Novel Food Information - Dicamba Tolerant Soybean MON 87708

Health Canada has notified Monsanto Canada Inc. that it has no objection to the sale of food derived from Dicamba Tolerant Soybean MON 87708. 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.

Background

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

1. Introduction

Monsanto Canada developed Dicamba Tolerant Soybean MON 87708 by modifying a proprietary, commercial soybean variety (A3525) using recombinant DNA techniques to introduce the dicamba mono-oxygenase (dmo) coding sequence derived from the bacterium Stenotrophomonas maltophilia. The sequence codes for the DMO protein which catalyzes the demethylation of the herbicide Dicamba to the non-herbicidal compounds 3,6-dicholorosalicylic acid (DCSA) and formaldehyde.

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 (e.g., Codex Alimentarius). The assessment considered: how Dicamba Tolerant Soybean MON 87708 was developed; how the composition and nutritional safety of this variety compares to non-modified varieties; and what the potential is for this variety to be toxic or cause allergic reactions. Monsanto has provided data to support that Dicamba Tolerant Soybean MON 87708 is safe for use 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 Dicamba Tolerant Soybean MON 87708 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

1. 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 Dicamba Tolerant Soybean MON 87708 and molecular biology data that characterizes the genetic change that confers tolerance to the herbicide dicamba. Event MON 87708 was produced using Agrobacterium-mediated transformation of commercial soybean variety A3525 with the transformation vector PV-GMHT4355.

The transformation vector PV-GMHT4355 was constructed to contain two T-DNA cassettes (T-DNA I and T-DNA II). The T-DNA I cassette contained the gene of interest, dmo, and the associated regulatory sequences. The integration of this cassette into the host genome confers the desired tolerance to Dicamba herbicides. T-DNA II cassette contained the gene cp4 epsps and its associated regulatory sequences. The integration of this cassette into the host genome confers tolerance to the herbicide glyphosate. This cassette was used by the petitioner as a selectable marker for transformed cells.

Meristem tissue of the non-transgenic soybean variety, A3525, was transformed using vector PV-GMHT4355 transferred via Agrobacterium tumefaciens. After co-culturing with the Agrobacterium carrying the vector, the meristems were placed on selection medium containing glyphosate, carbenicillin, cefotaxime, and ticarcillin/clavulanate acid mixture, to inhibit the growth of untransformed plant cells and excess Agrobacterium. The meristems were then placed in media conducive to shoot and root development. Rooted plants with normal phenotypic characteristics were selected and transferred to soil for growth (R0).

The R0 plants generated through this transformation were self-pollinated to produce R1 plants. A non-lethal dose of glyphosate was applied to R1 plants and those plants with minor herbicide injury were selected for further analyses, whereas plants showing no injury, indicating that they contained the cp4 epsps coding sequence from T-DNA II, were eliminated from further development. Subsequently, plants that were homozygous for T-DNA I were identified by quantitative polymerase chain reaction (qPCR) analysis. The result of this process was the selection of MON 87708 as a lead event for further charterization. This event contains only the introduced genetic elements from T-DNA I, as any elements from T-DNA II would have been elminated through the selective breeding.

The transferred T-DNA I contains the promoter for the Full-Length Transcript (FLt) of peanut chlorotic streak caulimovirus (PC1SV) , the 5' non-translated region from the Tobacco Etch virus (TEV), transit peptide and the first 24 amino acids of the mature protein of the RbcS gene from Pisum sativum (RbcS), the coding sequence of the dicamba mono-oxygenase (dmo) gene, and the terminator region from the RbcS2 gene of Pisum sativum (pea) (E9).

The dmo gene is derived from the bacterium Stenotrophomonas maltophilia strain DI-6, isolated from soil at a dicamba manufacturing plant. S. maltophilia is an aerobic, environmentally ubiquitous gram negative bacterium commonly present in aquatic environments, soil, and plants.S. maltophilia has been ubiquitously associated with plants and has been isolated from the rhizosphere of wheat, maize, grasses, beet, cucumber, chicory, potato, strawberry, sugarcane, and rapeseed, as well as from cotton seed, bean pods, and coffee.S. maltophilia is also widespread in the home environment and can be found around dishwashers, sponges, toothbrushes, flowers, plants, fruits, vegetables, frozen fish, milk, and poultry. Strains of S. maltophilia have been found in the transient flora of hospitalized patients as a commensal organism. S. maltophilia can be found in healthy individuals without causing any harm to human health and infections in humans caused by S. maltophilia are uncommon.

