Soybean Event DAS-44406-6

Health Canada has notified DOW AgroSciences Canada Inc. that it has no objection to the food use of herbicide tolerant soybean event DAS-44406-6. The Department conducted a comprehensive assessment of this variety according to its Guidelines for the Safety Assessment of Novel Foods. These Guidelines are based upon internationally accepted principles for establishing the safety of foods with novel traits.

Background:

The following provides a summary of the notification from DOW AgroSciences Canada Inc. and the evaluation by Heath Canada and contains no confidential business information.

1. Introduction

DOW AgroSciences Canada Inc. has developed soybean event DAS-44406-6 to be tolerant to the herbicides 2,4-dichlorophenoxyacetic acid (2,4-D), glyphosate and glufosinate. Recombinant DNA techniques were used to confer tolerance to the herbicides.

The safety assessment performed by Food Directorate evaluators was conducted according to Health Canada’s Guidelines for the Safety Assessment of Novel Foods. These Guidelines are based on harmonization efforts with other regulatory authorities and reflect international guidance documents in this area (e.g., Codex Alimentarius).  The assessment considered: how soybean event DAS-44406-6 was developed; how the composition and nutritional quality of soybean event DAS-44406-6 compared to non-modified varieties; and the potential for soybean event DAS-44406-6 to be toxic or cause allergic reactions. Dow AgroSciences Canada Inc. has provided data that demonstrates that soybean event DAS-44406-6 is as safe and of the same nutritional quality as traditional soybean varieties used as food in Canada.

The Food Directorate has a legislated responsibility for pre-market assessment of novel foods and novel food ingredients as detailed in the Food and Drug Regulations (Division 28). Food use of herbicide tolerant soybean event DAS-44406-6 is considered a novel food under the following part of the definition of novel foods: “c) a food that is derived from a plant, animal or microorganism that has been genetically modified such that

1. (i) the plant, animal or microorganism exhibits characteristics that were not previously observed in that plant, animal or microorganism.”

2. Development of the modified plant

The petitioner has provided information describing the methods used to develop DAS-44406-6 soybean and data that characterize the genetic modification that confers tolerance to the 2,4-D, glufosinate, and glyphosate herbicides through the expression of the AAD-12, 2mEPSPS, and PAT proteins.

DAS-44406-6 soybean was produced via Agrobacterium-mediated transformation of soybean cotyledonary node explants using the plasmid pDAB8264.  The T-DNA insert in the plasmid contains the 2mepsps gene from Zea mays, a synthetic plant-optimized sequence of the aad-12 gene from Delftia acidovorans, and the pat gene from Streptomyces viridochromogenes.

3. Characterization of the Modified Plant

Southern blot analysis was used to determine the number of insertion sites and copies of the integrated T-DNA as well as the presence or absence of plasmid backbone sequence.  This analysis demonstrated the T-DNA inserted at a single site and at a single locus of the soybean genome, and a single functional copy of the T-DNA was integrated.  DAS-44406-6 soybean genomic DNA probed for backbone sequences showed no detectable hybridization bands, thus DAS-44406-6 soybean does not contain backbone sequences from the transformation vector pDAB8264.

In addition to Southern blot analysis, genomic DNA extracted from DAS-44406-6 soybean was subjected to DNA sequencing analysis.  Sequencing of the insert and the flanking genomic DNA confirmed the arrangement and linkage of the various elements within the insert.  This analysis also included investigation of potential new open reading frames (ORF’s) within the soybean genome.  Bioinformatics analysis determined there to be no concerns associated with the hypothetical protein expression of new potential ORF’s formed as a result of the transgene insertion in DAS-44406-6 soybean.

Southern blot analysis was performed using five generations of DAS-44406-6 soybean.  The digested genomic DNA of DAS-44406-6 soybean showed the expected banding pattern across the generations tested.  The segregation data further confirmed the stability of the insert, showing that it segregates according to Mendelian inheritance principles.  These results were also consistent with the molecular characterization data that indicated a single genomic insertion site.

4. Product Information

Three new proteins are expected to be expressed in DAS-44406-6 soybean based on the characterization of the inserted genetic material.  The 2mepsps gene expressed in DAS-44406-6 soybean encodes a 47.5 kDa protein, the aad-12 gene encodes a 32 kDa protein, and the pat gene encodes a 21 kDa protein.

Characterization of the 2mEPSPS protein in DAS-44406-6 soybean was conducted using SDS-PAGE and Western blot analysis, N- and C- terminal sequencing analysis, MALDI-TOF mass spectrum analysis, and glycosylation analysis.  The protein analysis demonstrated that the soybean produced 2mEPSPS is equivalent to the microbe (P. fluorescens) produced protein that was used in the toxicology studies.

