Decision Document 2012-95 Determination of the Safety of BASF Canada Inc.'s Soybean (Glycine max (L.) Merr.) Event CV127

(publié aussi en français) November 2, 2012

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

The Canadian Food Inspection Agency (CFIA) has evaluated information submitted by BASF Canada Inc. in regard to soybean event CV127, which is tolerant to imidazolinone herbicides. This plant with a novel trait (PNT) was assessed by the Plant and Biotechnology Risk Assessment Unit (PBRA) of the CFIA for its environmental risk, including human health, having regard to weediness potential, gene flow, plant pest potential, impact on non-target organisms and impact on biodiversity. Taking into account the intended use of this product, which does not include cultivation in Canada, the risk from incidental release of soybean event CV127 into the environment was determined to be minimal.

The Animal Feed Division of the CFIA has determined that imidazolinone tolerant soybean event CV127 does not present livestock feed safety concerns when compared to currently commercialized soybean varieties.

Taking into account the conclusion of the risk assessment and recognizing that incidental release into the environment could occur when this PNT is used as intended, unconfined release^Footnote 1 into the environment^Footnote 2 of soybean event CV127 and of any lines derived from it is therefore authorized by the Plant Biosafety Office of the Plant Health and Biosecurity Directorate as of November 2, 2012 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 evaluation, use as livestock feed of soybean event CV127 and of any lines derived from it is authorized by the Animal Feed Division of the Animal Health Directorate on the condition that they meet the restrictions specific to imazapyr and imazapic treated feeds set out in this authorization.

In addition to the above conditions, authorizations of the soybean lines derived from soybean event CV127 are conditional on the following: (i) no inter-specific crosses are performed, (ii) the intended use(s) are similar, (iii) it is known, following thorough characterization, that these plants do not display any additional novel traits and are substantially equivalent to soybean event CV127 in terms of their specific use and safety for the environment, including human health, as well as livestock safety and (iv) the novel gene is expressed at a level similar to that of the authorized line.

Soybean event CV127 is subject to the same phytosanitary import requirements as its unmodified counterparts. Soybean event CV127 is required to meet the requirements of other jurisdictions; including but not limited to, the Food & Drugs Act, and the Pest Control Products Act.

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.

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:

Plant Biosafety Office
Plant Health and Biosecurity Directorate
59 Camelot Drive, Ottawa
Ontario K1A 0Y9
(613) 773-5000

Animal
Animal Health Directorate
59 Camelot Drive, Ottawa
Ontario K1A 0Feed Division Y9
(613) 225-2342

Table of Contents

• Brief Identification of the Modified Plant
• Background Information
• Description of the Novel Trait
  1. Development Method
  2. Tolerance to Imidazolinone Herbicides
  3. Stable Integration into the Plant Genome
• Criteria for the Environmental Assessment
  1. Potential of Soybean Event CV127 to Become a Weed of Agriculture or be Invasive of Natural Habitats
  2. Potential for Gene Flow from Soybean Event CV127 to Sexually Compatible Plants Whose Hybrid Offspring May Become More Weedy or More Invasive
  3. Altered Plant Pest Potential of Soybean Event CV127
  4. Potential Impact of Soybean Event CV127 on Non-Target Organisms
  5. Potential Impact of Soybean Event CV127 on Biodiversity
• Criteria for the Livestock Feed Assessment
  1. Potential Impact of Soybean Event CV127 on Livestock Nutrition
  2. Potential Impact of Soybean Event CV127 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
• New Information Requirements
• Regulatory Decision

I. Brief Identification of the Modified Plant

II. Background Information

BASF Plant Science, L.L.C. and the Brazilian Agricultural Research Corporation (EMBRAPA) developed jointly, through the use of recombinant DNA technology, a soybean designated as CV127 that is tolerant to applications of imidazolinone herbicides imazapyr and imazapic, with no significant injury at normal field application rates. This will allow post-emergent use of these imidazolinone herbicides in soybean event CV127 crops, thus providing an alternative means of weed control in soybean production. Soybean event CV127 was developed for cultivation primarily in Brazil and Argentina, where the major weeds in soybean crops are sensitive to imazapyr and imazapic.

