Novel Food Information - Herbicide Tolerant Canola Lines 73496 and 61061

Health Canada has notified Pioneer Hi-Bred Production Ltd. that it has no objection to the food use of Herbicide Tolerant Canola Lines 73496 and 61061. The Department conducted a comprehensive assessment of these varieties 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 Pioneer Hi-Bred Production Ltd. and the evaluation by Heath Canada and contains no confidential business information.

1. Introduction

Pioneer Hi-Bred Production Ltd. has developed two herbicide tolerant canola (Brassica napus L.) lines containing events DP-Æ73496-4 and DP-Æ61Æ61-7 (i.e., 73496 and 61061) respectively. These lines were developed through microprojectile bombardment of embryonic microspores with a 2.1-kb DNA fragment (i.e., PHP28181A). This fragment contains the coding sequence and required regulatory elements to express the GAT4621 protein: an engineered glyphosate acetyltransferase (GAT) capable of converting glyphosate to a non-phytotoxic form, N-acetylglyphosate. The GAT4621 protein has been subject to a previous Health Canada assessment study in Pioneer Hi-Bred Production Ltd.`s Corn Event 98140.

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 herbicide tolerant canola lines 73496 and 61061 were developed; how the composition and nutritional quality of these two lines compared to conventional varieties; and the potential for these two lines to be toxic or cause allergic reactions. Pioneer Hi-Bred Production Ltd. has provided data that demonstrates that herbicide tolerant canola lines 73496 and 61061 are as safe and of the same nutritional quality as conventional 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 the Food and Drug Regulations (Division 28). Herbicide tolerant canola lines 73496 and 61061 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 herbicide tolerant canola lines 73496 and 61061, in addition to the molecular biology data that characterize the genetic change, which results in the improved tolerance to glyphosate herbicides. This phenotype was achieved by microprojectile bombardment of the conventional canola variety 1822B with a 2.1-kb DNA fragment (i.e., PHP28181A) containing the coding sequence and required regulatory elements to express the GAT4621 protein. Embryonic microspores from variety 1822B were bombarded with gold particles coated with the PHP28181A DNA fragment. Successful transformants were selected on media containing glyphosate. Plants regenerated from transformation and tissue culture were selected for further development based on molecular analysis, herbicide efficacy, and agronomic evaluations. From these plants, two lines (i.e., 73496 and 61061) were selected.

3. Characterization of the Modified Plant

Southern blot analysis of herbicide tolerant canola lines 73496 and 6061 demonstrated the insertion of a single copy of the gat4621 coding sequence and its required regulatory elements at a single locus for each line. Analysis also verified the absence of any extraneous sequence related to the plasmid from which the PHP28181A DNA fragment was derived.

Generational stability of the single insert was determined across multiple generations of both canola lines (i.e., 73496 and 61061). DNA from five generations of each respective line were analysed by Southern blot analysis and the presence of the single insert was confirmed in each generation of each line. Analysis of the inheritance pattern of the gat4621 gene (i.e., coding sequence and regulatory elements) and the herbicide tolerance phenotype within multiple segregating generations of canola lines 73496 and 61061 were consistent with the finding of a single site of insertion that segregates according to Mendelian laws of genetics.

4. Product Information

Herbicide tolerant canola lines 73496 and 61061 differ from their conventional counterpart by the addition of the gat4621 coding sequence and its required regulatory elements. The insertion of the gat4621 gene results in the expression of the GAT4621 protein: an engineered glyphosate acetyltransferase (GAT) capable of converting glyphosate to a non-phytotoxic form, N-acetylglyphosate. The canola plants expressing the GAT4621 protein are thus tolerant to the herbicide glyphosate.