The expression levels of the 2mEPSPS protein in leaf, grain, root, and forage tissues of DAS-44406-6 soybean were examined.  Average expression ranged from 21.86 ng/mg dry weight in grain to 2583.46 ng/mg dry weight in V10-12 stage leaf tissue.  Expression levels were comparable for all treatments (2,4-D, glufosinate, glyphosate, and all three herbicides combined).

Characterization of the AAD-12 protein in DAS-44406-6 soybean was conducted using SDS-PAGE and Western blot analysis, MALDI-TOF mass spectrum, and glycosylation analysis.  The protein analysis demonstrated that the soybean produced AAD-12 is equivalent to the microbe (P.fluorescens) produced protein that was used in the toxicology studies.

The expression levels of the AAD-12 protein in leaf, grain, root, and forage tissues of DAS-44406-6 soybean were examined. Average expression ranged from 23.52 ng/mg dry weight in R3 stage root to 121.22 ng/mg dry weight in V10-12 stage leaf tissue.  Expression levels were comparable for all treatments (2,4-D, glufosinate, glyphosate, and all three herbicides combined).

Characterization of the PAT protein in DAS-44406-6 soybean was conducted using SDS-PAGE and Western blot analysis, and an enzyme-linked immunosorbent assay (ELISA).  The protein analysis demonstrated that the soybean produced PAT is equivalent to the microbe (E.coli) produced protein, and the characterization results are consistent with the protein expressed in other transgenic crops.

The expression levels of the PAT protein in leaf, grain, root, and forage tissues of DAS-44406-6 soybean were examined. Average expression ranged from 1.56 ng/mg dry weight in R3 stage root to 10.59 ng/mg dry weight in V10-12 stage leaf tissue.  Expression levels were comparable for all treatments (2,4-D, glufosinate, glyphosate, and all three herbicides combined).

5. Dietary Exposure

The genetic modification of DAS-44406-6 soybean is not intended to alter any of its nutritional aspects. Therefore, the use of DAS-44406-6 soybean and products derived from it will be similar to traditional soybean varieties. The introduction of DAS-44406-6 soybean will only replace a fraction of current soybean varieties and is not anticipated to result in a change in the dietary intake of soybean and soybean-derived products.

6. Nutrition

A crop composition study with DAS-44406-6 soybean, a near-isogenic non-transgenic control and six non-transgenic commercial reference lines were grown in 2010 at ten sites located in the United States.  Reference lines were of similar maturity to the control and test substance and were randomized across sites in a balanced incomplete-block design with three references at each site and each reference line present at five sites.  Plots were arranged in a randomized complete block (RCB) design, with a unique randomization at each site.  The entire trial site was surrounded by a minimum of 4 rows of non-transgenic soybean cultivar of similar maturity.  Appropriate insect, weed, and disease control practices were applied to produce an agronomically acceptable crop.  The DAS-44406-6 soybeans were either unsprayed, sprayed with 2,4-D only, sprayed with glufosinate only, sprayed with glyphosate only, or sprayed with all three herbicides.

The compositional analytes measured in the DAS-44406-6 and non-transgenic control soybean seeds were: proximate content (protein, fat, ash, moisture, carbohydrate), fiber (ADF, NDF, total dietary fiber), minerals (calcium, copper, iron, magnesium, manganese, phosphorus, potassium, selenium, sodium, zinc), amino acids (alanine, arginine, aspartic acid, cystine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), fatty acids, (caprylic, capric, lauric, myristic, myristoleic, pentadecanoic, pentadecenoic, palmitic, palmitoleic, heptadecanoic, heptadecenoic, stearic, oleic, linoleic, linolenic, γ-linolenic, arachidic, eicosenoic, eicosadienoic, eicosatrienoic, arachidonic, and behenic)vitamins (A,B1,B2,B3,B5,B6,B9,C,E, β-tocopherol, γ-tocopherol, δ-tocopherol, and total tocopherol), anti-nutrients (lectin, phytic acid, trypsin inhibitor), oligosaccharides (raffinose, stachyose), and isoflavones (total daidzein equivalent, total genistein equivalent, total glycitein equivalent). 

Results indicate that several analytes (17 of 88) in the DAS-44406-6 soybean showed a statistically significant overall treatment effect. However, these differences are acceptable for one the following reasons: the analyte values were within the ranges published in the Draft OECD 2011 Soybean Document or within ranges of additional literature reviewed by the evaluator; or the magnitude of difference was too small to impact dietary intakes and pose a nutrition safety concern.

DAS-44406-6 herbicide tolerant soybean has similar composition to its non-transgenic control. 