Soybean event CV127 was developed using recombinant DNA technology, resulting in the introduction of the csr1-2 gene from Arabidopsis thaliana. The csr1-2 gene encodes an A. thaliana acetohydroxyacid synthase (AtAHAS) enzyme that is tolerant to imidazolinone herbicides due to a point mutation that results in a single amino acid substitution. This amino acid substitution renders the enzyme, and thus soybean event CV127, tolerant to imidazolinone herbicides while retaining its ability to function as a key enzyme in the synthesis of branched-chain amino acids. In addition to the csr1-2 gene cassette, the DNA used in the transformation of soybean event CV127 contained a gene encoding the gamma subunit of the SEC61 protein from A. thaliana (AtSEC61γ). The SEC61 protein is a multimeric transport protein of the endoplasmic reticulum that is ubiquitous in all plants and other eukaryotes. The presence of this gene in soybean event CV127 has no feed or environmental significance.

BASF Canada Inc. has provided data on the identity of soybean event CV127, 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 and allergenicity of the novel protein to humans and to livestock. Data were provided for the evaluation of herbicide residues in the feed commodities derived from the crop, following the intended herbicide application.

Soybean event CV127 was field tested at seven locations in Brazil in the 2006/2007 growing season and at six locations in Brazil during the 2007 short growing season. This germplasm, which has been developed for cultivation primarily in Brazil, is not suitable for cultivation in the soybean-growing regions of Canada as it would not reach maturity due to the environment. Consequently, studies were not conducted in a Canadian or similar environment. A risk assessment was conducted taking into account that the seed of soybean event CV127 will not be authorized for sale in Canada.

Agronomic characteristics of soybean event CV127 such as seed germination and seed dormancy, seedling vigour, days to flowering and maturity, susceptibilities to various soybean pests and pathogens, and seed production were compared to those of an unmodified control.

Nutritional components of soybean event CV127, such as proximates, amino acids and fatty acids, were compared with those of an unmodified control.

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 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 CV127 to become a weed of agriculture or be invasive of natural habitats;
• the potential for gene flow from soybean event CV127 to sexually compatible plants whose hybrid offspring may become more weedy or more invasive;
• the potential for soybean event CV127 to become a plant pest;
• the potential impact of soybean event CV127 or its gene products on non-target species, including humans; and
• the potential impact of soybean event CV127 on biodiversity.

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

The AFD has considered both intended and unintended effects and similarities and differences between the modified plant and its counterpart relative to the safety and efficacy of feed ingredients derived from soybean event CV127 for their intended purpose; including:

• potential impact of soybean event CV127 on livestock nutrition and
• potential impact of soybean event CV127 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 CV127 meet the definitions and requirements of feeds as listed in Schedule IV of the Feeds Regulations.

BASF Canada Inc. has provided the CFIA with a method for the detection and identification of the soybean event CV127.

III. Description of the Novel Trait

1. Development Method

Soybean event CV127 was developed through biolistic-mediated transformation of soybean somatic embryos with a DNA fragment containing the crs1-2 gene plus its regulatory sequences. Transformed cells were selected on the basis of tolerance to imazapyr, an imidazolinone herbicide.

2. Tolerance to Imidazolinone Herbicides

Imidazolinone herbicides target the AHA. enzyme in plants, a key enzyme in the synthesis of the essential branched-chain amino acids isoleucine, leucine, and valine, resulting in inhibition of protein synthesis. The csr1-2 gene from A. thaliana, which was introduced into soybean event CV127, encodes an imidazolinone herbicide-tolerant AtAHA. enzyme. The herbicide tolerance is due to a single point mutation of guanine to adenine in the coding sequence for the AtAHA. protein that results in a substitution of serine with asparagine at residue 653 (S653N). This amino acid change prevents the binding of imidazolinone herbicides to the AtAHA. protein and thereby results in tolerance of soybean event CV127 to these herbicides with no effect on the normal biosynthetic function of the enzyme or feedback regulation by branched-chain amino acids.

