Novel Food Information - Herbicide Tolerant Cotton DAS-81910-7
Health Canada has notified DOW AgroSciences Inc. that it has no objection to the sale of food derived from Herbicide Tolerant Cotton DAS-81910-7. The Department conducted a comprehensive assessment of this cotton 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 Inc. and the evaluation by Heath Canada. This document contains no confidential business information.
DOW AgroSciences Inc., developed Herbicide Tolerant Cotton DAS-81910-7 (hereafter DAS-81910-7) using recombinant DNA techniques to introduce the coding sequence for the aryloxylkanoate dioxygenase (aad-12) protein derived from Delftia acidovorans and the coding sequence for phosphinothricin acetyl-transferase (pat) derived from Streptomyces viridochromogenes. As a result, DAS-81910-7 expresses both the AAD-12 and PAT proteins. The expressed AAD-12 protein catalyzes the conversion of 2,4-D to 2,4-Dichlorophenol (DCP) a herbicidally inactive compound. The expressed PAT protein acetylates L-phosphinothricin, the active compound in glufosinate herbicides, rendering it inert.
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 reflects international guidance documents in this area (eg., Codex Alimentarius). The assessment considered: how DAS-81910-7 was developed; how the composition and nutritional quality of DAS-81910-7 compared to non-modified cotton varieties; and what the potential is for DAS-81910-7 to be toxic or cause allergic reactions. DOW AgroSciences Inc. has provided data which demonstrates that DAS-81910-7 is as safe and of the same nutritional quality as traditional cotton 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 Division 28 of Part B of the Food and Drug Regulations (Novel Foods). Foods derived from DAS-81910-7 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
- (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 Herbicide Tolerant DAS-81910-7 cotton (hereafter referred to as DAS-81910-7) and molecular biology data that characterize the genetic change that confers herbicide tolerance. DAS-81910-7 was produced using Agrobacterium tumefaciens (A. tumefaciens) mediated transformation of cotton variety Coker 310 with the transformation vector pDAB4468. This transformation vector was constructed to contain a single transfer DNA (T-DNA) region, which contained two gene expression cassettes. The pDAB4468 transformation plasmid used to transform DAS-81910-7 is the same plasmid used in the transformation of the previously approved DAS-68416-4 soybean variety.
The elements contained in the pDAB4468 T-DNA were as follows: the matrix attachment region from Nicotiana tobacum (RB7-MAR), the Promoter, 5’ untranslated region and intron from the Arabidopsis thaliana polyubiquitin UBQ10 gene (AtUBI10), the Synthetic plant optimized version of an aryloxyalkanoate dioxygenase from Delftia acidovorans (aad-12), the 3’ untranslated region comprising the transcriptional terminator and polyadenylation site of the open reading frame 23 (ORF23) of Agrobacterium tumefaciens pTi15955 (AtuORF23), the promoter and 5’ untranslated region derived from the cassava vein mosaic virus (CsVMV), the synthetic, plant optimized version of the phophinothricin N-acetyl transferase gene from Streptomyces viridochromogenes (pat) and the 3’ untranslated region comprising the transcriptional terminator and polyadenylation site of the open reading frame 1 (ORF1) of Agrobacterium tumefaciens pTi15955 (AtuORF1).
The first expression cassette contains the coding region that confers tolerance to 2,4-D herbicides. This coding region produces an AAD-12 protein derived from Delftia acidovorans. The gene, aad-12, is a synthetic copy of the native coding sequence that has been codon optimized for expression in plants. This coding sequence results in an AAD-12 protein that is 80% homologous to the wild-type protein.
The second expression cassette contains the coding region that confers tolerance to PAT herbicides. This coding region produces a PAT protein derived from Streptomyces viridochromogenes. The gene, pat, is copy of the native coding sequence that has been codon optimized for expression in plants. Numerous previously reviewed genetically modified crops have used PAT as the source of glufosinate herbicide tolerance and no evidence, to date, has shown it be unsafe for human consumption.
Cotton seeds were germinated on basal media and hypocotyl segments were isolated and infected with A. tumefaciens carrying the pDAB4468 plasmid. The hypocotyl segments were then grown on a series of media containing carbenicillin and glufosinate ammonium to eliminate excess A. tumefaciens and untransformed cells. Gene specific PCR was performed on embrogenic calli to identify transgenic lines containing both genes. Calli identified as containing both genes were then transferred to culture media that stimulates root regeneration. Rooted plants (T0) where then transferred to soil mixtures in high humidity growth chambers for 2-4 weeks before being transferred to the greenhouse.
