TruFlex Roundup Ready Canola MON 88302

Health Canada has notified Monsanto Canada Inc. that it has no objection to the sale of food derived from MON 88302 canola (TruFlex™ Roundup Ready® Canola). The Department conducted a comprehensive assessment of this canola 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 MON 88302 canola through genetic modification using recombinant DNA techniques to introduce the CP4 5-enolpyruvylshikimate-3-phosphate synthase (cp4 epsps) coding sequence derived from the common soil bacterium Agrobacterium sp. strain CP4. The sequence codes for the CP4 EPSPS enzyme which is functionally equivalent to the native canola EPSPS, except that it is tolerant to inhibition by glyphosate herbicide.

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 MON 88302 canola was developed; how the composition and nutritional quality of MON 88302 canola compared to non-modified varieties; and what the potential is for MON 88302 canola to be toxic or cause allergic reactions. Monsanto has provided data which demonstrates that MON 88302 canola is as safe and of the same nutritional quality as traditional canola 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 MON 88302 canola 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 MON 88302 canola and molecular biology data that characterizes the genetic change which confers tolerance to glyphosate herbicides. Commercial canola variety Ebony was genetically modified using a Agrobacterium tumefaciens transformation. The transforming plasmid PV-BNHT2672 carried a transfer DNA (T-DNA) sequence comprised of a cp4 epsps gene cassette.

The cp4 epsps gene cassette contained a chimeric promoter containing the promoter of the tsf1 gene from Arabidopsis thaliana combined with the enhancer sequences from the Figwort Mosaic virus S35 promoter (P-FMV/TSF1), the untranslated leader (exon1) sequence from the A. thaliana tsf1 (L-TSF1), the intron from tsf1 gene (I-TSF1), the chloroplast transit peptide sequence from A. thaliana (TS-ctp2), the coding region of cp4 epsps from Agrobacterium sp. strain (CR-cp4 epsps) and the nontranslated region of the Pisum sativum rbcE9 gene (T-E9). This cassette is identical to one of two cassettes contained in the previously approved cotton line MON 88913 and is also identical to the cassette used in the development of Roundup Ready 2 Yield® Soybean (MON 89788).

3. Characterization of the Modified Plant

Southern blot analysis of MON 88302 canola demonstrated the insertion of a single copy of the cp4epsps cassette in the canola genome at a single locus. Southern blot analysis also demonstrated the integrity of the cp4 epsps gene and its regulatory elements. Southern blot analysis also demonstrated, as expected, the absence of any plasmid derived sequences outside the T-DNA region, such as the spectinomycin resistance gene found in the plasmid backbone. The elements contained in the cp4 epsps cassette have been shown to be stable with no rearrangements through Southern, PCR and sequence analysis. These analyses of the insert show that the cassette is entirely integrated into the genome and that all the elements are intact.

The stability of the inserted cp4 epsps cassettes was evaluated in five generations of canola grown at six separate locations. The results of Southern blot analysis and segregation data demonstrated the stability of MON 88302 canola at the genomic level in different environments.

Confirmation of the cp4 epsps protein identity and equivalence to the E. coli-produced protein that was in the toxicology studies was generated using sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and densitometry, N-terminal sequence analysis, Matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry, Immunoblotting, an enzymatic activity assay, and glycosylation analysis. The results of these analyses demonstrated that the plant-produced cp4 epsps is equivalent to the E. coli-produced protein and that no glycosylation of the protein occurs. Protein expression levels determined through ELISA showed that CP4 EPSPS represented 0.01% of the total protein of MON 88302 grain.

4. Product Information

MON 88302 canola differs from conventional canola by the insertion of the novel gene cp4 epsps and associated regulatory elements. The insertion of this gene results in the expression of the novel protein CP4 EPSPS. The expression of this protein confers tolerance to glyphosate The CP4 EPSPS protein is originally derived from Agrobacterium sp. strain CP4, a common soil-borne bacterium. The CP4 EPSPS protein is functionally identical to the endogenous plant EPSPS enzymes, including that naturally found in canola, with the exception of comparatively reduced affinity to glyphosate. In conventional plants, glyphosate binding to EPSPS enzymes results in the blockage of synthesis of the 5-hydroxyl of shikimate-3-phosphate, a precursor of essential aromatic amino acids and other aromatic metabolites necessary for growth and development. In the case of the CP4 EPSPS, the reduced affinity to glyphosate results in continued production of aromatic amino acids in plants containing the novel cp4 epsps gene.

The petitioner has provided data to demonstrate the level of expression of the CP4 EPSPS in the altered canola. This study used plant samples from six field sites planted in the 2009 growing season in the major canola growing locations in the United States and Canada. Each site was planted in four 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 (collected twice root-1 and root-2) and forage.

The quantities of CP4 EPSPS protein were determined by an 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 CP4 EPSPS protein quantities across locations for OSL1, OSL2, OSL3, OSL4,seed, roo1, root-2 and forage tissues were 180, 180, 230, 210, 27, 82, 38, and 170 μg/g dwt, respectively. Based on the data collected in this study the petitioner has calculated that the CP4 EPSPS accounts for approximately 0.01% of the total protein in MON 88302 seed on a dry weight basis.

The petitioner has indicated that, while the levels of protein were determined, the primary use of canola in Canada is for the generation of food grade oil. Food grade canola oil is a highly refined, bleached and deodorized product, containing little to no protein. Therefore, the expected human exposure to the protein through food sources is expected to be very minimal. However, the petitioner has indicated that the total protein content in canola oil is approximately <0.2ppm (0.00002%) and that based on the total protein calculations above, CP4 EPSPS could be estimate to make up a maximum of 0.01% of that 0.2ppm of protein.