In addition to the S653N mutation, a second mutation was discovered in the crs1-2 gene integrated in the genome of soybean event CV127. This mutation, in which an arginine residue at position 272 is replaced by lysine (R272K), does not impact the enzymatic function of the AtAHA. protein or its herbicide-tolerance properties.

AtAHA. expression in soybean event CV127 is driven by the native A. thaliana promoter. Samples of soybean event CV127 tissues were collected from plants at various growth stages grown at distinct locations across Brazil during two growing seasons. Levels of AHA. protein expression were evaluated by enzyme-linked immunosorbent assays. Due to the high level of amino acid similarity, the AHAS-specific antibodies used in the assay were not capable of distinguishing between the AtAHA. expressed by the csr1-2 gene and endogenous soybean AHA. proteins, and therefore total AHA. levels were measured. The average AHA. protein expression was generally higher in soybean event CV127 tissues than in tissues from the unmodified control, which is consistent with the expression of the csr1-2 gene in soybean event CV127. Average AHA. levels across all stages, sites, and growing seasons, expressed in nano-grams AHA. protein per gram dry weight tissue (ng/g dwt), ranged from below the limit of quantification to 314 ng/g dwt in whole plant, below the limit of quantification to 714 ng/g dwt in leaves, below the limit of quantification to 50 ng/g dwt in roots, below the limit of quantification to 125 ng/g dwt in flowers, and below the limit of quantification to 14 ng/g dwt in grain.

AHA. proteins are ubiquitous proteins present in bacteria, fungi, algae and plants. The AHA. protein expressed in soybean event CV127 is highly similar to AHA. proteins found in many crop species, including canola, soybean, corn and wheat. The source of the AtAHA. protein expressed in soybean event CV127, A. thaliana, is not a known human or animal pathogen and is not known to cause allergic reactions in humans.

BASF Canada Inc. provided a bioinformatics evaluation of the AtAHA. amino acid sequence, which confirmed the lack of relevant similarities between the AtAHA. amino acid sequence and sequences of known allergens and toxins. In vitro digestive fate studies showed that the AtAHA. protein is rapidly degraded in simulated gastric fluid unlike protein allergens, which are normally resistant to digestion. The AtAHA. protein produced in soybean event CV127 is not glycosylated, unlike many known allergens, providing additional evidence that the AtAHA. protein expressed in soybean event CV127 does not have the properties of known allergens.

In addition to the csr1-2 gene cassette, the DNA used in the transformation of soybean event CV127 contained the coding and regulatory sequences for the gamma subunit of the SEC61 protein from A. thaliana (AtSEC61γ). The SEC61 protein is a multimeric transport protein of the endoplasmic reticulum that is ubiquitous in all plants and other eukaryotes. DNA sequencing revealed that the insert in soybean event CV127 is truncated 18 bp upstream from the 5' UTR of the AtSEC61γ gene, indicating that it is unlikely that an intact promoter is present.

Experiments were performed to determine whether the AtSEC61γ gene is expressed in soybean event CV127. The results showed that the AtSEC61γ gene is weakly transcribed in soybean event CV127, but in an immunoblot analysis, the AtSEC61γ subunit protein could not be detected in leaves or grain of soybean event CV127. The AtSEC61γ subunit protein does not possess any attributes of known allergens or toxins, and the source of the AtSEC61γ protein, A. thaliana, is not a known human or animal pathogen and is not known to cause allergic reactions in humans. AtSEC61γ is homologous to SEC61γ proteins present in crop plants with a history of safe use in food and feed. It does not share sequence similarity with any known allergens or toxins. It is also rapidly digested in simulated gastric fluid, which is uncharacteristic of known allergens. Therefore, given that levels of AtSEC61γ protein in soybean event CV127 are expected to be low, if present at all, and that AtSEC61γ does not demonstrate any toxic or allergenic characteristics, the introduction of the AtSEC61γ gene in soybean event CV127 does not present a safety concern.