3. Characterization of the Modified Plant
The petitioner used Southern blots to confirm the number of copies of each functional element within the insert. To do this, the petitioner used a variety of restriction enzymes and six different probes, each specific for one or more functional elements in the T-DNA. For all the Southern blots the petitioner provided the testing results for the T2, T3, T4, T5 and BC1F2 generations of DAS-81910-7 each done in triplicate. A negative control of the non-transformed cotton variety Coker 310 was included in each Southern. Also included was a positive control of Coker 310 genomic DNA spiked with pDAB4468 plasmid DNA. The Southern blot data presented by the petitioner demonstrated that presence of a single, intact copy of the T-DNA region.
The petitioner also performed a series of Southern blots to demonstrate the absence of any plasmid backbone sequences. Using 4 separate probes covering the entire plasmid backbone, the petitioner probed DAS-81910-7 genomic DNA. From the evidence presented by the petitioner no plasmid backbone has been transferred into DAS-81910-7.
Generational stability of the single insert was determined across five generations of DAS-81910-7 (T2, T3, T4, T5 and BC1F2). Southern blot analysis was presented for each generation confirming the presence of the T-DNA cassette and its stability across generations. Similarly, the petitioner performed the same methodology to demonstrate a lack of backbone sequences across generations. The evidence presented confirmed a lack of backbone sequences.
The petitioner also undertook testing on the BC1F2 generation using lateral flow strips and event specific PCR to analyze the generation to demonstrate that the insert is inherited in the expected manner for an insert at a single locus. Based on the evidence presented by the petitioner both methods demonstrated the expected segregation pattern. The petitioner also tested the T1 and BC1F2 generations using a PAT protein and AAD-12 zygosity assay. This testing demonstrated the traits were inherited in the expected ratio.
To determine if any putative reading frames were created by the integration of the T-DNA insert into DAS-81910-7, the petitioner analyzed the DNA sequence of the insert and its flanking border regions. Putative reading frames were conservatively defined as sequences that begin and end with a stop codon and are greater than or equal to eight amino acids in length.
A total of eleven putative reading frames spanning the junctions between the insert and its flanking border regions in DAS-81910-7 cotton were identified and subjected to BLASTp search for sequence similarity with known toxin proteins. The search against the GenBank non-redundant protein sequence dataset (updated March 07, 2013) did not detect any significant protein sequence similarity with toxic proteins harmful to humans or animals.
The petitioner also undertook an evaluation of the potential allergenicity of the 11 identified ORFs using two methods. As recommend by Codex Alimentarius, the first method involved a search over 80-amino-acids or longer stretches to detect >35% identity between a query protein and known allergens. Of the 11 putative proteins, 5 were identified as being below the cut-off (29 amino acids in length) for this analysis. The remaining 6 putative protein sequences showed no similarity to known allergens.
The second method employed by the petitioner involved evaluating short amino acid stretches for identity between the query protein and known allergens. Each putative reading frame was analyzed for any matches of eight contiguous amino acids and compared with the FARRP allergen database. Since all eleven putative reading frames identified in DAS-8191Ø-7 cotton were greater than or equal to eight amino acids in length, all were subjected to this analysis. None of the eleven putative reading frames analyzed against the peer reviewed FARRP allergen database generated a significant amino acid sequence similarity to known allergens.
The petitioner has conducted the toxicological assessment using AAD-12 and PAT proteins expressed in and purified from Pseudomonas fluorescens. To ensure that the results of the toxicological studies are applicable to the proteins expressed in DAS-81910-7 cotton, equivalence studies (i.e., SDS-PAGE, western blot analysis, glycoprotein staining, MALDI-TOF MS, N-terminal amino acid sequence analysis and specific enzymatic activity) were conducted to confirm that the protein produced in P. fluorescens used for toxicology studies is representative of the protein produced in the modified cotton. Based on the results of these studies, the proteins were determined to be equivalent with respect to their physical properties, immunological staining properties and sequencing. The petitioner has also demonstrated that the microbially produced proteins are equivalent to or are from the same batch as used in previously submitted toxicology studies.
