Novel Food Information Arctic Fuji Apple Event NF872
Health Canada has notified Okanagan Specialty Fruits Inc. that it has no objection to the food use of Arctic Fuji apple event NF872. 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.
The following provides a summary of the notification from Okanagan Specialty Fruits Inc. and the evaluation by Heath Canada and contains no confidential business information.
Okanagan Specialty Fruits Inc. developed Arctic Fuji apple event NF872 to be non-browning when exposed to mechanical damage such as slicing or bruising. Recombinant DNA techniques were used in order to make the apples non-browning.
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 Arctic Fuji apple event NF872 was developed; how the composition and nutritional quality of Arctic Fuji apple event NF872 compared to non-modified varieties; and the potential for Arctic Fuji apple event NF872 to be toxic or cause allergic reactions. Okanagan Specialty Fruits Inc. provided data that demonstrates that Arctic Fuji apple event NF872 is as safe and of the same nutritional quality as traditional apple varieties used as food in Canada.
In 2015, Health Canada indicated no objection to the sale of two other non-browning apples, Arctic Golden (Event GD743) and Arctic Granny (Event GS784), which were transformed with the same vector used in the development of Arctic Fuji apple event NF872.
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 Arctic Fuji apple event NF872 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
- 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 Arctic Fuji apple event NF872 and the data that characterize the genetic change which makes the apple event non-browning through the expression of a polyphenol oxidase (PPO) enzyme suppression cassette which down-regulates enzyme levels through RNA interference (RNAi). In addition, these apple lines also express a selectable marker sequence, nptII, which codes for the neomycin phosphotransferase protein (NptII) which confers resistance to kanamycin in plants.
Arctic Fuji apple event NF872 was genetically modified using Agrobacterium-mediated transformation of apple leaf tissue with the vector GEN-03. The transfer DNA (T-DNA) from the transforming plasmid contains the PPO suppression cassette and the coding sequence for the NptII protein. The PPO suppression cassette consists of four apple genes. The nptII gene sequence is from the E. coli Transposon Tn5.
3. Characterization of the Modified Plant
Whole genome sequencing analysis of Arctic Fuji apple event NF872 was used to determine 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 three T-DNA insertions and there were multiple GEN-03 fragments at each location. The insertions in NF872 also included one partial copy of vector backbone, however there are no functional elements of the backbone present.
Commercial apples are vegetatively propagated and therefore do not involve processes that are responsible for genetic variation such as meiosis, recombination, or segregation. Cultivated apple trees are expected to be genetically and phenotypically stable. The Arctic Fuji apple event NF872 was developed in 2003 and vegetatively propagated through multiple generations in tissue culture prior to entry into field trials in 2005. In 2016, the presence of the transgene in a propagation block of Arctic Fuji apples was confirmed using PCR, which was consistent for transgene stability in vegetatively propagated trees.
4. Product Information
Arctic Fuji apple event NF872 differs from conventional apples by the insertion of the PPO suppression cassette and its associated regulatory elements, as well as the nptII gene and its associated regulatory elements. The apples have delayed browning as compared to conventional apples.
5. Dietary Exposure
The genetic modification of Arctic Fuji apple event NF872 is not intended to alter any of its nutritional aspects when compared to undamaged, non-genetically modified apples. It is expected that Arctic Fuji apples will be used in applications similar to conventional apples and thus no change in the food use of apples is anticipated.
The nutritional composition of Arctic Fuji apple event NF872 and its control counterpart (NF) was obtained from field trials in New York (NY) and Washington (WA) in 2015 and 2016. For each year, at the NY site, mature apples from three NF872 and two control trees were harvested. At the WA site, mature apples from two NF872 and two control trees were harvested. At each location, six NF872 and six NF apples were randomly selected for analysis of proximates and other nutrients, and the same number was selected for analysis of total phenolic compounds. USDA and other literature sources of apple composition were consulted as references. Field trials were not conducted in Canada; however, data was provided demonstrating that the US geographical locations, average temperature and precipitation levels are comparable to the primary Canadian apple growing regions of Ontario and British Columbia.
The nutritional and compositional analytes measured in the apple samples included: moisture, protein, fat, carbohydrate, sugar, ash, dietary fibre, potassium, vitamin C, and phenolics. These nutrients were assessed in apples from event NF872 and compared to the control NF apples to determine if they were nutritionally equivalent.
Of all the analytes measured, only the vitamin C and potassium levels in the NF872 apples were significantly different (higher) compared to controls (P< 0.05). The lower vitamin C levels in control apples could be expected, as it is a substrate in the browning process. Following damage to the apple (e.g., slicing for nutritional analysis), the PPO enzyme (normally restricted to plastids) and substrates (phenolic compounds normally restricted to the vacuole) come into contact and react in the presence of oxygen. This results in the formation of o-quinones which can undergo spontaneous polymerization to produce high molecular weight compounds such as brown pigments (melanins). Vitamin C acts as a natural browning inhibitor by reducing o-quinones back to phenolic compounds before they form brown pigments. When the apple samples were sliced and exposed to oxygen for as long as 24 hours prior to testing, vitamin C would be depleted in the control samples, while in the NF872 samples, vitamin C would be conserved. Also, the vitamin C level in the NF872 apples (i.e., 4.3 mg/100 g) was similar to the USDA nutrient database value (4.6 mg/100 g) and within the range reported in the literature (0.2780 mg/100 g).