In S. maltophilia the degradation of Dicamba is the result of an enzyme system that serves as an electron transport chain involving a reductase, a ferredoxin and the terminal component DMO. The ferrodoxin component of this system closely resembles the ferrodoxin naturally found in plant choloroplasts. To take advantage of this naturally occurring source of reduced ferredoxin (and eliminate the need to introduce the bacterial ferredoxin and reductase components) the petitioner included a chloroplast transit peptide to allow for transport of the DMO protein to the chloroplast.

3. Characterization of the Modified Plant

Southern blot analysis of Dicamba Tolerant Soybean MON 87708 demonstrated the insertion of a single copy of T-DNA I cassette in the soy genome at a single locus. Analysis also verified the absence of any extraneous sequences related to the PV-GMHT4355 plasmid backbone. Southern blots were also used to demonstrate the expected absence of any genetic material from T-DNA II in Dicamba Tolerant Soybean MON 87708.

The petitioner conducted further analysis of the insertion through PCR and sequencing. Using PCR primers, two overlapping PCR products encompassing the inserted cassette and the flanking genomic DNA, the PCR products were sequenced to confirm that no sequence alterations had occurred in the inserted cassette. Based on the sequencing the T-DNA I intergrated into the soybean genome matches the sequence contained in the plasmid and is 3003 bp in length.

Generational stability of the single insert was determined across five generations of Dicamba Tolerant Soybean MON 87708. Southern blot analysis was present for each generation confirm the presence of T-DNA I.

The petitioner also provided the results of segregation analysis from three generations to indicate that this trait is inherited in the expected Mendelian fashion. From the data provided no differences from the expected ratios are shown and therefore the segregation is considered to occur in the expected manner. This is further evidence that the insert is stable in the soybean genome and is evidence that it is inherited in the expected Mendelian fashion.

In MON 87708, the introduced DMO proteins are active in the chloroplast, a plastid organelle, where it can interact with the ferredoxin required for its function. MON 87708 contains a dmo expression cassette that encodes for a single MON 87708 DMO precursor protein targeted to the plant's chloroplasts. The MON 87708 DMO precursor protein contains 84 amino acids at the N-terminus of the protein that were added to target the protein to the chloroplast. These additional amino acids correspond to a 57 amino acid chloroplast transit peptide (CTP) from pea Rubisco small subunit and the first 24 amino acids from the N-terminus of the mature Rubisco small subunit, which are incorporated to improve the targeting of the precursor protein to the chloroplast, and three amino acids encoded by an intervening sequence were used for cloning purposes. Typically, transit peptides are precisely removed from the precursor protein following delivery to the targeted plastid resulting in the full-length protein.

Analysis of mature seed extracts from MON 87708 by western blot demonstrated the presence of two immunoreactive bands. Characterization of these two bands revealed that the precursor protein had been processed into two forms of the protein. The lower molecular weight band corresponded to the full-length protein that was the result of the removal of the CTP, the 24 amino acids from pea Rubisco small subunit and the amino acids from the intervening sequence (MON 87708 DMO). The second higher molecular weight band corresponds to the MON 87708 DMO protein plus 27 amino acids on the N-terminus originating from the pea Rubisco small subunit and intervening sequence that were not cleaved (MON 87708 DMO+27). The petitioner provided examples in the literature of alternatively processed forms of a protein targeted to a plant's chloroplast, where part of the transit peptide remains.

The petitioner provided a complete characterization of both MON 87708 DMO and MON 87708 DMO+27, using 1) immunoblot analysis to establish identity and immunoreactivity of the MON 87708 DMO protein and the MON 87708 DMO+27 protein using an anti-DMO antibody, 2) N-terminal sequence analysis of the MON 87708 DMO protein and the MON 87708 DMO+27 protein, 3) matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to generate a tryptic peptide map of the MON 87708 DMO protein and the MON 87708 DMO+27 protein, 4) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to establish the apparent molecular weight of the MON 87708 DMO protein and the MON 87708 DMO+27 protein, 5) glycosylation status of the MON 87708 DMO protein and the MON 87708 DMO+27 protein, and 6) MON 87708 DMO activity analysis to demonstrate functional activity. The results of these analyses confirmed the identity and functional activity of both forms of the DMO protein and confirmed that the proteins are not glycosylated in MON 87708 soybean.