3. Stable Integration into the Plant Genome

Based on Southern blot analysis and sequence data, it was determined that a single intact copy of the csr1-2 gene cassette has been inserted in soybean event CV127. In addition, a single copy of the entire coding sequence and the intact 5' and 3' untranslated regions of the AtSEC61γ subunit gene has also been inserted. A 376 bp repeat of the coding sequence from the csr1-2 gene is also integrated at the 3' junction between the insert and the flanking soybean genomic sequence. This insertion created a new open reading frame of 501 bp that extends into the flanking soybean genomic DNA; however RT- PCR experiments indicated no detectable transcription of this open reading frame.

The stability of the inserted DNA was demonstrated by Southern blot analysis across four generations of selfed soybean event CV127. The results of the analysis are consistent with the finding of a single locus of insertion that is stably inherited across generations. The inheritance pattern of the csr1-2 gene and imidazolinone tolerant phenotype has been studied in two generations of soybean event CV127. Trait inheritance studies confirmed the expected trait segregation ratios, further confirming that the imidazolinone tolerance trait in soybean event CV127 is conferred by a single functional copy of the csr1-2 gene that segregates according to Mendelian laws of genetics.

IV. Criteria for the Environmental Assessment

1. Potential of Soybean Event CV127 to Become a Weed of Agriculture or be Invasive of Natural Habitats

The biology of soybean, described in the CFIA biology document BIO1996-10, "The Biology of Glycine max (L.) Merr. (Soybean)", indicates that unmodified plants of this species are not invasive of unmanaged habitats in Canada due to traits such as the lack of seed dormancy and the poor competitive ability of seedlings.

Soybean event CV127 was tested in seven locations in Brazil in the 2006/2007 growing season and at six locations in Brazil during the 2007 short growing season . The following agronomic characteristics were evaluated: seed germination, dormancy, initial plant stand, final plant stand, seedling vigour, plant height, green stem, pod shattering, degree of lodging, days to full flower, days to full maturity, seed size (weight of one hundred seeds), and grain yield. The agronomic characteristic data showed no biologically meaningful differences between soybean event CV127 and the unmodified control (an unmodifed soybean variety that shares the same genetic background as soybean event CV127), or a selection of conventional soybean varieties, and support a conclusion of phenotypic equivalence to conventional soybean varieties. No significant differences were detected in percent germinated seed, percent dead seed, and percent viable firm swollen seed. Viable hard seeds (defined as seeds that did not imbibe water) were detected in soybean event CV127 as well as in the unmodified control at less than 1% total seed (viable hard seed is associated with seed dormancy).

The introduction of the imidazolinone tolerance trait did not make soybean event CV127 weedier or more invasive of natural habitats since none of the soybean's growth characteristics were modified. Reproductive characteristics and soybean event CV127's tolerance to abiotic and biotic stresses were unchanged as well (further detail provided in Section 2: Potential for Gene Flow from Soybean Event CV127 to Sexually Compatible Plants Whose Hybrid Offspring May Become More Weedy or More Invasive and Section 3: Altered Plant Pest Potential of Soybean Event CV127, respectively). No competitive advantage was conferred to soybean event CV127, other than that conferred by tolerance to imidazolinone herbicides.

Cultivation of soybean event CV127 would require implementation of a herbicide tolerance management plan specific to imidazolinone. Since soybean event CV127 is not intended to be cultivated in Canada, a herbicide tolerance management plan specific to this product is not required.

Soybean event CV127 is not intended to be cultivated in Canada and seed will not be authorized for sale in Canada to ensure its intended use. Therefore, environmental exposure to soybean event CV127 plants will be minimal.

Based on the above information, the CFIA has concluded that soybean event CV127 is unlikely to become a weed of agriculture or be invasive of natural habitats as a result of incidental environmental release.

2. Potential for Gene Flow from Soybean Event CV127 to Sexually Compatible Plants Whose Hybrid Offspring May Become More Weedy or More Invasive

Soybeans exhibit a high percentage of self-fertilization. Cross pollination is usually less than one percent, especially when plants are more than one metre apart, and pollen flow from cultivated soybeans to other soybeans or to related species is minimal.

