Novel Food Information - Sugarcane CTC175-A

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 CTC175-A. 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.

Background

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 CTC175-A to be insect-resistant to help control sugarcane borer infestations. Recombinant DNA techniques were used in order to make the sugarcane insect-resistant. The sugarcane is to be cultivated only in Brazil and the sugarcane-derived food ingredients to be 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 CTC175-A was developed; how the composition and nutritional quality of raw and refined sugars derived from sugarcane CTC175-A compared to non-modified varieties; and the potential for sugarcane CTC175-A to be toxic or cause allergic reactions. Centro de Tecnologia Canavieira provided data that demonstrates that raw and refined sugar derived from sugarcane CTC175-A 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 CTC175-A 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

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 sugarcane CTC175-A and the data that characterize the genetic change which makes the sugarcane event insect-resistant through the expression of the insecticidal protein Cry1Ab expression cassette. In addition, this sugarcane line also expresses a selectable marker sequence, nptII, which codes for the neomycin phosphotransferase protein (NptII) which confers resistance to kanamycin in plants.

Sugarcane CTC175-A was produced though biolistic transformation applied to embryogenic cells (calluses) of the sugarcane primordial leaves (heart) using a DNA fragment containing the expression cassettes for the cry1Ab and nptII genes purified from vector pGH-CTC2.nptII. The first cassette contains the modified coding sequence of the cry1Ab gene from Bacillus thuringiensis which is regulated by the PEP carboxylase promoter from Zea mays (PepC) and the transcriptional terminator sequence of the nopaline synthase (nos) gene. The second cassette includes the nptII gene sequence which is regulated by the ubiquitin promoter from Zea. mays (ubi-1) and the 3’ end of the nopaline synthase (nos) gene terminator sequence.

3. Characterization of the Modified Plant

Southern blot, qPCR 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. This analysis demonstrated 7 insertion sites with 6 copies of cry1Ab and 9 copies of nptII in sugarcane CTC175-A. The analysis detected no vector backbone sequences present in sugarcane CTC175-A.

The multiple T-DNA insertion sites were mapped using genome sequencing data to examine the potential formation of new open reading frames (ORFs) within the multiple insertion sites in the sugarcane genome. A total of 9 junction sequences between the inserted DNA and the genome were found in the analysis of the sugarcane CTC175-A event. Bioinformatic analysis of these sequences indicated no evidence of any translated open reading frames spanning the insert to plant junctions. The T-DNA insertions in sugarcane CTC175-A are highly unlikely to cause the translation of new potential ORFs.

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 four vegetative propagations was performed and the results demonstrated the stability of the insert.

4. Product Information

Two new proteins would be expected to be expressed in sugarcane CTC175-A based on the characterization of the inserted genetic material. The Cry1Ab and NptII proteins produced by the insertion of the two cassettes were characterized. This characterization included western blot analysis which demonstrated the presence and integrity of the Cry1Ab and NptII proteins in sugarcane CTC175-A. The petitioner also provided data demonstrating that the plant expressed proteins in sugarcane CTC175-A are equivalent to previously assessed Cry1Ab and NptII proteins.

5. Dietary Exposure

The genetic modification of sugarcane CTC175-A is not intended to alter any of its nutritional aspects when compared to conventional non-genetically modified varieties. It is expected that refined sugar from sugarcane CTC175-A 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 as a human food or food ingredient, so the food or food ingredient consumed by Canadians that would be derived from sugarcane event CTC175-A is refined sugar. Nutritional composition analysis of refined sugar is not informative as it is almost entirely composed of sucrose (99.93%), the remaining impurities being water, inverted or reducing sugar (glucose and fructose), ash, coloured components, and other organic non-sugar compounds. Canadians do consume a small amount of sugarcane stalk as a confection or food ingredient in Oriental cuisine but sugarcane is not a significant source of other nutrients and sugarcane generally has low digestibility due to its high fibre content.

The compositional parameters chosen for nutrient analysis were based on the recommendations of the Organization for Economic Co-Operation and Development (OECD) “Consensus Document on Compositional Considerations for New Varieties of Sugarcane (Saccharum ssp. hybrids): Key Food and Feed Nutrients, Anti-nutrients and Toxicants” (2011). Specifically, the petitioner measured the levels of proximates (i.e., moisture, protein, total fat, ash, crude fibre) and sucrose. In addition, the petitioner measured Neutral Detergent Fiber (NDF) and Acid Detergent Fiber (ADF) compositions due to relevance to the transformed events.

Data from six field trials over the 2014 to 2015 growing season in sugarcane growing areas of Brazil was analyzed. The field trials utilized a randomized block design with four replicates per block. Each block contained the test cultivar (CTC175-A), the control cultivar (isoline CTC 20), and four reference varieties (CTC 20 null, CTC9001, CTC4 and CTC15) grown under identical conditions.

