Novel food information: Canola (Brassica napus) Event CLB-1

Health Canada has notified BASF Canada that it has no objection to the sale of food derived from herbicide tolerant canola CLB-1 BN02-131. 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 BASF Canada and the evaluation by Heath Canada and contains no confidential business information.

Table of Content

• BACKGROUND
  1. Introduction
  2. Product Development
  3. Characterization of the Modified Plant
  4. Product Information
  5. Dietary Exposure
  6. Nutrition
  7. Toxicology
  8. Allergenicity
• CONCLUSION

1. Introduction

BASF Canada has developed canola event CLB-1 using oligonucleotide-directed mutagenesis and conventional breeding procedures in order to confer tolerance to the imidazolinone herbicides through the expression of an AHASL1A protein. The expression of this protein, which contains two different single-point mutations, allows for the plant to continue branched chain amino acid synthesis in the presence of imidazolinone herbicides, which are used as a broad weed killer.

The assessment considered: how CLB-1 canola was developed; how the composition and nutritional quality of CLB-1 canola compared to non-modified canola varieties; and what the potential is for CLB-1 canola to be toxic or cause allergic reactions. BASF has provided data which demonstrates that CLB-1 canola is as safe as traditional canola varieties used as food in Canada.

The Food Directorate has a legislated responsibility for the 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 canola CLB-1 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) a substance, including a microorganism that does not have a history of safe use as a food.

2. Product Development

The petitioner has provided information describing the methods used to develop CLB-1 canola and data that characterizes the introduced point mutations that confer tolerance to the imidazolinone herbicides through the expression of the AHASL1A protein. CLB-1 canola was produced via oligonucleotide-directed mutagenesis and conventional breeding procedures. The use of oligonucleotide-directed mutagenesis allows for targeted site-specific mutations to be introduced. This is in contrast to the random nature of mutations using the method of chemical mutagenesis.

Gene repair oligonucleotides (GRON's) were introduced into protoplasts of conventional spring oilseed rape line BN02 using a PEG treatment. The GRONs pair specifically with either the coding or non-coding strand of the ahasL1A gene with the exception of a deliberate mismatch at the target mutation site. The elements of the GRON attract the endogenous DNA repair mechanisms of the cell and depending on the design of the GRON an insertion, deletion, or replacement of a nucleotide occurs. The GRONs are composed of deoxyribonucleotides, are single-stranded, contain blocking substituents at both the 5' and 3' ends, and are synthesized using standard oligonucleotide synthesis methods. Once the GRON has induced the mutation it unbinds from the DNA along with the repair mechanism and is broken down into its elements. Also, the GRONs as a whole and their elements would not be expected to remain in the final commercial line due to the plant breeding pedigree that follows the development of the initial mutated line.

Two targeted mutations were introduced into the AHASL1A gene using site-specific GRON's. The mutations were introduced one at a time, with the first amino acid change being the replacement of an alanine residue with a threonine corresponding to position 122 relative to the Arabidopsis thaliana AHASL protein; the second amino acid change is the replacement of a serine residue with an asparagine corresponding to position 653 relative to the A. thaliana AHASL protein. The gene containing these mutations is referred to as the ahasL1A-A122(At)T/S653(At)N gene.

3. Characterization of the Modified Plant

DNA sequencing confirmed the ahasL1A-A122(At)T/S653(At)N gene contained the mutations as expected and also demonstrated the lack of any mutations in the ahasL1C gene that is also present in canola and has high sequence similarity. This demonstrates the specificity of the oligonucleotide-directed mutagenesis only towards the L1A gene.

DNA based diagnostic testing using real-time PCR for single-nucleotide polymorphism (SNP) identification was performed. The results demonstrated the stable presence of the CLB-1 trait across multiple generations. The segregation data further confirmed the stability of the trait, showing that it segregates according to Mendelian inheritance principles.

3. Characterization of the Modified Plant

CLB-1 canola expresses one new protein based on the method of development and characterization of the mutation. The ahasL1a gene with the two point mutations encodes for an acetohydroxyacid synthase large subunit (AHASL) protein. The AHASL protein is involved in the synthesis of the branched-chain amino acids valine, leucine, and isoleucine.

Characterization of the AHASL1A protein in CLB-1 canola was conducted using an activity assay which compared the sensitivity of AHAS from CLB-1, the BN02 near isogenic line, and the conventional B. napus line BNS-2 plants to imazamox herbicide. The activity of the CLB-1 AHAS in the absence of herbicide was equivalent to the near isogenic control, as well as the conventional line. In the presence of the imidazolinone herbicide, the CLB-1 AHAS was significantly less sensitive than the near isogenic control.