The coding sequence of the GAT4621 protein (i.e., gat4621) is a derivative of three native gat genes from the common soil bacterium Bacillus licheniformis. The gat4621 coding sequence was generated by 11 rounds of gene shuffling: a genetic technique that facilitates the combination of gene portions to generate chimeric forms with unique properties. B. licheniformis is frequently present in food and water. Due to its long history of apparent safe use in food production without noted safety concerns, the European Food Safety Authority (EFSA) (2009) included B. licheniformis on the Qualified Presumption of Safety (QPS) list of source organisms that may be safely used for food and feed. Further, various strains of B. licheniformis are approved as source organisms for food enzymes (e.g., amylase, protease, and pullulanase) as per Health Canada's Food and Drug Regulations and associated Marketing Authorizations for Food Additives (List of Permitted Food Enzymes). As such, it is unlikely that the use of B. licheniformis as a source organism for gat genes will pose a pathogenic, toxic, or allergenic concern.

The petitioner has provided data to demonstrate the level of expression of the GAT4621 protein in herbicide tolerant canola lines 73496 and 61061. This study used plant samples from six field trial locations in Canada and the United States in the 2009 growing season. All locations are relevant canola-growing regions and represent a range of environmental conditions typically encountered in the production of canola. At each field site, both transgenic canola lines (i.e., 73496 and 61061) and near-isoline comparator were planted using a randomized block field design. Tissues of the canola lines that were collected for GAT4621 protein quantification included: seed, roots, and whole plants. Tissue samples were collected from impartially selected, healthy individual plants from each block at the following growth stages: BBCH15 - five true leaves unfolded (whole plant samples); BBCH33 - three visibly extended internodes (whole plant samples); BBCH65 - full flowering, 50% flowers open on main raceme, older petals falling (whole plant and root samples); and BBCH90 - senescence (seed samples). Additionally, concentrations of the GAT4621 protein were quantified in samples of toasted meal and hulls produced from bulked seed samples harvested from the field-grown canola lines (i.e., 73496 and 61061).

The concentration of GAT4621 protein was determined by enzyme-linked immunosorbent assay (ELISA). Protein concentrations for the tissues were calculated on a microgram (µg) per gram (g) dry weight (dwt) basis. Expression of the GAT4621 protein is controlled by the constitutive UBQ10 promoter from the Arabidopsis thaliana UBQ10 polyubiquitin gene. For both transgenic canola lines, quantifiable amounts of GAT4621 protein were detected in each plant tissue tested. The range of mean concentrations in whole plant samples across growth stages was 5.2-6.9 µg/g dwt for canola line 73496 and 7.9-12 µg/g dwt for canola line 61061. Mean concentrations of GAT4621 protein measured in root samples from each transgenic canola line were similar at 6.6 and 5.2 µg/g dwt for canola lines 73496 and 61061, respectively. Mean concentrations of GAT4621 protein measured in seed samples were similar at 6.2 and 5.1 µg/g dwt for canola lines 73496 and 61061, respectively.

The concentration of GAT4621 protein in processed products derived from the seeds of canola lines 73496 and 61061 were also analysed. Quantifiable levels of GAT4621 were measured in all tested processed fractions including seed hulls, toasted meal processed using hulled seed, and toasted meal produced using unhulled seed. The concentration of GAT4621 protein measured in toasted meal produced from hulled seed (8.1 and 3.0 µg/g dwt for canola lines 73496 and 61061, respectively) were higher than corresponding values measured for toasted meal produced from unhulled seed (2.5 and 2.2 µg/g dwt for canola lines 73496 and 61061, respectively), particularly for canola line 73496-derived material. The petitioner stated that this may have been due to different toasting processes employed for meal from hulled versus unhulled seed and/or variation due to small-batch processing that could have affected the extent of protein denaturation and thus the ability to be detected by ELISA. It is the opinion of the evaluators that this is an acceptable rationale to explain these differences.

5. Dietary Exposure

It is expected that herbicide tolerant canola lines 73496 and 61061 will be used in all applications similar to that of conventional canola varieties. The only fraction of canola that is consumed by humans is refined, bleached, deodorized (RBD) oil. Due to the expression of the GAT4621 protein, canola lines 73496 and 61061 have increased levels of N-acetylated amino acids present in seed tissue (primarily N-acetylaspartate and N-acetylglutamate). The petitioner also stated that acetylated amino acids naturally occur in canola and other commonly consumed foods (e.g., eggs, turkey, and beef) and are thus safe to consume. Further, the increased levels of acetylated amino acids are not expected to affect the sole product prepared for human consumption (i.e., RBD oil), as all amino acids will be removed during the manufacturing process to levels below the limit of detection. The petitioner has stated that with the introduction of herbicide canola lines 73496 and 61061, they do not expect a significant change in the food use of RBD canola oil.