The potassium level in the NF872 apples (126 mg/100 g) was higher than the USDA value (109 mg/100 g), however, when adjusted for moisture differences there was less than a 6% difference in potassium levels, and this small difference is not a nutritional concern.
The nutrient composition of Arctic Fuji apple event NF872 has been shown to be similar in composition to other apples on the Canadian market.
The toxicological impacts of the novel molecular products produced by Arctic Fuji apple event NF872 were examined. These products were considered to be the small interfering RNA molecules (siRNAs) expressed by the suppression sequence, as well as the protein selection marker, NptII. The potential for exogenous siRNA to silence human protein expression is considered to be the possible hazard of RNAi-based novel foods. Data investigating this potential was provided.
The dietary exposure to the novel siRNAs in Arctic Fuji apple was estimated by quantifying the amount of total small RNA in Arctic Fuji apple event NF872. This amount of total small RNA was refined to provide an estimate of exposure to a unique, transgene-derived siRNA (i.e., novel siRNA). The exposure to novel siRNAs was reported on a basis of copies of siRNA per cell in the human body, and more conservatively, in the human liver. According to the literature, the exposure to 100 copies of small RNA or greater per cell is required to result in repression of targeted transcripts. Dietary exposure to a novel unique siRNA expressed by Arctic Fuji apple was estimated to be 3.2 copies/hepatocyte for the general population and 6.0 copies/hepatocyte for children. This is below the threshold of 100 copies per cell and therefore not considered to pose a hazard.
The human body presents several physical barriers and chemical conditions which would likely prevent the novel siRNA molecules from being absorbed at the levels of dietary exposure presented above. However, if the novel siRNAs were absorbed at sufficient levels for repression, these molecules would not be expected to generate off-target silencing of human protein expression. The bioinformatics analyses data demonstrated that the predicted siRNA molecules produced by Arctic Fuji apple event NF872 do not have statistically significant homology to protein-coding genomic regions or to human transcript sequences.
The protein selection marker gene, nptII, has previously been approved for several genetically modified crops, and a history of safe use has been demonstrated for the NptII protein. Furthermore, NptII protein is expressed at negligible levels and does not accumulate in mature Arctic apple fruit, as demonstrated previously. Expression of NptII in Arctic Fuji apple is not considered to represent a toxicological concern.
Apples are not considered a priority allergen in Canada. While allergenic responses to apples do occur, severe reactions are rare and are restricted to exposure with the fresh fruit. The impact of the genetic modification in Arctic Fuji apple on the allergenicity of these apples as compared to conventional varieties was examined. The suppression sequence coding for novel siRNAs does not express any novel protein products and therefore will not impact the allergenicity of Arctic Fuji apple event NF872.
Arctic Fuji apples, like other Arctic Apples, have a decreased level of polyphenol oxidase. Under normal conditions, polyphenol oxidase produces oxidized phenols which bind to allergenic proteins in apples. Theoretically a decreased level of polyphenol oxidase could lead to an increase in the availability of allergenic proteins. However, there is no evidence to support this theoretical increase. In the submission for Arctic Apple events GD743 and GS784, field trials demonstrated that the total protein and phenol content of Arctic Apples were within apple literature ranges, which suggests the amount of allergen has not changed. In addition, influenced by environmental factors, the levels of endogenous allergens in apples are naturally highly variable, and minor changes to allergen content would not be considered biologically significant. It was concluded that the potential increase in the availability of allergenic proteins in Arctic Fuji apple is not likely to result in an additional health risk.
The expressed protein, selection marker, NptII, does not pose a concern for increased allergenicity of Arctic Fuji apple as compared to conventional apples. The history of safe use established for this protein and the negligible levels of NptII expressed in Arctic Apples serve to support this claim.
Based on the above, Arctic Fuji apple event NF872 is not considered to pose a greater toxicological or allergenic risk than conventional apple varieties.
Health Canada's review of the information presented in support of the food use of Arctic Fuji apple event NF872 does not raise concerns related to food safety. Health Canada is of the opinion that food derived from Arctic Fuji apple event NF872 is as safe and nutritious as food from current commercial apple varieties.
Health Canada's opinion deals only with the food use of Arctic Fuji apple event NF872. Issues related to its use as animal feed have been addressed separately through existing regulatory processes within 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 Arctic Fuji apple event NF872 destined for commercial sale.
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.
For further information, please contact:
Novel Foods Section
Health Products and Food Branch
Health Canada, PL2204E
251 Frederick Banting Driveway
Ottawa, Ontario K1A 0K9
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