4. Product Information
Herbicide Tolerant Cotton DAS81910-7 differs from conventional cotton through the introduction of the coding sequence for the aryloxylkanoate dioxygenase (aad-12) protein derived from Delftia acidovorans and the coding sequence for phosphinothricin acetyl-transferase (pat) derived from Streptomyces viridochromogenes. As a result, DAS-81910-7 expresses both the AAD-12 and PAT proteins. The expressed AAD-12 protein catalyzes the conversion of 2,4-D to 2,4-Dichlorophenol (DCP) a herbicidally inactive compound. The expressed PAT protein acetylates L-phosphinothricin, the active compound in glufosinate herbicides, rendering it inert.
A field expression study was conducted in the U.S. during 2012. Six sites (Alabama, Georgia, Louisiana, Missouri, North Carolina, and Texas) were planted with DAS-8191Ø-7 cotton and the non-transgenic control. Two treatments of the DAS-8191Ø-7 cotton (unsprayed or sprayed with 2,4-D and glufosinate-ammonium) were tested and plant tissue was collected from leaf, squares, bolls, pollen, flower, whole plant, root, and seed.
AAD-12 protein was extracted from the collected DAS-8191Ø-7 cotton tissues and the soluble, extractable AAD-12 protein from each tissue was measured using a draft enzyme-linked immunosorbent assay (ELISA) method. AAD-12 protein levels for all tissue types were calculated on ng/mg dry weight basis. The mean AAD-12 protein levels were highest in the leaf at 71.17 ng/mg followed by pollen at 70.71 ng/mg, squares at 38.33 ng/mg, flower at 30.63 ng/mg, seed at 18.75 ng/mg, bolls at 17.17 ng/mg, whole plant at 16.42 ng/mg, and root at 10.74 ng/mg. AAD-12 expression levels were comparable for both sprayed and unsprayed treatments.
Similarly, PAT protein was extracted from the collected DAS-8191Ø-7 cotton tissue and the soluble, extractable PAT protein was measured using an enzyme-linked immunosorbent assay (ELISA) method. The mean PAT protein levels were highest in the leaf at 13.29 ng/mg, followed by squares at 7.91 ng/mg, flower at 5.30 ng/mg, seed at 3.85 ng/mg, bolls at 3.16 ng/mg, root at 1.67 ng/mg, whole plant at 0.97 ng/mg and pollen at 0.11 ng/mg. PAT expression levels were comparable for both sprayed and unsprayed treatments.
5. Dietary Exposure
Humans may consume cotton products as a food, although they constitute a minor component of daily dietary intake. Cottonseed, which remains after the cotton is ginned, is used to produce cottonseed oil, which is used in shortening, margarine and cooking oils. Cotton linters are also used in the production of meat casings or as thickeners in ice cream and salad dressing. Neither of these products is used in great amounts and previous submissions have demonstrated that these products contain negligible amounts of protein. Cottonseed from DAS-81910-7 will not alter this pattern of dietary exposure.
A crop composition study with DAS-81910-7 cotton, a near-isogenic non-transgenic control and six non-transgenic commercial reference lines (3 reference varieties per site) were grown in 2012 at eight different sites in the US (one site each in Alabama, Georgia, Louisiana, Mississippi, Missouri and North Carolina and two sites in Texas). A randomized complete block design with 4 replicate plots of each entry at each site was used. DAS-81910-7 cotton (sprayed and un-sprayed with 2,4-d and glufosinate) were compared to unsprayed controls and reference varieties.
Because cotton is very susceptible to injury from 2, 4-D applications, to avoid injuring control cotton and the reference varieties, drift from 2, 4-D had to be reduced or eliminated. As a result, in the study with 2, 4-D, the test substance DAS-81910-7 which was sprayed with 2, 4-D had to be separated from the comparators. To accommodate this requirement, the study was divided into two sub-experiments. Sub-experiment 1 contained entries that did not receive 2, 4-D applications, and sub-experiment 2 contained DAS-81910-7 sprayed with 2, 4-D and glufosinate and an additional entry of DAS-81910-7 unsprayed.
These entries were arranged in a randomized complete block design within each sub-experiment, and the two sub-experiments were separated by 100 ft. to prevent herbicide drift from injuring the controls. Both sub-experiments were present at all field testing sites. Appropriate insect, weed, and disease control practices were applied to produce an agronomically acceptable crop.