5. Dietary Exposure

It is expected that MON 88302 canola will be used in applications similar to that derived from conventional canola varieties. The only fraction of canola that is consumed by humans is refined, bleached, deodorized (RBD) oil, containing little to no protein. Therefore, the expected human exposure to the protein through food sources is expected to be very minimal. However, the petitioner has indicated that the total protein content in canola oil is approximately <0.2ppm (0.00002%) and that based on their total protein calculations, CP4 EPSPS could be estimated to make up a maximum of 0.01% of that 0.2ppm of protein.

6. Nutrition

The nutrient data for this submission was obtained from test (MON 88302), non-transgenic control (Ebony), and seven commercial canola varieties (reference) grown in 2009 at two USA locations and three Canadian locations in a randomized complete block design with four replicate plots for each test, control, and reference. MON 88302 received glyphosate treatment. Analytical data from the seed samples was combined across all sites and used to establish a 99% tolerance interval for each composition component for each of the commercial canola. The field trial experiments were acceptable. All analysis was done using approved scientific and appropriate statistical methods.

The nutritional and compositional analytes measured in the MON 88302 (± glyphosate) and non-transgenic control canola seed were: proximate content (protein, total fat, ash, moisture, carbohydrate), fiber (ADF, NDF, and total dietary fiber), and minerals (calcium, copper, iron, magnesium, manganese, phosphorus, potassium, sodium and 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 (C8 to C24), vitamin (vitamin E as α-tocopherol), and anti-nutrients ( phytic acid, sinapic acid, alkyl glucosinolates, indolyl glucosinolates, and total glucosinolates).

The nutritional composition results showed that the range of values for all tested analytes for the control and MON 88302 (± glyphosate) were within the tolerance intervals established for the commercial canola lines grown concurrently. The mean values of most analytes for MON 88302 (± glyphosate) were not significantly different when compared to the mean values of the control canola line. For most of the statistically significant findings, the magnitude of the relative differences between the mean values of the control and canola MON 88302 were less than 20% (with the exception of one anti-nutrient listed below).

Specifically, for the combined locations, the mean analyte values for 9 analytes in whole seed were statistically different between unsprayed MON 88302 and the unsprayed control:

1. 18:2 linoleic acid, 18:3 linolenic acid, 20:1 eicosenoic acid and calcium were observed to be higher (9.60%, 18.19%, 4.91% and 7.33% , respectively);
2. 18:0 stearic acid, 18:1 oleic acid, 20:0 arachidic acid, 22:0 behenic acid and phytic acid were observed to be lower (13.92%, 4.55%, 9.14%, 4.58%,and 8.54%, respectively).

Similarly, for the combined locations, the mean values for 9 analytes in whole seeds were statistically significant between the sprayed MON 88302 and the unsprayed control:

1. Total dietary fiber, 18:2 linoleic acid, and 18:3 linolenic acid were observed to be higher (13.81%, 8.98%, 20.01%, respectively);
2. 16:1 palmitoleic acid, 18:0 stearic acid, 18:1 oleic acid, 20:0 arachidic acid, 22:0 behenic acid, and alkyl glucosinolate were observed to be lower (7.56, 15.06, 4.52, 10.68, 6.01, 27.59%, respectively).

For the particular analytes where a statistical difference was determined, the difference was considered to be small when compared to the natural variability as determined by the 99% tolerance interval. All mean values differences were within the 99% tolerance intervals therefore these differences were not considered to be nutritionally important.

7. Toxicology

The novel gene present in MON 88302 was not isolated from a known pathogen. The petitioner provided the results of acute oral toxicity studies, which demonstrated no treatment -related changes in survival, clinical observations, body weight gain, food consumption or gross pathology. Based on this study, the petitioner reported a No Observable Adverse Effect Level (NOAEL) of 572 mg/kg bw, which was the maximum dose tested. No margin of exposure was calculated, based on the likely low level of exposure to the protein.

The petitioner also provided the results of a bioinformatic analysis of the protein against know toxins. From this analysis the novel protein bears no biologically significant homology to any known toxin. Additionally, this protein has been incorporated in other transgenic plants for a number of years with no reported cases of toxicity associated with the consumption of food products derived from these transgenic plants.

Conventional canola is known to contain erucic acid and glucosinolates, as well as the anti-nutrient factors phytic acid and sinapine. MON 88302 was not found to differ from conventional canola varieties with respect to the presence or content of anti-nutrients or endogenous toxins.

The novel protein is not considered likely to be an allergen since it does not share the characteristics of protein that are food allergens. Unlike many food allergens, the novel protein constitutes a negligible amount of the protein in foods that contain it. The amino acid sequence of the novel protein has no significant homology to any known allergen. The novel protein is rapidly degraded in a simulated gastric fluid and in simulated intestinal fluid systems, as detected by western blot analysis. These results suggest that the novel protein would be digested in the mammalian digestive tract.

The petitioner also provided the results of a bioinformatic analysis of the protein against know allergens. From this analysis the novel protein bears no biologically significant homology to any known allergen.

Conclusion:

Health Canada's review of the information presented in support of the food use of MON 88302 canola concluded that derived food products do not raise concerns related to safety. Health Canada is of the opinion that MON 88302 canola is similar to conventional commodity canola in terms of being an acceptable food source.

Health Canada's opinion deals only with the human food use of MON 88302 canola. Issues related to the environmental safety of MON 88302 canola in Canada and its use as livestock feed have been addressed separately through existing regulatory processes in the Canadian Food Inspection Agency.

Roundup Ready® and TruFlex™ are trademarks of Monsanto Technology LLC, Monsanto Canada, Inc. licensee. ©2013 Monsanto Canada Inc.

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