The expression levels of the AHASL protein in leaf and seed tissues were determined from enzymatic activity measurements. In all cases, the AHASL protein was present at low levels comprising approximately 0.0001466% of tissue weight in leaves and 0.0000720% of tissue weight in seed of CLB-1 canola.

5. Dietary Exposure

The modification of CLB-1 canola is not intended to alter any of its nutritional aspects. The consumption of canola products is limited to the refined oil. The oil derived from CLB-1 canola is to be used in similar applications as canola oils currently available in the Canadian market. Canola oil is used in both salad and cooking oil products, and is also acceptable in hydrogenated products, such as margarine and shortenings. The development of CLB-1 canola will not result in any change in the consumption pattern for these products.

6. Nutrition

The petitioner provided results of a compositional study as well as results of a quantitative comparison of protein profiles using High Performance Liquid Chromatography (HPLC) of grain samples from B. napus CLB-1 BN02-131 and other canola lines used as comparators.

In the compositional study, levels of key nutrients and antinutrients were measured in grain samples derived from the following canola lines: B. napus CLB-1 BN02-131, BN02 near-isogenic line, as well as BNS-2 and Topas conventional lines (comparators). All lines were grown in 2009 at three sites located in the United States in a randomized complete block design with four replicate plots per site. The petitioner mentioned that the field sites and the cultivation practices used in the trials are representative of environmental conditions and agronomic practices used in the B. napus-producing regions of Canada. Grain samples from three replicate plots were systematically selected and sent to Eurofins Scientific, Inc. for compositional analysis. In addition, the petitioner also grew B. napus CLB-1 BN02-131 plants treated with the herbicide imazamox (Solo^®) concurrently at the same sites as the other test lines. However, grain samples from the herbicide treated B. napus CLB-1 BN02-131 were analysed only for some selected analytes (proximates and amino acids). The petitioner used an analysis of variance and the Tuckey-HSD test to compare the means obtained for each measured analyte among the tested lines.

The petitioner provided the results of the compositional study for more than 80 analytes including proximates (moisture, protein, crude fat, crude fiber, ash, acid detergent fiber, neutral detergent fiber, and total dietary fibre), amino acids (18), fatty acids (33), vitamins (9), minerals (9), and antinutrients (3). With the exception of saturated fatty acid lignoceric (C24:0), alpha-tocopherol and vitamin B6, no significant statistical differences were found between grain samples from the untreated B. napus CLB-1 BN02-131 and the BN02 near-isogenic line. The statistical analysis showed differences between B. napus CLB-1 BN02-131 and the conventional lines used as comparators, but the mean differences were found to be of small magnitude and/or did not pose nutritional concerns. In addition, with the exception of amino acid threonine, no significant statistical differences were found between herbicide treated and non-treated B. napus CLB1 BN02-131. The petitioner concluded that grain from B. napus CLB-1 BN02-131 is compositionally equivalent to, and as nutritious as, grain from the BN-02 near-isogenic line as well as other B. napuscomparators either included in this study or reported in literature.

The petitioner also performed a quantitative comparison of protein profiles using High Performance Liquid Chromatography (HPLC) to further confirm substantial equivalence of the grain from B. napus CLB-1 BN02-131 to grain from the control BN02 near-isogenic line. The petitioner found no significant differences in peak areas between chromatograms of imidazolinone tolerant B. napus CLB-1 BN02-131 and near isogenic line BN02.

The quantity and the quality of the information/data provided by the petitioner were deemed satisfactory. The compositional analysis for grain samples did not raise any nutritional or safety concerns. The few differences observed between canola line B. napus CLB-1 BN02-131 and the near-isogenic line BN02 and/or the other B. napuscomparators were considered quantitatively small, not nutritionally important and/or within the literature range of commercial canola.

7. Toxicology

The Pre-Market Toxicology Assessment Section (PTAS) assessed information provided by the petitioner on the potential toxicity of CLB-1 canola (B. napus) intended for the production of canola oil. This included information pertaining to the safety of the novel AHASL1A protein (containing two point mutations, A122(At)T and S653(At)N) from previous Health Canada approvals, digestion assays, heat stability assays and bioinformatics data.

Health Canada has previously approved one of the two mutations, S653(At)N, in another member of the acetohydroxyacid synthase multigene family, AHAS1C, expressed in Canola event Clearfield 45A71. To date, exposure to canola oil derived from plants with an AHAS protein carrying the S653(At)N mutation has occurred in Canadian consumers without incident and is not associated with any toxicity.