6. Nutrition

Nutritional composition of herbicide tolerant canola lines 73496 and 61061 was evaluated through data generated from field trials conducted at six sites representative of major canola growing areas in Canada and the United States during the 2009 growing season. The transgenic canola lines (i.e., 73496 and 61061) were grown with the control line (a near-isogenic line not encoding the gat4621 gene) in a randomized block design, using 4 blocks per site. Plots containing canola lines 73496 and 61061 were treated with glyphosate while plots of the control canola line were left untreated. Seed samples were collected from all canola lines and analyzed for key nutritional components in accordance with the Organization for Economic Co-operation and Development (OECD) consensus document Consensus Document on Key Nutrients and Key Toxicants in Low Erucic Acid Rapeseed (Canola) including analysis of proximates, fibre, fatty acids, amino acids, vitamins, minerals, glucosinolates, secondary metabolites, phytosterols, and anti-nutrients, resulting in approximately 100 analytes assessed. Statistical analyses were conducted using SAS software (version 9.2) to generate means, confidence intervals, and statistical comparisons. Tolerance intervals were also used to assess the nutritional relevance of statistically significant differences between the control canola line and canola lines 73496 and 61061. These intervals were built using data from other previous studies including conventional commercial canola lines grown and processed under similar conditions.

Analytes that were statistically different for canola line 73496 with a greater than 10% difference between the mean values of the control and transgenic line were: vitamin B2 and delta-tocopherol; glucosinolates such as progoitrin, glucoalyssin, and neoglucobrassicin; secondary metabolite tannins-insoluble; and cholesterol.

Analytes that were statistically different for canola line 61061 with a greater than 10% difference between the mean values of the control and transgenic line were: magnesium; delta-tocopherol; glucosinolates such as progoitrin, glucobrassicin, glucoalyssin, neoglucobrassicin; secondary metabolite tannins-insoluble; and cholesterol.

The differences noted between the control and transgenic canola lines do not present nutritional safety concerns for the following reasons: differences between the control and transgenic lines were noted for some glucosinolates such as progoitrin, glucobrassicin, glucoalyssin, and/or neoglucobrassicin however the mean values of the total glucosinolate levels were not statistically different between the control and transgenic lines; tannins are typically associated with anti-nutrient effects as they form soluble and insoluble complexes with proteins however lower levels of tannins do not present any nutritional concerns; the range of values for all tested analytes are within the tolerance intervals made from conventional commercial canola lines; and many nutrients will be lost in the process of making refined, bleached, deodorized (RBD) oil, the main derived food product from canola. Lastly, components such as vitamin B, minerals, and glucosinolates are not major constituents RBD canola oil.

7. Chemistry/Toxicology

To produce sufficient quantities of the GAT4621 protein for toxicology and allergenicity studies, an Escherichia coli expression system was used. The petitioner provided sufficient data to demonstrate that the microbial-derived GAT4621 is biochemically, structurally, and functionally equivalent to the GAT4621 protein produced by canola lines 73496 and 61061. Thus, the experimental data obtained from studies using microbial-derived GAT4621 protein can be extrapolated to the plant-produced GAT4621 protein in canola lines 73496 and 61061. Demonstration of equivalency was based on a comparison of protein molecular weights, immunoreactivities, N-terminal amino acid sequences, and tryptic peptide mass maps.

A 14-day acute toxicity mouse study was performed. Five male and five female Crl:CDÒ-1(ICR) BR mice were administered either 1640 mg microbial-derived GAT4621 protein preparation (95% purity)/kg body weight, 1640 mg bovine serum albumin/kg body weight, or vehicle control (i.e., deionized water) by gavage and observed for 14 days. Body weights were measured at 0, 1, 2, 7, and 14 days after treatment. No adverse effects, changes in body weight, or changes in food consumption levels were observed in treated animals when compared to protein or vehicle controls. No treatment-related changes were observed upon gross examination at necropsy (conducted on day 14). A no observable effect level (NOEL) of 1640 mg/kg body weight/day was established based on the highest dose tested.