In addition, the petitioner provided a complete analysis of the specificity of DMO (both MON 87708 DMO and MON 87708 DMO+27 proteins isolated from MON 87708 soybean). The petitioner tested the specificity of the proteins when incubated with o-anisic acid, the endogenous compound that has the greatest structural similarity to dicamba. From these studies, DMO was found not to act on o-anisic acid. Additionally, studies conducted with wild-type DMO analysed the protein's specificity on o-anisic acid and a variety of other compounds which share varying levels of similarity with dicamba. In all these cases, no metabolism of the substrates was observed, further indicating the specificity of DMO.

The safety assessment of crops derived through biotechnology includes characterization of the functional and physicochemical properties, and confirmation of the safety of the introduced protein. MON 87708 DMO (both MON 87708 DMO and MON 87708 DMO +27) was purified in sufficient quantities directly from the seed of MON 87708 and used in the safety assessment. Typically protein safety assessments are conducted on proteins produced in heterologous expression systems, such as E. coli. Since the MON 87708 DMO used in the safety assessment was purified directly from MON 87708, equivalency evaluations between plant-produced and bacterially-produced MON 87708 DMO were not necessary.

4. Product Information

Dicamba Tolerant Soybean MON 87708 differs from conventional soybeans by the insertion of the novel gene dmo and associated regulatory elements. The insertion of this gene results in the expression of the novel proteins MON 87708 DMO and MON 87708 DMO+27. The expression of these proteins confer tolerance to dicamba herbicides.

The petitioner has provided data to demonstrate the level of expression of the MON 87708 DMO (this again includes MON 87708 DMO, MON 87708 DMO+27 and all trimaeric forms) in the altered soybean. This study used plant samples from five field sites planted in the 2008 growing season in the major soybean growing locations in the United States. Each site was planted in three replicated plots using a randomized complete block design. Data is presented as mean data points over all sites for overseason leaf at four time points (OSL1, OSL2, OSL3 and OSL4), seed, root and forage.

The quantities of MON 87708 DMO protein were determined by a validated enzyme-linked immunosorbent assay (ELISA). Protein quantities for the tissues were calculated on a microgram (µg) per gram (g) fresh weight (fwt) basis. Moisture content was measured for all tissue types, and protein quantities from these tissues were converted to dry weight (dwt) values. The mean MON 87708 DMO protein quantities across locations for OSL1, OSL2, OSL3, OSL4, seed, root, and forage tissues were 17, 31, 44, 69, 47, 6.1, and 53 µg/g dwt, respectively. Based on the data collected in this study the petitioner has calculated that the MON 87708 DMO accounts for approximately 0.011% of the total protein in MON 87708 grain on a dry weight basis.

5. Dietary Exposure

It is expected that Dicamba Tolerant Soybean MON 87708 will be used in applications similar to that derived from other soybean varieties. Soybeans are the largest source of vegetable oil worldwide. Refined, bleached, and deodorized soybean can be further processed to produce cooking oils, shortening, margarine, mayonnaise, salad dressings, and a wide variety of products that are either based entirely on fats and oils or contain fat or oil as a principal ingredient. Soy protein isolate is also used in such foods as soups, sauce bases, energy bars, nutritional beverages, infant formula, and dairy replacements.

To calculate the level of exposure, the petitioner used the mean concentration of MON 87708 DMO found in grain, at 43 µg/g fwt. It was noted that on average, soybean protein content in products is concentrated during processing by 1.35 fold. Therefore, to ensure that the estimation was as conservative as possible, the petitioner used 58 µg/g fwt as the mean concentration, a 1.35 times the mean level found in the protein expression trials.

Based on this assumption at the 95 percentile of consumption, daily dietary intake would be estimated at 0.0056 and 0.2314 mg/kg bw/day for adults and non-nursing infants respectively. The petitioner has also provided a calculation of the margin of exposure (MOE), which is based on the estimated exposure and the acute NOEL determined taken from toxicological testing. This calculation determines that how many times more protein would required to be consumed to reach the NOEL level. The calculated MOE for MON 87708 for adults and non-nursing infants respectively is 24,800x and 600x.