BASF provided information regarding floral development in soybean event CV127, including days to full flowering and pollen characteristics. These observations were taken at the same locations as the other agronomic characteristics described earlier. No significant differences in time to full flowering, pollen quantity per flower, percent pollen germination, or pollen tube length were detected between soybean event CV127 and the unmodified control. This indicates that the reproductive characteristics of soybean event CV127 are unaltered with respect to those of unmodified soybean.

Natural hybridization between cultivated soybean and the wild annual species Glycine soja has been documented. G. soja is occasionally cultivated in research plots in North America, but is not naturalized here and there are no reports of its escape from such plots to unmanaged habitats. The imidazolinone tolerance trait in soybean event CV127 would not confer a fitness advantage in the absence of imidazolinone herbicides, in the event that a natural hybrid occurred between soybean event CV127 and G. soja. No other sexually compatible species are present in North America.

Soybean event CV127 is not intended to be cultivated in Canada and seed will not be authorized for sale in Canada to ensure its intended use. Therefore, environmental exposure to soybean event CV127 plants will be minimal.

Based on the above information, the CFIA has concluded that the potential for gene transfer from soybean event CV127 to soybean relatives in managed ecosystems is negligible, and that there is no potential for gene transfer to related sexually compatible plant species in Canada as a result of incidental environmental release.

3. Altered Plant Pest Potential of Soybean Event CV127

The novel trait (tolerance to imidazolinone herbicides) is unrelated to plant pest potential (i.e. the potential for the plant to harbour new or increased populations of pathogens or pests). Ecological evaluations of soybean event CV127 did not show any increase or decrease in susceptibility to any insect or disease stressor that was outside of the range of conventional soybean varieties grown at the same locations.

Symptom severity from five pathogens was evaluated at four times during the growing season at 13 location-years. No meaningful differences between soybean event CV127 and the unmodified control were detected. The pathogens included Asian soybean rust (Phakopsora pachyrhizi), downy mildew (Peronospora manshurica), powdery mildew (Erysiphe diffusa), brown spot (Septoria glycines), and soybean leaf spot (Cercospora kikuchii).

Insect pest abundance was also observed at four times during the growing season at 13 location-years. The insects in each soybean plot were identified to the taxonomic rank of Order, but based on the familiarity with this herbicide tolerance trait and other components of the assessment (e.g. lack of toxicity, history of safe use, etc.) it is unlikely that any significant differences could be expected at a taxonomic rank of greater precision (e.g. family, genus, or species). The insect Orders that were assessed included Coleoptera (e.g. Diabrotica, Aracathus), Lepidoptera (Anticarsia, Agrotis, Pseudoplusia, Spodoptera) and Hemiptera (Euschistus, Piezodorus, Nezara). Damage caused by defoliation or fluid feeding from insect pests was also observed. Leaf, stem, and pod damage from insect feeding was below 2% for soybean event CV127 and the unmodified control.

Soybean event CV127 is not intended to be cultivated in Canada and seed will not be authorized for sale in Canada to ensure its intended use. Therefore, environmental exposure to soybean event CV127 plants will be minimal.

Based on the above information, the CFIA has concluded that soybean event CV127 would not be expected to display altered plant pest potential as a result of incidental environmental release.

4. Potential Impact of Soybean Event CV127 on Non-Target Organisms

The novel herbicide tolerance trait introduced into soybean event CV127 is unrelated to potential impact on non-target organisms interacting with soybean. Soybean event CV127 expresses an AtAHA. protein that differs from the wild type AHA. from A. thaliana by two amino acid substitutions. The AtAHA. protein expressed in soybean event CV127 is closely related to the AHA. proteins of crop plants that have a long history of safe use, such as soybean, wheat and corn. In addition, data provided by BASF Canada Inc. confirmed that the At AHA. protein expressed in soybean event CV127 does not possess any attributes of known allergens or toxins. Therefore, the expression of the AtAHA. protein is very unlikely to cause negative impacts on organisms interacting with soybean event CV127.

