Novel Food Information - Soybean Producing Aryloxyalkanoate dioxygenase-12 (AAD-12) and phosphinothricin acetyltransferase (PAT) - DAS-68416-4
Health Canada has notified Dow Agrosciences Canada, Inc. that it has no objection to the food use of herbicide tolerant soybean event DAS68416-4. The Department conducted a comprehensive assessment of this soybean event 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.
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.
DAS-68416-4 soybean produces aryloxyalkanoate dioxygenase-12 (AAD-12), an enzyme which confers resistance to 2,4-dichlorophenoxyacetic acid (2,4-D), and phosphinothricin acetyltransferase (PAT), which inactivates the herbicide phosphinothricin (glufosinate). The herbicide tolerance was achieved through transformation of a conventional soybean variety.
The assessment performed by Food Directorate evaluators determined: how DAS-68416-4 soybean was developed; how the composition and nutritional quality of DAS-68416-4 soybean compared to non-modified varieties; and what the potential is for DAS-68416-4 soybean to be toxic or cause allergic reactions. Dow Agrosciences has provided data that demonstrates DAS-68416-4 soybean is as safe and of the same nutritional quality as traditional soybean varieties used as food in Canada.
The Food Program has a legislated responsibility for pre-market assessment of novel foods and novel food ingredients as detailed in Division 28 of Part B of the Food and Drug Regulations (Novel Foods). Foods derived from DAS-68416-4 soybean are considered novel foods 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
- the plant, animal or microorganism exhibits characteristics that were not previously observed in that plant, animal or microorganism."
2. Development of the modified plant
DAS-68416-4 soybean was genetically modified using Agrobacterium tumefaciens-mediated DNA transfer of a transformation plasmid. The transferred DNA (T-DNA or insert) region of the plasmid contains the plant-optimized coding sequences for both the aad-12 and pat genes as well as regulatory elements to control expression. Tissue isolated from germinated soybean seeds from the elite "Maverick" cultivar line was infected with the Agrobacterium containing the transformation plasmid. This tissue was screened for the herbicide resistance trait and used to regenerate whole plants, which were further screened to confirm resistance to both glufosinate and 2,4-D.
3. Characterization of the Modified Plant
Southern blot analysis of DAS-68416-4 was presented to demonstrate that an intact copy of the gene cassette was inserted at a single locus in the soybean genome. The integrity of the aad-12 and pat genes and regulatory elements was shown, and the absence of plasmid backbone DNA, including the antibiotic resistance gene, in DAS-68416-4 was confirmed. The insertion locus was also sequenced to confirm that the inserted sequence was exactly as expected and that no endogenous genes were potentially disrupted by the insertion. The stability and inheritance of the inserted cassette was also evaluated in several generations of DAS-68416-4 by Southern blot analysis. The results demonstrated the apparent molecular stability of the inserted cassette following a traditional breeding program. As well, the genetic segregation data showed that the AAD-12 and PAT traits were inherited and expressed in the fraction of progeny plants expected for a single locus model.
The identity of the AAD-12 protein was confirmed using mass spectroscopy techniques, peptide fingerprinting, N- and C-terminal sequencing, electrophoresis, and immunological staining. PAT identity was ascertained using electrophoresis and immunological staining.
The expression levels of soluble, extractable AAD-12 and PAT in DAS-68416-4 plant tissues were determined using validated quantitative enzyme-linked immunosorbent assays (ELISAs) on samples of plants grown in the field. All tissues tested positive for both AAD-12 and PAT in this assay, including leaf, root, forage, and grain. The portion of the plant that enters the food supply is the grain, which is subject to processing. Grain harvested from DAS-68416-4 plants contained on average 16.52 ng AAD-12 /mg of dry weight tissue (range: 16.21 - 16.94 ng/mg) and 2.77 ng PAT/mg of dry weight tissue (2.73 - 2.82 ng/mg). The levels of AAD-12 and PAT appear to be in a similar range in plants treated with different combinations of the herbicides glufosinate and 2,4-D.
4. Product Information
DAS-68416-4 soybean differs from traditional soybean by the addition of the coding sequences of aad-12 derived from Delftia acidovorans and pat derived from Streptomyces viridochromogenes. Both donor species are soil bacteria that are not known to be human pathogens. Gene regulatory elements are included in the inserted DNA and control expression of the two enzymes. The aad-12 gene in DAS-68416-4 is identical to its counterpart in D. acidovorans with the following exceptions: codons were modified to optimize gene expression in plants without altering the amino acid sequence; and an additional alanine residue was inserted at position 2 to facilitate cloning. The pat gene was also modified by silent mutations to optimize codons for plant expression without altering the amino acid sequence. Expression of AAD-12 imparts resistance to 2,4-dichlorophenoxyacetic acid (2,4-D) by catalyzing its degradation into herbicidally-inactive phenols. PAT in turn catalyzes the inactivation of phosphinothricin (glufosinate) herbicide. The PAT trait from S. viridochromogenes has been reviewed in eight previously approved agricultural lines.