The petitioner provided the results of an in silico search which compared the amino acid sequences of the mutated AHASL1A to amino acid sequences of known toxins. The results of the search determined that the mutant AHASL1A did not share significant sequence similarity with any known toxins.

The results of in vitro pepsin and trypsin digestion assays demonstrated that total AHASL1A protein extracted from CLB-1 canola seed, which included both mutant and non-mutant proteins, was digested within 5 minutes of treatment. This suggests that ingested mutant AHASL1A would not cause systemic toxicity as it will be rapidly digested in the gastrointestinal tract and would not be absorbed into the circulation as an intact protein.

The results of the heat stability test showed that total AHASL1A protein extracted from CLB-1 canola seed, which included both mutant and non-mutant protein, was inactivated at 100oC (1 minute) as determined by enzymatic activity assay. This result suggests that mutant AHASL1A will be denatured at temperatures commonly involved in canola oil processing.

CLB-1 canola expresses mutant AHASL1A at very low levels (approximately 0.02 ug total AHASL/g fresh weight canola seed, only a small fraction of which is mutant protein). Total AHASL protein accounts for only 8x10-6 % of the total protein content of canola seeds. Further, since CLB-1 canola is intended for oil production, the final product will contain negligible amounts of total protein due to extensive processing. This indicates that the exposure to active, mutant AHASL1A protein from CLB-1 canola oil will be remote.

Based on the available data, CLB-1 canola is considered to be as safe as conventional canola for the production of canola oil and would not be expected to pose a health concern when consumed.

8. Allergenicity

The Pre-Market Toxicology Assessment Section (PTAS) assessed information provided by the petitioner on the potential allergenicity of CLB-1 canola (B.napus) intended for the production of canola oil. This included information pertaining to the safety of the novel AHASL1A protein (containing two point mutations, A122(At)T and S653(At)N) from previous Health Canada approvals, digestion assays, heat stability assays and bioinformatics data.

Health Canada has previously approved one of the two mutations, S653(At)N, for another member of the acetohydroxyacid synthase multigene family, AHAS1C, expressed in canola event Clearfield 45A71. To date, exposure to canola oil derived from plants with an AHAS protein carrying the PM1 mutation has occurred in Canadian consumers without incident and is not associated with any allergenicity.

The petitioner provided the results of an in silico search which compared the amino acid sequences of the mutated AHASL1A to amino acid sequences of putative or known allergens. The results of the search determined that mutant AHASL1A did not share significant sequence similarity with any putative or known allergens.

The results of in vitro pepsin and trypsin digestions assays demonstrated that total AHASL1A protein extracted from CLB-1 canola seed, which included both mutant and non-mutant protein, was digested within 5 minutes of treatment. This suggests that mutant AHASL1A will be rapidly digested in the gastrointestinal tract following ingestion, and would not be expected to prompt an immune response.

The results of the heat stability test showed that total AHASL1A protein extracted from CLB-1 canola seed, which included both mutant and non-mutant protein, was inactivated at 100oC (1 minute) as determined by enzymatic activity assay. This result suggests that mutant AHASL1A will be denatured at temperatures commonly involved in canola oil processing.

CLB-1 canola expresses mutant AHASL1A at very low levels (approximately 0.02 ug total AHASL/g fresh weight canola seed, only a fraction of which is mutant protein). Total AHASL protein accounts for only 8x10-6 % of the total protein content of canola seeds. Further, since CLB-1 canola is intended for oil production, the final product will contain negligible amounts of total protein due to extensive processing. This indicates that the exposure to active, mutant AHASL1A protein from CLB-1 canola oil will be negligible.

Based on the available data, CLB-1 canola is considered as safe as conventional canola used for the production of canola oil and would not be expected to pose an allergenic health concern when consumed.

CONCLUSION:

Health Canada's review of the information presented in support of the food use of CLB-1 canola concluded that derived food products do not raise concerns related to safety. Health Canada is of the opinion that CLB-1 canola is as safe and nutritious as food from current commercial canola varieties.

Health Canada's opinion deals only with the human food use of canola event CLB-1 BN02-131. Issues related to the environmental safety of CLB-1 canola in Canada and its use as livestock feed have been addressed separately through existing regulatory processes in the Canadian Food Inspection Agency (CFIA). 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 imidazolinone tolerant CLB-1 canola destined for commercial sale.