrease outside of the reference range was observed in any insect, disease, or abiotic stressor in 305423 soybean.

The insertion of the gm-fad2-1 gene cassette and the gm-hra gene cassette did not make 305423 soybean weedy or invasive of natural habitats since none of the soybean's reproductive or growth characteristics were modified and 305423 soybean's tolerance to abiotic and biotic stresses was unchanged. The emergence of 305423 soybean volunteer plants, should they arise, can easily be managed by mechanical means and other available chemicals currently used to control conventional soybeans.

On the basis of this information, the CFIA concluded that the soybean event 305423 has no increased weediness or invasiveness potential compared to currently commercialized soybean varieties.

2. Potential for Gene Flow from Soybean Event 305423 to Wild Relatives Whose Hybrid Offspring May Become More Weedy or More Invasive

The biology of soybean, as described in CFIA Biology Document BIO1996-10, shows that soybeans exhibit a high percentage of self-fertilization and cross pollination is usually less than one percent, suggesting that any pollen flow from cultivated soybeans to related species is minimal. Natural hybridization between cultivated soybean and the wild annual species Glycine soja can occur. G. soja is not naturalized in North America, and although this species could occasionally be grown in research plots, there are no reports of its escape from such plots to unmanaged habitats.

The CFIA has therefore concluded that the potential for gene transfer from soybean event 305423 to soybean relatives in managed ecosystems is negligible, and that there is no potential for gene transfer to related wild species in Canada.

3. Altered Plant Pest Potential of Soybean Event 305423

Soybean is not considered a plant pest in Canada. The novel traits (increased oleic acid levels in seed and tolerance to ALS-inhibiting (Group 2) herbicides) are unrelated to plant pest potential. Ecological evaluations of 305423 soybean did not show any increase or decrease in susceptibility to any insect or disease stressor compared to lines sharing identical genetic background and commercial soybean varieties grown at the same locations. These stressors included 8 pest insects and 7 diseases.

The CFIA has, therefore, determined that the soybean event 305423 does not display any altered pest potential compared to currently commercialized soybean varieties.

4. Potential Impact of Soybean Event 305423 on Non-Target Organisms

No protein is produced from the gm-fad2-1 gene cassette. The only novel protein expressed in 305423 soybean is GM-HRA. Due to the previous history of exposure to and safe use of organisms containing proteins similar to GM-HRA, as well as the results of the safety assessments conducted on the GM-HRA protein produced in soybean event 305423, no negative impacts resulting from exposure of organisms to soybean event 305423 are expected.

The overall lipid content of 305423 soybean grain remained unchanged compared to conventional soybean grain. Soybean 305423 grain contains increased levels of oleic acid, and lower levels of linoleic acid and linolenic acid, and to a lesser extent palmitic acid. A trace amount (0.3% of the total fatty acids) of the 9,15-linoleic acid isomer and an increase in heptadecenoic and heptadecanoic acids (0.8% and 1.2% of the total fatty acids, respectively) were also detected in 305423 soybean grain. These 3 minor fatty acids are also constituents of many edible sources of fat, such as vegetal oils, butter and meat. There is no evidence to indicate that exposure to these fatty acids is associated with adverse effects on environment or human health. Therefore, the modified fatty acid profile of 305423 soybean seeds is not expected to negatively impact organisms interacting with soybean.

Composition analyses showed that the levels of key nutrients in 305423 soybean grain (other than certain fatty acids) and forage are comparable to those in commercial soybean varieties, confirming that the modified fatty acid profile is limited to the seed of 305423 soybean and the gross composition of the plant green matter is unchanged. The levels of anti-nutrients in soybean 305423 grain are comparable to the levels of anti-nutrients in commercial soybean varieties. Due to silencing of the endogenous trypsin inhibitor KTi3 gene, the level of trypsin inhibitor was lower in 305423 soybean compared to a control line sharing the same genetic background. However, other soybean varieties contain levels of trypsin inhibitor that are comparable to the level of trypsin inhibitor in 305423 soybean seed.

Ecological evaluations of 305423 soybean did not show any increase in resistance to insects or pathogens compared to commercial soybean varieties.

Soybean is one of the 8 major foods known to cause allergic effects. Pioneer Hi-Bred Production Ltd. conducted a study using the sera from clinically reactive soy allergic patients (both children and adults) to assess whether the genetic modification used to generate soybean event 305423 altered the endogenous allergen content of soybean. The study compared seed protein extracts from the 305423 soybean and its non-transgenic counterpart by identifying both the relative reactivity of the extracts using ELISA inhibition assays and specific proteins that bind IgE using IgE immunoblot analysis. The results of these immunoassays indicated that the levels of endogenous allergens in soybean event 305423 are comparable to those in non-transgenic soybean.