BASF Canada Inc. showed that the gamma subunit of the SEC61 protein from A. thaliana (AtSEC61γ) is not expressed at detectable levels in soybean event CV127 leaves and grain. The SEC61 protein is a multimeric transport protein of the endoplasmic reticulum that is ubiquitous in all plants and other eukaryotes. In addition, BASF Canada Inc. showed that the AtSEC61 γ protein does not possess any attributes of known food allergens or toxins. Therefore, in the unlikely event that the AtSEC61 γ protein is present in soybean event CV127 tissues at very low levels, this protein would not pose any environmental safety concerns.

The detailed compositional analysis showed that the levels of key nutrients and anti-nutrients in soybean event CV127 grain and forage are comparable to those in soybean varieties cultivated in Brazil. In addition, investigation of the soybean allergen content in soybean event CV127 seeds using two-dimensional gel electrophoresis showed that soybean event CV127 seeds do not contain increased levels of endogenous allergens compared to the unmodified control. In addition, broiler chickens were not adversely affected by the consumption of a diet containing 30-40% toasted meal from soybean event CV127. Collectively, these results indicate that the composition of soybean event CV127 seeds is similar to that of conventional soybean seeds. Therefore, it is very unlikely that the genetic transformation may have caused unintended changes to the composition of soybean event CV127 tissues that would negatively impact organisms interacting with soybean event CV127.

In addition, field evaluations of soybean event CV127 did not show any increase in resistance to soybean pathogens commonly found in Brazil compared to conventional soybean varieties. The numbers of coleopteran, lepidopteran and hemipteran insects collected on soybean event CV127 plants showed no biologically meaningful difference from numbers collected on the unmodified control and two other conventional soybean varieties (see Section 3: Altered Plant Pest Potential of Soybean Event CV127).

Soybean interacts with a plant-associated nitrogen-fixing bacterium (Bradyrhizobium japonicum), which results in the fixation of atmospheric nitrogen to a plant-available form inside root nodules. Soybean event CV127 was compared with the unmodified control and two other conventional soybean varieties with respect to interaction with B. japonicum and nitrogen fixation capacity. No biologically meaningful differences were observed between soybean event CV127 and the unmodified control or the conventional soybean varieties, indicating that the symbiotic relationship between the bacterium B. japonicum and soybean event CV127 was not altered by the introduction of the novel trait.

Soybean event CV127 is not intended to be cultivated in Canada and seed will not be authorized for sale in Canada to ensure its intended use. Therefore, environmental exposure to soybean event CV127 plants will be minimal.

Based on the above information, the CFIA has concluded that soybean event CV127 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 CV127 on Biodiversity

Soybean event CV127 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 imidazolinone herbicide tolerance trait from soybean event CV127 to plants in unmanaged environments.

Soybean event CV127 does not pose a risk to interacting with non-target organisms. In addition, BASF Canada Inc. provided data showing that the introduction of the imidazolinone tolerance trait does not alter the symbiotic relationship between the bacterium B. japonicum and soybean event CV127 compared to conventional soybean.

Soybean event CV127 is not intended to be cultivated in Canada and seed will not be authorized for sale in Canada to ensure its intended use. Therefore, environmental exposure to soybean event CV127 plants will be minimal.

Based on the above information the CFIA has concluded that the potential impact on biodiversity 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 CV127, 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 CV127 meet the definitions and requirements of feeds as listed in Schedule IV of the Feeds Regulations.

1. Potential Impact of Soybean Event CV127 on Livestock Nutrition

Nutrient and anti-nutrient composition:

The compositional equivalence of soybean event CV127 to its unmodified control and other conventional soybean varieties was assessed from several sites in Brazil during the 2006/2007 and 2007/2008 growing season. Seed samples were collected from replicated plots over two seasons and analysed for proximate (protein, crude fat, moisture and ash), crude fibre (CF), acid detergent fibre (ADF), neutral detergent fibre (NDF), total dietary fibre (TDF), amino acids, fatty acids, minerals, vitamins, isoflavones (diadzein, genistein and glycitein), phospholipids and anti-nutrients (phytic acid, lectin, urease, trypsin inhibitor, raffinose and stachyose). Forage samples collected in 2007/2008 showed no statistically significant differences between soybean event CV127 and the unmodified control for proximate, ADF and NDF. Statistically significant differences were observed between soybean event CV127 seeds and the unmodified control for protein (season 1 only), fat, ADF, NDF, however all the means were within the range of conventional soybean varieties. No statistically significant differences were observed between soybean event CV127 and unmodified control for all amino acids analyzed (season 1). On the other hand, significant differences were found between soybean event CV127 and the unmodified control for alanine, histidine, and tyrosine in season 2. All means for these amino acids were within the range of conventional soybean varieties or published literature values.

Except for palmitic acid (season 1), stearic acid (season 2), oleic, linoleic, linolenic acids (both seasons), no statistically significant differences were observed between soybean event CV127 and the unmodified control for the other fatty acids. All means for these nutrients in soybean event CV127 were within the range of conventional soybean varieties or literature values. Statistically significant differences were observed between soybean event CV127 and the unmodified control for iron (season 1), phosphorus (season 2), magnesium and potassium (both seasons), however all means were within conventional soybean varieties and/or literature ranges. Significant differences were observed between soybean event CV127 and the unmodified control soybeans for all vitamins (except total tocopherol) in season 1 and for tocopherols, vitamin B1 and E in season 2. All means were however within conventional soybean varieties and/or literature ranges. Total diadzein, genistein and glycitein were statistically significantly lower in soybean event CV127 seed compared to the unmodified control, but the means of soybean event CV127 were within conventional soybean varieties and/or literature ranges. Except for phosphatidyl inositol and phosphatidyl choline (season 2), statistically significant differences were observed between soybean event CV127 and the unmodified control for all other phospholipids measured however the means were within those of the conventional soybean varieties ranges. No statistically significant differences were observed between soybean event CV127 and the unmodified control defatted- toasted meal for proximates, ADF and NDF.

There were no statistically significant differences observed between soybean event CV127 seed and the unmodified control for phytic acid, lectin, urease and trypsin inhibitor. Statistically significant differences were observed between soybean event CV127 and the unmodified control for raffinose and stachyose in both seasons, however the means were within conventional soybean ranges. For soybean meal, no statistical significant differences were observed between soybean event CV127 and the unmodified control soybean meal for raffinose, stachyose, lectin, urease and phytic acid. Trypsin inhibitor levels in soybean event CV127 soybean meal were significantly higher than the unmodified control, but comparable to reported literature ranges.

Broiler chicken performance:

To assess the wholesomeness of soybean meal from soybean event CV127, a 42-day broiler feeding study using 576 birds was undertaken. Soybean event CV127 and three conventional soybeans (Conquista (isoline), Monsoy 8001 and Coodetec 217) were grown at a site in Brazil during the 2006/2007 growing season. Soybean meal from the various soybean varieties (144 birds per treatment) were incorporated at 40% in the initial to 30% in the final stages of the trial. There were no statistically significant differences between birds fed feed containing soybean event CV 127 meal and those fed meal from the conventional varieties in terms of body weight gain, feed intake and feed conversion efficiency.

Conclusion

The evidence provided by BASF supports the conclusion that the nutritional composition of soybean event CV127 is compositionally equivalent to conventional soybean varieties. No negative effects were observed on the health or performance of broilers consuming CV127 soybean diets.

2. Potential Impact of Soybean Event CV127 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 csr1-2 gene encodes an AtAHA. enzyme that contains a single amino acid change that results in tolerance of the enzyme to imidazolinone herbicides. The assessment of soybean event CV127 evaluated the impact of the following potential hazards relative to the safety of feed ingredients derived from this event:

• The presence of novel protein AtAHA.
• The chemical pesticide residue profile.

