Novel food information: Sugarcane CTC75064-3

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Health Canada has notified Centro de Tecnologia Canavieira that it has no objection to the food use of raw and refined sugar derived from sugarcane event CTC75064-3. The Department conducted a comprehensive assessment of this variety 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 Centro de Tecnologia Canavieira and the evaluation by Heath Canada, and contains no confidential business information.

1. Introduction

Centro de Tecnologia Canavieira developed sugarcane CTC75064-3 to be insect-resistant in order to help control sugarcane borer infestations. The sugarcane is to be cultivated only in Brazil and the sugarcane-derived food ingredients imported into Canada are raw and refined sugars.

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 sugarcane CTC75064-3 was developed; how the composition and nutritional quality of raw and refined sugars derived from sugarcane CTC75064-3 compared to non-modified varieties; and the potential for sugarcane CTC75064-3 to be toxic or cause allergic reactions. Centro de Tecnologia Canavieira provided data that demonstrates that raw and refined sugar derived from sugarcane CTC75064-3 is as safe and of the same nutritional quality as traditional sugarcane 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 B.28). Food use of sugarcane CTC75064-3 is considered a novel food 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

2. Development of the Modified Plant

The petitioner provided information describing the methods used to develop sugarcane CTC75064-3 and data that characterize the genetic modification that results in the insect-resistance through the expression of Cry1Ac. Sugarcane CTC75064-3 was developed by Agrobacterium tumefaciens mediated transformation of the sugarcane variety RB867515 with transformation plasmid pCTC146. Those cells that were successfully transformed with the pCTC146 plasmid were selected for tolerance to the aminoglycoside geneticin, which was imparted by the presence of the neomycin phosphotransferase protein encoded by the nptII gene. The event CTC75064-3 was selected from multiple transformants based on both in vitro and field assays for resistance to damage from the target pest, and for favourable agronomic characteristics.

The first expression cassette contains the cry1Ac gene from Bacillus thuringiensis subsp. kurstaki, strain HD73, a ubiquitous soil bacterium. The expression of cry1Ac gene is controlled by the double enhanced 35S (2x35S) promoter from the cauliflower mosaic virus (CaMV35S), the L-Cab leader sequence from wheat, an intron of the rice OsAct1 gene (I-OsAct1), and the 35S terminator. The second cassette contains the selectable marker gene nptII from E.coli under the control of the maize ZmUbi1 promoter and nos terminator from Agrobacterium tumefaciens. The cry1Ac gene sequence found in CTC75064-3 sugarcane encodes a 615 amino acid insecticidal protein with a molecular weight of 68.7 kDa. The nptII gene encodes a 265 amino acid neomycin phosphotransferase protein with a molecular weight of 29 kDa.

3. Characterization of the Modified Plant

Southern blot, PCR and DNA sequence analysis was performed in order to characterize the number of insertion sites and copies of the integrated T-DNA as well as the presence or absence of plasmid backbone sequence. These analyses demonstrated the genomic integration of a single copy of each cassette at a single locus in sugarcane CTC75064-3. The analysis also detected no vector backbone sequences present in sugarcane CTC75064-3.

Commercial sugarcane relies upon vegetative (clonal) propagation and therefore it would be expected that stability of the inserted T-DNA is maintained over many generations. Southern blot analysis of leaves from multiple vegetative propagations was performed and the results demonstrated the insert stability in sugarcane CTC75064-3.

4. Product Information

Two new proteins, Cry1Ac and NPTII, are expressed in sugarcane CTC75064-3 based on the characterization of the inserted genetic material. A bioinformatics analysis was conducted on the sequences spanning the junctions between the inserted DNA and the genomic DNA in sugarcane CTC75064-3 to confirm the absence of other unintended proteins as a result of the T-DNA insertion. This analysis showed no unintended protein translation and no interruption of endogenous protein coding sequences resulting from the insertion of the T-DNA. Therefore, no unexpected protein or fusion protein would be encoded at the T-DNA insertion site in sugarcane CTC75064-3.