7. Chemistry/Toxicology

The potential toxicity of the PAT protein has previously been evaluated in several other novel foods including corn (NF 122, 75, 34, 32, 29, 26, 13), soybean (NF 207, 60), cotton (NF 183, 105, 101) and canola (NF 31, 7) crop varieties. The safety of this protein is well established therefore no additional studies need to be reviewed at this time. However, the petitioner submitted a new in-silico study for amino acid sequence similarities between the PAT protein and known toxins using the most up-to-date database (GenBank 2011). The results of this study were consistent with previous assessments and demonstrated that the amino acid sequence of the PAT protein does not match the sequence of any known toxins.

The AAD-12 protein has previously been evaluated in a soybean crop (NF-207) and no safety issues were identified from a toxicological perspective. The safety studies submitted by the petitioner for this protein were the same studies evaluated in NF-207. These included an acute oral toxicity study, an in-vitro study on its stability in simulated gastric fluid (SGF), a heat stability assay, and a search for amino acid sequence similarity with known toxins and allergens. No safety issues were identified in the previous assessment therefore the review of additional safety studies was not required. However, the petitioner submitted a new in-silico study for amino acid sequence similarities between the AAD-12 protein and known toxins using the most up-to-date database (GenBank 2011). The results of this study were consistent with previous assessments and demonstrated that the amino acid sequence of the AAD-12 protein does not match the sequence of any known toxins.

The 2mEPSPS protein has also been previously evaluated by PTAS in a cotton seed crop (NF 157) and no safety issues were identified. However the petitioner submitted new safety studies (completed in 2011) for this protein and these were reviewed by PTAS. New studies included an acute oral toxicity study, an in-vitro study on its stability in simulated gastric fluid (SGF), a heat stability assay, and a search for amino acid sequence similarity with known toxins and allergens. These new studies further supported the safety of the 2mEPSPS protein.

A margin of exposure (MOE) was calculated for each protein based on the NOAEL of acute oral toxicity studies in mice, and the estimated daily intake (EDI) from the consumption of products derived from DAS-44406-6 soybean using North American data obtained from the Continuing Survey of Food Intake for Individuals (CSFII). The MOE values for the most sensitive population (infants aged 0-2 years) were 174,825 for the PAT protein, 5,546 for the AAD-12 protein, and 16,694 for the 2mEPSPS protein. These MOE values are sufficiently large to support the safety of each protein for the proposed use.

Based on the evidence presented above, PTAS expressed no toxicological concerns regarding DAS-44406-6 soybean expressing the PAT, AAD-12 and 2mEPSPS proteins.

The allergenic safety of the PAT, AAD-12 and 2mEPSPS proteins has previously been assessed by PTAS in other genetically modified crop varieties. It was demonstrated that these proteins have no homologies with known allergens and that they are readily denatured when exposed to high temperatures and degraded in simulated mammalian gastric fluids (SGF). This suggests that even if a small amount of these proteins were to be ingested, it is unlikely that they would be absorbed into the bloodstream and cause an allergic reaction. Furthermore, new and updated safety studies on allergenicity were submitted by the petitioner. These included a heat stability study and digestibility study for the 2mEPSPS protein (as mentioned in section 4), and studies comparing the amino acid sequence of each protein to known allergens using the latest protein database (FARRP 2011). These new studies provided further evidence that the three test proteins do not pose an allergenic risk.

Since soybean is one of the 8 most allergenic foods in Canada, the petitioner conducted an additional study to determine if the genetic modification used to generate DAS-44406-6 altered the endogenous allergen content compared to a non-transgenic control. A serum sample pooled from 10 clinically reactive soy allergic patients was analyzed by SDS-PAGE and an ELISA inhibition analysis. It was demonstrated that the genetic modification of DAS-44406-6 did not alter the endogenous allergenicity of soybean in soy allergenic patients compared to the non-transgenic control.

Conclusion:

Health Canada’s review of the information presented in support of the food use of herbicide tolerant soybean event DAS-44406-6 does not raise concerns related to food safety. Health Canada is of the opinion that food derived from soybean event DAS-44406-6 is as safe and nutritious as food from current commercial soybean varieties.

Health Canada's opinion deals only with the food use of soybean event DAS-44406-6. Issues related to its use as animal feed have been addressed separately through existing regulatory processes in the Canadian Food Inspection Agency (CFIA).  From their assessment, the CFIA concluded that there are no concerns from an environmental and feed safety perspective. This perspective is applicable to the food and feed products derived from soybean event DAS-44406-6 destined for commercial sale.

This Novel Food Information document has been prepared to summarize the opinion regarding the subject product provided by the Food Directorate, Health Products and Food Branch, Health Canada. This opinion is based upon the comprehensive review of information submitted by the petitioner according to the Guidelines for the Safety Assessment of Novel Foods.

(Également disponible en français)

For further information, please contact:

Novel Foods Section
Food Directorate
Health Products and Food Branch
Health Canada, PL2204A1
251 Frederick Banting Driveway
Ottawa, Ontario K1A 0K9
novelfoods-alimentsnouveaux@hc-sc.gc.ca