The nutritional and compositional analytes measured in the DAS-81910-7 cottonseed, non-transgenic control and reference varieties were: proximate content (protein, fat, ash, moisture, carbohydrate), fiber (Acid Detergent Fiber, Neutral Detergent Fiber, total dietary fiber and crude fiber), minerals (calcium, copper, iron, magnesium, manganese, phosphorus, potassium, selenium, sodium, sulfur, and zinc), amino acids (alanine, arginine, aspartic acid, cysteine, 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 (B1, B2, B3, B6, B9, and E (alpha-tocopherol) and, beta-carotene) and anti-nutrients (dihydrosterculic acid, malvalic acid, sterculic acid, free gossypol and, total gossypol).
Of all the analytes measured, the following showed a statistically significant (p<0.05) difference in DAS-81910-7 compared to control: ash (lower in DAS 81910-7), carbohydrates (higher in DAS-81910-7), protein (lower in DAS 81910-7), calcium (higher in DAS 81910-7), copper (lower in DAS 81910-7), manganese (lower in DAS 81910-7), arginine (lower in DAS 81910-7), myristic acid (lower in DAS 81910-7), palmitic acid (lower in DAS 81910-7), palmitoleic acid (lower in DAS 81910-7), oleic acid (lower in DAS 81910-7), linoleic acid (higher in DAS 81910-7), free gossypol (lower in DAS 81910-7), total gossypol (lower in DAS 81910-7), and malvalic acid (higher in DAS 81910-7).
No trends were noted in the statistically significant differences reported above and all the analyte values were within the ranges published in the OECD 2009 cotton document, reference variety ranges and/or within literature ranges. In addition, in some cases the differences were of benefit (i.e., lower levels of the anti-nutrients free gossypol and total gossypol in DAS-81910-7). Overall, there are no nutritional concerns with the composition of DAS-81910-7.
PAT and AAD-12 proteins have been previously evaluated by Health Canada. Based on the safety of the donor organisms, the absence of acute oral toxicity at high doses, the lack of amino acid homology to known toxic proteins, and the rapid digestion of the proteins in simulated gastric or intestinal fluids, both proteins were previously concluded to be safe for human consumption at the levels of exposure.
The petitioner submitted new data which served to further support the safety of the PAT and AAD-12 proteins. The new studies included updated searches of the publicly available databases assessing the amino acid sequence similarity of these proteins to known toxins. These updated bioinformatics analyses indicated that neither the PAT nor AAD-12 proteins demonstrated significant sequence homology with known toxic proteins.
The presence of toxins endogenous to cotton was assessed for DAS-81910-7 cotton. Mean levels of gossypol and cyclopropenoid fatty acids were found to be within the range of conventional cotton and therefore did not pose a health concern at the levels present.
A conservative dietary exposure assessment based on the consumption of cotton seed commodities by the US population was provided. With this information, the difference between the estimated dietary exposure and the acute oral NOAELs for both PAT and AAD-12 proteins were calculated and was considered sufficiently large in both cases (greater than 2,000,000 times) to support the safety of DAS-81910-7 cotton.
PAT and AAD-12 proteins were evaluated previously by Health Canada. Based on the lack of homology to known allergens and the rapid digestion of the proteins in simulated gastric or intestinal fluid, both proteins were concluded to be safe for human consumption from an allergenic perspective.
New data submitted by the petitioner included updated searches of the publicly available databases assessing the amino acid sequence similarity of PAT and AAD-12 to known allergens. In addition, a new thermal stability study conducted with the AAD-12 protein. This data further supports the safety of the PAT and AAD-12 proteins. The bioinformatic analyses indicated that neither PAT nor AAD-12 proteins shared significant amino acid sequence identity with known and putative allergens or celiac-induction proteins.
Health Canada’s review of the information presented in support of the food use of Herbicide Tolerant Cotton DAS-81910-7 concluded that derived food products do not raise concerns related to safety. Health Canada is of the opinion that Herbicide Tolerant Cotton DAS-81910-7 is similar to conventional cotton in terms of being an acceptable food source.
Health Canada's opinion deals only with the human food use of Herbicide Tolerant Cotton DAS-81910-7. Issues related to the environmental safety of Herbicide Tolerant Cotton DAS-81910-7 in Canada and its use as livestock 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.
(Également disponible en français)
For further information, please contact:
Novel Foods Section
Health Products and Food Branch
Health Canada, PL2204E
251 Frederick Banting Driveway
Ottawa, Ontario K1A 0K9
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