No statistically significant differences were observed in analytical results between CTC175-A and the control or the reference varieties. As well, all nutrient levels for all varieties were within the ranges of the literature values set out in the OECD (2011) sugarcane document, with the exception of crude fibre. The range of crude fibre content as a percentage of dry matter for the event bordered on the lower limit of the range listed in the OECD document, the other reference varieties (except for CT15) also were lower in crude fibre than the values reported by OECD, the event’s values for Neutral Detergent Fibre and Acid Detergent Fibre were not significantly different than the OECD values, and the food use in Canada will be limited to refined sugar. Therefore, the event’s slightly lower content of crude fibre compared to OECD values has no nutritional significance.

Based on the available information, the use of CTC175-A sugarcane as a source of either refined sugar or sugarcane stalks as food is as safe with regard to nutrient composition as refined sugar and stalks from conventional sugarcane varieties.

7. Chemistry/Toxicology

The petitioner provided toxicological evidence and scientific safety rationale to demonstrate the safety of sugarcane Event CTC175-A expressing the Cry1Ab and NptII proteins. This safety rationale, supported by chemical analysis, was based on the fact that exposure to the novel proteins is expected to be negligible, and that the same proteins were previously approved in other genetically modified crops available for sale in Canada.

Through the chemical analysis the petitioner was able to demonstrate that exposure to the novel proteins in the finished food product (refined sugar) would be negligible. Raw and refined sugar are highly processed products subjected to high temperatures and fluctuating pH values. The petitioner referenced publicly available literature in which it was determined that no protein could be detected in store bought refined sugar with a limit of detection of 1ppm, or 1 µg/g. The petitioner also analysed samples of raw sugar derived from Event CTC175-A, and no protein was detectable in those samples^{Footnote 1}. It can therefore be expected that exposure to both the Cry1Ab and NptII proteins would be negligible.

Despite the evidence that refined sugar contains no measurable protein, a worst case exposure scenario was described by the petitioner where it was assumed that refined sugar contained the same amount of protein as is present in the unprocessed sugarcane stalk. Using consumption values for added sugars as described by the Canadian Sugar Institute^{Footnote 2}, it was estimated that exposure to the novel proteins would equal 308 ng Cry1Ab/kg bw/day and 16.6 ng NptII/kg bw/day.

Both of these proteins have previously been expressed in food crops approved for sale in Canada and evidence was provided demonstrating that the Cry1Ab and NptII proteins expressed in Event CTC175-A were identical to those expressed in previously evaluated and authorised by Health Canada.

For Cry1Ab an NOAEL of >4,000 mg/kg bw/day was established based on an acute oral toxicity study conducted in mice. Based on the worst case exposure scenario generated by the petitioner and the acute NOAEL established in previous safety assessments, a margin of exposure (MOE) of 13,000,000 was calculated. From a safety perspective, this very large MOE value is considered sufficient to protect Canadian consumers.

Exposure to the inert NptII protein is expected to be negligible and food commodities derived from crops containing this protein have been available in Canada for 20 years with no reported adverse effects. On this basis, it was not expected to pose a safety concern.

A scientific rationale was provided, which was supported by previously reviewed studies, to support the allergenic safety of the Cry1Ab and NptII proteins expressed in Event CTC175-A.

In previous safety assessments of Cry1Ab, it was demonstrated that the protein was significantly degraded in simulated gastric fluids and that the protein sequence did not resemble any known allergens. Together with the fact that exposure to the intact protein is expected to be negligible from the consumption of raw or refined sugar, as well as the history of safe use in food commodities derived from crops containing Cry1Ab, there is no reason to believe that the presence expression of the protein in Sugarcane Event CTC175-A would pose an allergenic risk to consumers.

Based on its history of safe use in food commodities derived from crops containing the protein, as well as the anticipated negligible exposure, there is no reason to believe that the presence of the NptII protein in Sugarcane Event CTC175-A would pose an allergenic risk to consumers.

Based on the above, raw or refined sugar derived from sugarcane CTC175-A is not considered to pose a greater toxicological or allergenic risk than conventional sugarcane varieties.

Conclusion

Health Canada’s review of the information presented in support of the food use of raw and refined sugar derived from sugarcane CTC175-A does not raise concerns related to food safety. Health Canada is of the opinion that food derived from sugarcane CTC175-A is as safe and nutritious as food from current commercial sugarcane varieties.

Health Canada's opinion deals only with the food use of sugarcane CTC175-A.

For further information, please contact:

Novel Foods Section
Food Directorate
Health Products and Food Branch
Health Canada, PL2204E
251 Frederick Banting Driveway
Ottawa, Ontario K1A 0K9
novelfoods-alimentsnouveaux@hc-sc.gc.ca