Novel AtAHA. protein

AHA. enzymes are found in a wide variety of plants and micro-organisms. The AtAHA. protein in CV127 shares no significant biologically relevant sequence homology with any known toxins or allergens and lacks modes of action that suggest it may be intrinsically toxic. This protein is also heat labile and rapidly degraded under conditions similar to those encountered in the gastrointestinal tract. No adverse effects were noted in a single-dose oral toxicity study in mice, using a similar AHA. purified from E. coli, at 2620 mg/kg-bw. These factors support the lack of intrinsic toxicity of the AHA. protein.

Chemical pesticide residue profile

The safety of herbicide residues and metabolites in soybean event CV127, following application of herbicides, was also evaluated as part of the feed safety assessment.

Herbicide tolerant soybean for use with imazapyr or imazapic has not at this time been the subject of any prior AFD Decisions. The safety of imazapyr and imazapic residues in feed ingredients after the application of imazapyr or imazapic to soybean event CV127 has not been fully assessed, as there were no regulatory points of reference available at the time of the evaluation. The AFD has therefore placed a provisional restriction on feed commodities derived from soybean event CV127 following the application of imazapyr or imazapic until an authorization for imazapyr or imazapic application on soybean event CV127 has been granted by Health Canada's Pest Management Regulatory Agency (PMRA).

Conclusions

Feed ingredients derived from soybean event CV127, without the application of imazapyr or imazapic are considered to meet present ingredient definitions for soybean and as such are approved for use as livestock feed in Canada.

VI. New Information Requirements

If at any time BASF Canada Inc. becomes aware of any information regarding risk to the environment, including risk to human or animal health, which could result from release of soybean event CV127 in Canada or elsewhere, BASF Canada Inc. will immediately provide such information to the CFIA. On the basis of such new information, the CFIA will re-evaluate the potential impact of soybean event CV127 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 CV127.

VII. Regulatory Decision

Based on the review of the data and information submitted by BASF Canada Inc. and other relevant information, the Plant and Biotechnology Risk Assessment Unit of the Plant Health Science Directorate, CFIA has determined that soybean event CV127 does not present altered environmental risk when compared to currently commercialized soybean varieties in Canada.

Based on the review of data and information submitted by BASF Canada Inc., including comparisons of soybean event CV127 with an unmodified control, the Animal Feed Division of the Animal Health Directorate, CFIA, has concluded that the novel gene and its corresponding trait will not confer to soybean event CV127 any characteristic that would raise any concerns regarding the safety or nutritional composition of soybean event CV127. 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 CV127 has been found to be as safe as and as nutritious as traditional soybean varieties.

Please note that the safety of imazapyr and imazapic residues in feed ingredients after the application of imazapyr and imazapic to soybean event CV127 was not fully assessed. Any feed ingredient(s) derived from the combination of soybean event CV127 and imazapyr or imazapic may not be manufactured or sold in Canada, or imported into Canada, until an authorization for imazapyr or imazapic application on soybean event CV127 has been granted by Health Canada's Pest Management Regulatory Agency (PMRA).

Recognizing that incidental release into the environment could occur when this PNT is used as intended, unconfined release^Footnote 1 into the environment^Footnote 2 of soybean event CV127 and of any lines derived from it are therefore authorized by the Plant Biosafety Office of the Plant Health and Biosecurity Directorate as of November 2, 2012 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.

Use as livestock feed of soybean event CV127 and of any lines derived from it is authorized by the Animal Feed Division of the Animal Health Directorate on the condition that they meet the restrictions specific to imazapyr and imazapic treated feeds set out in the this authorization.

In addition to the above conditions, authorizations of the soybean lines derived from soybean event CV127 are conditional on the following: (i) no inter-specific crosses are performed, (ii) the intended use(s) are similar, (iii) it is known, following thorough characterization, that these plants do not display any additional novel traits and are substantially equivalent to soybean event CV127 in terms of their specific use and safety for the environment, including human health, as well as livestock safety and (iv) the novel gene is expressed at a level similar to that of the authorized line.

Soybean event CV127 is subject to the same phytosanitary import requirements as its unmodified counterparts. Soybean event CV127 is required to meet the requirements of other jurisdictions; including but not limited to, the Food & Drugs Act, and the Pest Control Products Act.