The Cry1Ac and NPTII proteins expressed in this sugarcane line were of the expected apparent molecular weight, as determined by Western blot analysis. Results of a MALDI-TOF analysis of the plant-expressed Cry1Ac and NPTII protein showed 100 % amino acid sequence identity between the proteins in the leaves of sugarcane CTC75064-3 and the sequences for Cry1Ac (previously reviewed for sugarcane line CTC91087-6) and NPTII (previously reviewed for sugarcane line CTC175-A). Based on the provided information, the plant expressed Cry1Ac and NPTII proteins in CTC75064-3 sugarcane are equivalent to the previously assessed proteins found in CTC91087-6 (Cry1Ac) and CTC175-A (NPTII).

5. Dietary Exposure

The genetic modification of sugarcane CTC75064-3 is not intended to alter any of its nutritional aspects when compared to conventional non-genetically modified sugarcane varieties. It is expected that refined sugar from sugarcane CTC75064-3 will be used in applications similar to refined sugar from conventional sugarcane varieties and thus no change in the food use of refined sugar is anticipated.

6. Nutrition

The sugarcane-derived food ingredients imported into Canada from Brazil are raw sugar (~95%) and refined sugar (~5%). Upon importation, all raw sugar is required to be refined before use for human consumption. This means that the food or food ingredient derived from sugarcane event CTC75064-3 that Canadians would consume is refined sugar.

Following the recommendations of the Codex Standard for Sugars (1999) Footnote 1, nutrient analyses were conducted for the following compositional parameters: moisture, polarization, reducing sugars, conductometric ashes, colour, starch, dextran, phenolic compounds and sulphite. While the petitioner measured the levels of proximates, raw data for moisture, crude protein, crude fat, crude fibre, ash, sucrose, glucose, fructose or other analytes to be assessed in a compositional analysis was not provided. Instead, the petitioner bridged data from their other recently approved sugarcane crops (CTC175-A and CTC91087-6) and publicly available literature. This approach was considered acceptable given that sugarcane CTC75064-3 is highly comparable to the previously approved lines CTC175-A and CTC91087-6.

Polarization differences of events CTC75064-3 and RB867515 were statistically significant, however, their mean values were within the range for raw sugar (97-99.5% sucrose), per Division 18 of the Food and Drug Regulations. Statistically significant differences were also reported in moisture, reducing sugars and conductometric ashes, although the values for CTC75064-3 and the control were within the range for the reference varieties. The remaining parameters (colour, phenolic compounds, starch, dextran and sulphite) were not statistically different from the control variety and are within the range of values observed for the commercial reference varieties grown at the same site, using the same agronomic conditions.

Based on the information provided on the composition of sugarcane CTC75064-3, there aren't any nutritional safety concerns with the sale of foods derived from sugarcane CTC75064-3 compared to conventional sugarcane.

7. Chemistry/Toxicology

The focus of the safety evaluation for the refined sugar derived from the genetically modified sugarcane crop relates to the Cry1Ac and NPTII proteins expressed from event CTC75064-3. The petitioner provided a safety rationale based on the fact that the same proteins were previously approved (in two other genetically modified sugarcane crops from the same petitioner), and further demonstrated a lack of exposure to these two proteins in the final product (i.e., refined sugar).

To demonstrate the lack of exposure to the Cry1Ac and NPTII proteins from event CTC75064-3, the petitioner analyzed raw sugar from four separate production plots of this crop. Neither the Cry1Ac nor the NPTII proteins were detectable at the limit of detection (i.e., 0.9 ng/g sugar and 0.6 ng/g sugar, respectively).

As raw sugar is further processed and purified into refined sugar (> 99.8% sucrose), any residual protein in raw sugar is expected to be further removed and/or degraded during the extra processing steps. Refined sugar is a highly processed product, whereby the raw form is subjected to filtration, high temperatures and fluctuating pH. Therefore exposure to the proteins expressed from event CTC75064-3 is not expected in refined sugar.