Based on the above, the CFIA has determined that the unconfined release of soybean event 305423 will not result in altered impacts on interacting organisms, including humans, compared to current commercial soybean varieties.

5. Potential Impact of Soybean Event 305423 on Biodiversity

Soybean event 305423 has no novel phenotypic characteristics that would extend its range beyond the current geographic range of soybean production in Canada. Since soybean has no wild relatives with which it can outcross in Canada, there will be no transfer of the high oleic acid and herbicide tolerance traits from 305423 soybean to plants in unmanaged environments. Soybean event 305423 does not pose an increased risk to interacting non-target organisms. Soybean event 305423 was not bred for tolerance to commercial levels of ALS-inhibiting herbicides. Should Pioneer Hi-Bred Production Ltd. market 305423 soybean as an alternative tool for weed control in soybean production, the impact on local weed species biodiversity and possibly other trophic levels that utilize these weed species will be minimal as reduction in weed biodiversity in agricultural fields is not unique to the use of plants with novel traits, and is a common practice in virtually all modern agricultural systems.

The CFIA has therefore concluded that the potential impact on biodiversity of soybean event 305423 is equivalent to that of currently commercialized soybean varieties.

V. Criteria for the Livestock Feed Assessment

1. Potential Impact of Soybean Event 305423 on Livestock Nutrition

Nutritional Composition:

The compositional equivalence of soybean event 305423, to its non transgenic, isoline control was determined from replicated trials at four sites in the U.S. and two sites in Canada. Forage and grain samples were collected at each site and analysed for proximates, acid detergent fibre (ADF) and neutral detergent fibre (NDF). Grain samples were also analyzed for fatty acids, amino acids, minerals, vitamins, oligosaccharides and isoflavones. There were no statistically significant differences between soybean event 305423 and the control for proximates and fibre in forage samples. The ash content in soybean event 305423 grain was significantly lower than control, however the means were within the tolerance interval and literature ranges. As expected, oleic acid (C18:1) content in soybean event 305423 was four times higher than the control and outside the tolerance/literature ranges, while linoleic acid (C18:2) in soybean event 305423 was 15 times lower than the control and also outside the tolerance interval /literature ranges. The mean levels of myristic, palmitic, stearic and linolenic acids were statistically significantly lower in soybean event 305423 than the control, but the means were within the tolerance interval and/or literature values. The mean levels of palmitoleic, heptadecenoic, heptadecanoic, arachidic, eicosenoic acid and lignoceric acid were statistically significantly higher in soybean event 305423 than the control. However, the means were with the tolerance interval of commercial varieties and/or literature values, except for heptadecenoic and heptadecanoic acids. Pioneer Hi-Bred Production Ltd. demonstrated that the terminal metabolic substrate (propionyl-CoA) in odd chain fatty acids enters into the Krebs cycle upon further metabolism as succinyl-CoA for energy production, and therefore the higher levels of these 2 minor fatty acids presents no risk to livestock. Furthermore, livestock consuming mostly defatted toasted soybean meal will not have a higher exposure to these fatty acids.

No statistically significant differences were observed between soybean event 305423 and control for all amino acids measured. The mean values for all amino acids were within the tolerance interval and/or literature ranges. No statistically significant differences were observed between soybean event 305423 and control for all minerals and vitamins, except for calcium and γ-tocopherol, but the means were within the tolerance interval and/or literature ranges. There were no statistically significant differences between soybean event 305423 and the control for genistin, genistein, daidzein, glycitin, glycitein and malonylglycitin. The mean values for malonylgenistin, daidzin and malonyldaidzin were statistically significantly different between soybean event 305423 and control soybean, however the means were within the tolerance interval and/or literature values.

Anti-nutrients:

Phytic acid, trypsin inhibitor, lectins, stachyose and raffinose were analyzed in soybean event 305423 grain and compared to isoline control. No statistically significant differences were observed between soybean event 305423 and the control for stachyose, raffinose, lectins and phytic acid. Trypsin inhibitor in soybean event 305423 was significantly lower than the control, however the mean was within the range of the tolerance interval and literature values.

Broiler Study:

To assess the wholesomeness of soybean event 305423 for use in animal feed, a 42- day broiler study was conducted. Diets containing soybean event 305423 were evaluated by comparing growth, organ and carcass yield of broiler chickens fed processed fractions (meal, hull and oil), to those fed diets containing the isoline control (091) and three commercial reference varieties. There were 10 broilers per pen (50% male and females) with 12 pens per treatment. One hundred twenty Ross x Cobb broilers were fed in 3 phases, starter (0-21d), grower (22-35d) and finisher (36- 42d) with the meal included at 26.5%, 23% and 21.5% of the diet, respectively. Soybean hulls and oil were added at 1% and 0.5%, respectively, in each phase. No statistically significant differences were observed between broilers consuming diets containing soybean event 305423 and control soybeans for mortality, weight gain, feed efficiency, organ and carcass yield variables. All means were within the tolerance interval calculated for the study.