6. Nutrition

The nutrient data for this submission was obtained from test (MON 87708), control (A3525) and commercial soybean varieties (reference varieties) from in 2008 at five USA locations in a randomized complete block design with three plots for each test, control and reference varieties. MON 87708 received dicamba herbicide at the ideal growth stage at the maximum permitted amount. Analytical data from the reference varieties was combined across all sites and used to establish a 99% tolerance interval for each composition component for commercial soybean.

All field trial experiments used to test dicamba herbicide tolerant soybean were acceptable. All analysis of test, control and reference varieties were done using approved scientific and appropriate statistical methods.

Nutrients and/or anti-nutrients were analyzed, in seed, as follows: Proximate(s): moisture, crude protein, crude fat, crude fibre, ash, carbohydrates (calculated); plus acid detergent fibre (ADF), neutral detergent fibre (NDF); Fatty Acid(s): C8-C22; Amino Acid(s): asparagine, threonine, serine, glutamic acid, proline, glycine, alanine, cysteine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, arginine, tryptophan; Vitamin(s): vitamin E; Anti-nutrient(s): lectin, phytic acid, raffinose, stachyose, trypsin inhibitors, isoflavones (daidzein, genistein, glycitein) and, in forage, as follows: Proximate(s): moisture, crude protein, crude fat, crude fibre, ash, carbohydrates (calculated); plus acid detergent fibre (ADF), neutral detergent fibre (NDF).

For the combined locations, statistical differences test (MON 87708) vs. control (A3525) were noted in 29 analytes. For nutrients and anti-nutrients where a statistical difference was determined, all mean values were within the 99% tolerance interval and/or literature ranges.

7. Toxicology

In a standard acute mouse oral toxicity study with a 14-day observation period, DMO protein prepartions did not result in any adverse effects at the highest dose tested, 140 mg/kg b.w. (NOEL). The estimated daily intake provided by the petitioner (0.23 mg/kg b.w./day) and the estimated daily intake calculated by PTAS (0.63 mg/kg b.w./day) for DMO proteins derived from MON 87708 in the most sensitive subpopulation, soy-based formula fed infants, were two orders of magnitude less than the NOEL. Thus, the level of consumption in the most sensitive subpopulation is well below the safe acute oral dose established in mammalian studies.

DMO proteins are both heat labile and readily digested in simulated gastric and intestinal fluid. As such it is unlikely that these proteins will be absorbed as active or intact enzymes in the large intestine.

DMO protein accounts for only 0.011% of the total protein content of MON 87708 soybean seeds. The DMO+27 sequence (which includes the DMO sequence) was not found to share significant homolgy with know allergens, gliadins, toxins or any proteins associated with safety concerns. As such, it is unlikely that DMO proteins will function as allergens, antinutrients, or toxins when consumed. Testing performed with human IgE show that the genetic modification in MON 87708 soybean did not increase the level of endogenous allergens when compared to conventional soybeans. Furthermore, the compositional analysis data for MON 87708 soybeans did not show increased levels of endogenous allergens or toxins when compared to conventional soybean. Taken together, these results suggest that the genetic alterations in MON 87708 did not elevate the levels of endogenous toxins, antinutrients or allergens that are normally present in soybeans.

DCSA and formaldehyde, which are generated from the DMO-mediated metabolism of dicamba herbicide, are not anticipated to exceed established regulatory limits for DCSA and are well within the range of formaldehyde concentrations measured for a variety of agricultural commodities. Therefore both metabolites are considered unlikely to pose a health concern.

Conclusion

Health Canada's review of the information presented in support of the food use of Dicamba Tolerant Soybean MON 87708 concluded that derived food products do not raise concerns related to safety. Health Canada is of the opinion that Dicamba Tolerant Soybean MON 87708 is similar to regular conventional commodity soybeans in terms of being an acceptable food source.

Health Canada's opinion deals only with the human food use of Dicamba Tolerant Soybean MON 87708. Issues related to the environmental safety of Dicamba Tolerant Soybean MON 87708 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
Food Directorate
Health Products and Food Branch
Health Canada, PL2204A1
251 Frederick Banting Driveway
Ottawa, Ontario K1A 0K9
novelfoods-alimentsnouveaux@hc-sc.gc.ca