Despite the substantial evidence demonstrating that refined sugar contains no measurable protein, a worst case exposure scenario was described by the petitioner. Using consumption values for added sugars described by the Canadian Sugar Institute (i.e., 50 g refined sugar/person per day), and assuming a 60 kg body weight, results in an intake of 0.83 g refined sugar/kg bw per day. Assuming that refined sugar contains an amount of protein equal to the aforementioned LODs, it was estimated that protein intake would be 0.747 ng Cry1Ac/kg bw per day and 0.50 ng NPTII/kg bw per day. These intake values are negligible, and are considered to be highly-conservative estimates.

Equivalency was confirmed for the Cry1Ac and NPTII proteins expressed from sugarcane CTC75064-3 to these same proteins in crops (from the same petitioner) that have been previously assessed and approved by Health Canada. Variations of these two proteins have also been approved for over 20 years, with no reports of adverse effects.

The petitioner provided data comparing the sequence homology of the currently notified Cry1Ac and NPTII proteins to known toxins identified in the NCBI protein database. Footnote 2The only hits identified for the Cry1Ac protein were within the Cry toxin family itself, and no structural or functional homologies to other toxins were reported. No hits were reported for the NPTII protein.

No new toxicology data was provided by the petitioner; however, updated toxicological literature reviews were performed for the two proteins, and no relevant reports were identified.Footnote 3, Footnote 4 Publicly available data also suggests that mammalian digestive cells do not have binding sites specific to the Cry1Ac protein, and thus are not susceptible to its insecticidal effects.

Based on the history of safe use and the available toxicological information, as well as the lack of expected exposure to these proteins, there are no toxicological concerns with consumption of refined sugar derived from sugarcane CTC75064-3.

The petitioner provided data comparing the sequence homology of the currently notified Cry1Ac and NPTII proteins to the sequences of putative and known allergens retrieved from the AllergenOnline™ Database. Footnote 5 For both proteins, there were no full-length homologies reported below the E score Footnote 6 threshold provided by the petitioner, nor was there > 35 % homology reported for the 80-amino acid sliding window; these results indicate no significant structural homologies between the Cry1Ac and NPTII proteins and known allergens. There were also no 8-amino acid exact matches, indicating that the proteins are unlikely to possess any known IgE epitopes.

Updated allergenicity-related literature reviews were performed for the two proteins, and no relevant reports were identified. Footnote 7, Footnote 8 Publicly available data suggests that Cry1Ac and NPTII proteins rapidly digest under conditions representative of the gastrointestinal tract.

Based on the history of safe use and the available allergenicity information, as well as the lack of expected exposure to these proteins, there are no allergenicity concerns with consumption of refined sugar derived from sugarcane CTC75064-3.


Health Canada's review of the information presented in support of the food use of raw and refined sugar derived from sugarcane CTC75064-3 does not raise concerns related to food safety. Health Canada is of the opinion that food derived from sugarcane CTC75064-3 is as safe and nutritious as food from current commercial sugarcane varieties.

Health Canada's opinion deals only with the food use of sugarcane CTC75064-3.

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


Footnote 1

Codex (1999) Codex standard for sugars. CODEX STAN 212-1999

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Footnote 2

Search conducted in Feb. 2020, number of known toxin proteins not reported.

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Footnote 3

Search criteria: PubMED: ["Cry1Ac" AND "tox*" OR "toxicity"], last 5 years.

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Footnote 4

Search criteria: PubMED: ["NPTII" OR "neomycin phosphotransferase II enzyme" AND "tox*" OR "toxicity"], last 5 years.

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Footnote 5; AllergenOnline database contained 2171 allergens (v20); search conducted in Feb. 2020.

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Footnote 6

The E score (expectation score) measures the likelihood that any observed similarities among sequences could have occurred by chance. A high E score indicates a low degree of similarity between the query sequence and the sequence from the database. According to the petitioner, alignments between two sequences must have an E score of less than 1 x 10-5 to conclude presence of significant homology (in which case an analysis of the percent homology would be investigated).

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Footnote 7

Search criteria: PubMED: ["Cry1Ac" AND "Allerg"], last 5 years.

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Footnote 8

Search criteria: PubMED: ["NPTII" OR "neomycin phosphotransferase II enzyme" AND "Allerg"], last 5 years.

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