The evidence provided by Pioneer Hi-Bred Production Ltd. supports the conclusion that the nutritional composition of soybean event 305423 is substantially equivalent to conventional soybean, except for the increase in oleic acid (C18:1), a decrease in linoleic acid (C18:2) and elevated levels of heptadecanoic (C17:0) and heptadecenoic (C17:1) acids. No detrimental effects of the altered levels of these analytes were observed in the health, growth and carcass performance of broilers consuming diets containing soybean event 305423.

2. Potential Impact of Soybean Event 305423 on Livestock and Workers/Bystanders

Recombinant DNA technology was used to introduce the gm-fad2-1 gene fragment and the gm-hra gene into soybean event 305423. The gm-hra gene encodes a modified ALS enzyme that was altered by site-directed mutagenesis to contain two amino acid differences from the wild type enzyme (i.e. the GM-HRA protein). ALS enzymes are found in a wide variety of plants and micro-organisms. ALS enzymes are not known toxins or allergens and the two amino changes are not expected to change this. The modified ALS enzyme did not have homology with any known allergens or toxins. It was also heat labile and rapidly degraded under conditions similar to those encountered in the gastrointestinal tract. This information suggests that the modified ALS is unlikely to be a novel toxin or allergen.

Introduction of the gm-fad2-1 fragment resulted in the silencing of the FAD2-1 gene and an altered soybean fatty acid profile, but not in production of a novel protein. This alteration of fatty acid profile is unlikely to result in toxic effects on livestock or humans. Introduction of the gm-fad2-1 fragment caused an unintended suppression of the endogenous Kunitz trypsin inhibitor KTi3 gene in soybean event 305423; the trypsin inhibitors are anti-nutritional factors. Based on the detailed characterization provided (nutritional composition and agronomic data of the modified plant compared to unmodified comparators) it is unlikely that secondary modifications causing unintended effects have occurred in soybean event 305423.

No adverse effects from the GM-HRA protein were observed in an acute oral toxicity study in mice using 100 times the highest predicted livestock dose level per kg body weight. No adverse effects on nutrition or health were observed in a poultry feeding trial comparing soybean event 305423 to conventional varieties of soy in the diet. Other than the altered fatty acid profile, the specific effects of suppressed genes were not well characterized. The lack of toxic effects in the poultry feeding trial, however, supports the safety of soybean event 305423.

The evidence provided by Pioneer Hi-Bred Production Ltd. indicates there is no potential impact of soybean event 305423 on livestock and workers/by-standers when compared to commercialized soybean lines.

VI. New Information Requirements

If at any time, Pioneer Hi-Bred Production Ltd. becomes aware of any information regarding risk to the environment, including risk to human or animal health, which could result from release of soybean event 305423 materials in Canada or elsewhere, Pioneer Hi-Bred Production Ltd. will 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 305423 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 305423.

VII. Regulatory Decision

Based on the review of the data and information submitted by Pioneer Hi-Bred Production Ltd., and through comparisons of soybean event 305423 with unmodified soybean counterparts, the Plant and Biotechnology Risk Assessment Unit of the Science Strategies Division, CFIA, has concluded that the novel genes and their corresponding traits do not confer to soybean event 305423 any characteristic that would result in unintended environmental effects following unconfined release.

Based on the review of submitted data and information by Pioneer Hi-Bred Production Ltd., including comparisons of soybean event 305423 with its unmodified soybean counterparts, the Animal Feed Division of the Animal Health Directorate, CFIA has concluded that the introduced genes and their corresponding traits will not confer to soybean event 305423 any characteristic that would raise any concerns regarding the safety or nutritional composition of soybean event 305423 . Grain soybean, its byproducts and soybean oil are currently listed in Schedule IV of the Feeds Regulations and are, therefore, approved for use in livestock feeds in Canada. Soybean event 305423 has been assessed and found to be as safe as and as nutritious as traditional soybean varieties. Soybean event 305423 and its products are considered to meet present ingredient definitions and are approved for use as livestock feed ingredients in Canada.

Unconfined release into the environment and use as livestock feed of soybean event 305423 is therefore authorized by Plant Biosafety Office of the Plant Health and Biosecurity Directorate and the Animal Feed Division of the Animal Health Directorate as of April 30, 2009. Any soybean lines derived from soybean event 305423 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, and it is known based on characterization, that these plants do not display any additional novel traits and are substantially equivalent to currently grown soybean varieties in Canada, in terms of their potential environmental impact and livestock feed safety.

Soybean event 305423 is subject to the same phytosanitary import requirements as its unmodified counterpart.

Please refer to Health Canada's Decisions on Novel Foods for a description of the food safety assessment of soybean event 305423.

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