BLASTP (2.2.13 algorithm) bioinformatics searches of the National Center for Biotechnology Information (NCBI; 2010) Entrez protein database (10,530,540 sequences) were performed using the GAT4621 protein amino acid sequence. The GAT4621 protein did not share structurally relevant similarities with any proteins that were associated with toxicological safety concerns.

8. Allergenicity

The GAT4621 protein amino acid sequence was compared with sequences retrieved from the Allergen Database (version 10.0, 2010) of the Food Allergy Research and Resource Program (FARRP) using the FASTA34 alignment tool (Pearson and Lipman, 1988). None of the 1471 sequences were found to share equal or greater than 35% amino acid identity with the GAT4621 protein. A search was also conducted using an 8-amino acid sliding window to screen for potential linear IgE-epitopes. The GAT4621 protein did not share sequence identity with any known or putative allergen sequences over 8 or greater contiguous amino acids. As such, the GAT4621 protein is not considered to have significant sequence identity with known allergens.

The mean concentrations of the GAT4621 protein in herbicide-treated canola lines 73496 and 61061 seed extracts were determined by ELISA to be 6.2 and 5.1 µg/g dwt, respectively. The data provided for the nutrient composition of canola lines 73496 and 61061 seed indicates that protein accounts for 25.9 and 25.8% of the canola seed dry weight, respectively. Therefore, the GAT4621 protein accounts for only 2.0-2.4·10-3% of the total canola seed protein in the transgenic canola lines. Normally, allergenic proteins perform a storage function and constitute 1-80% of the total proteins. Therefore, it is unlikely that the GAT4621 protein will function as an allergen as it is present in negligible quantities in the seed of canola lines 73496 and 61061.

Microbial-derived GAT4621 protein was digested in simulated gastric fluid and simulated intestinal fluid within 30 seconds and 5 minutes of exposure, respectively. As the majority of the GAT4621 protein is expected to be digested in the stomach, it is unlikely that functionally active GAT4621 protein derived from dietary intake will pose a human health concern.

The petitioner demonstrated that 50% of microbial-derived GAT4621 protein lost functional activity when heated at 49-52 °C for 15 minutes and was essentially inactivated at temperatures at or above 53 °C, as determined by continuous absorbance spectrophotometric assay.

The petitioner indicated that only the refined, bleached, deodorized (RBD) canola seed oil from canola lines 73496 and 61061 will be consumed by humans. As the RBD canola oil contains negligible amounts of protein (Canadian Nutrient File), and the protein that is present is denatured during the manufacturing process (e.g., seed cooking cycle at temperatures greater than 80 °C), it is unlikely that RBD canola oil products will be a significant source of exposure to the GAT4621 protein or indeed any protein from canola seed for Canadian consumers.

Conclusion

Health Canada's review of the information presented in support of the food use of herbicide tolerant canola lines 73496 and 61061 does not raise concerns related to food safety. Health Canada is of the opinion that food derived from herbicide tolerant canola lines 73496 and 61061 is as safe and nutritious as food from current commercial canola varieties.

Health Canada's opinion deals only with the food use of herbicide tolerant canola lines 73496 and 61061. Issues related to its use as animal feed have been addressed separately through existing regulatory processes in the Canadian Food Inspection Agency (CFIA). The CFIA evaluated information provided on the environmental, animal, and human health safety of herbicide tolerant canola lines 73496 and 61061 with the intended use in animal feed. 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 herbicide tolerant canola lines 73496 and 61061 destined for commercial sale.

It is the continuing responsibility of the food manufacturer or importer to ensure that their products are in compliance with all applicable statutory and regulatory requirements. Any new information obtained in relation to these products which have potential health and safety implications should be forwarded to Health Canada for our consideration in order to ensure the continued safety and integrity of all foods available in the Canadian marketplace. The sale of a food which poses a hazard to the health of consumers would contravene the provisions of the Food and Drugs Act.

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