5. Dietary Exposure
The genetic modification of DAS-68416-4 soybean is not intended to alter soybean consumption patterns. Therefore, the use of herbicide tolerant DAS-68416-4 soybean and products derived from it will be similar to traditional soybean varieties. The introduction of DAS-68416-4 soybean will only replace a fraction of current soybean varieties, and it is not anticipated to result in a change in the dietary intake of soybean and soybean-derived products.
Information on the nutritional composition and anti-nutrient levels in DAS-68416-4 soybean and non-transgenic control samples were reviewed from test crops grown in five major growing regions across North America. The data was collected using an appropriate study design and accepted analytical methods.
The nutritional and compositional analytes measured in the soybean samples included: proximate (moisture, protein, fat, carbohydrate, acid detergent fibre, and neutral detergent fibre), total dietary fibre, amino acids (18), fatty acids (22), minerals (13), vitamins (10, including 4 vitamin E tocopherol forms), anti-nutrients (trypsin, lectin, stachyose, raffinose, and phytic acid), and isoflavones (glycosides, including daidzin, genistin, glycitin, and aglycones such as daidzein, genistein, glycitein, and total aglycone equivalents).
Of all the analytes measured, the following showed a statistically significant (p<0.05) overall treatment effect: protein (lower in DAS-68416-4), carbohydrate (higher), potassium (higher), amino acids (11 lower), fatty acids (4 lower), gamma-tocopheral (higher), folic acid (lower), and glycitin expressed as the aglycone equivalent glycitein (higher). These differences between the transgenic DAS-68416-4 and non-transgenic control, however, are acceptable for two reasons. First, the analyte values for protein, carbohydrate, amino acids, fatty acids, and folic acid were within the published ranges for soybean. It was noted that for glycitin (expressed as the aglycone equivalent glycitein), the level was significantly higher than control, however the total glycitein equivalent (including both glycitein and glycitin expressed as aglycone equivalent) was within published ranges. The second rationale applies to the differences observed for potassium, palmitoleic fatty acid, and gamma-tocopherol. For these analytes, the observed differences were too small to impact dietary intakes and pose a nutritional safety concern. It was concluded that the petitioner demonstrated that DAS-68416-4 soybean had similar composition to its non-transgenic comparator.
In terms of chemical safety considerations, it is expected that DAS-68416-4 soybean would be no different from conventional soybean, assuming that its ability to uptake heavy metals and its susceptibility to mycotoxin-producing fungi have not been altered relative to conventional soybean.
The novel aad-12 and pat genes present in DAS-68416-4 were not derived from pathogens. The AAD-12 protein is not considered likely to be a toxin based on acute toxicity studies in mice which were administered the protein at levels orders of magnitude greater than the hypothetical intake level from this crop in the human diet. Furthermore, AAD-12 bears no biologically significant sequence similarity to any known toxin. Plants expressing PAT have been previously reviewed by Health Canada, including soybean varieties. Evidence for safety of PAT was provided through a rationale for familiarity with the trait, with reference to previous toxicity studies and updated database searches for potential toxins and allergens, which raised no issues of concern.
AAD-12 is also not considered to be an allergen as it does not share characteristics with food allergens. The amino acid sequence of AAD-12 does not bear similarity to any known allergens. AAD-12 is quickly degraded in simulated gastric fluid as well as at high temperatures similar to those that would be used during industrial processing and cooking. This suggests that an intact protein would not likely be consumed. It is unlikely that DAS-68416-4 soybean containing a small amount of AAD-12 protein would pose an allergenic risk to human health.
Health Canada's review of the information presented in support of the food use of DAS-68416-4 soybean does not raise concerns related to food safety. Health Canada is of the opinion that food derived from DAS-68416-4 is as safe and nutritious as food from current commercial soybean varieties.
Health Canada's opinion deals only with the food use of DAS68416-4 soybean. Issues related to its use as animal feed have been addressed separately through existing regulatory processes in the Canadian Food Inspection Agency.
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.
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For further information, please contact:
Novel Foods Section
Health Products and Food Branch
Health Canada, PL2204A1
251 Frederick Banting Driveway
Ottawa, Ontario K1A 0K9
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