Novel Food Information: Arctic® apple event PG451

On this page

• Background
• Introduction
• Development of the Modified Plant
• Characterization of the Modified Plant
• Product Information
• Dietary Exposure
• Nutrition
• Chemistry
• Toxicology
• Allergenicity
• Conclusion

Background

Health Canada has notified Okanagan Specialty Fruits (OSF) that it has no objection to the food use of Arctic^® apple event PG451 (PG451). The Department conducted a comprehensive assessment of this apple variety. As PG451 was transformed with the identical DNA sequence as a previously assessed genetically modified (GM) plant of the same species, OSF submitted their novel food notification of PG451 as a 'retransformant', for the safety of this apple variety to be assessed according to Health Canada's Guidance on the Pre-Market Assessment of Foods Derived from Retransformants, which are based upon the Department's Guidelines for the Safety Assessment of Novel Foods. These Guidelines are based upon internationally accepted principles for establishing the safety of foods with novel characteristics.

The tiered approach to assessing the safety of a retransformant takes into account the Department's prior knowledge regarding the level of risk associated with the related GM plants previously assessed by Health Canada.

The following provides a summary of the notification from OSF and the evaluation by Health Canada. This document contains no confidential business information.

Introduction

OSF has developed a novel apple (Malus × domestica) variety, PG451 that exhibits suppression of polyphenol oxidase (PPO) expression, resulting in resistance to enzymatic browning compared to conventional apple varieties. PG451 will be marketed as Arctic^® Gala.

PG451 was developed through the introduction of a chimeric suppression cassette (PGAS) comprising fragments of four apple polyphenol oxidase (PPO) genes (PPO2, GPO3, APO5 and pSR7), designed to suppress expression of the entire apple PPO gene family. The PGAS suppression cassette was introduced into PG451 using the identical transformation vector (i.e., GEN-03) that was used to develop Arctic^® apple events NF872 (approved by Health Canada in 2018), GS784 (approved by Health Canada in 2015), and GD743 (also approved by Health Canada in 2015).

The same T-DNA containing the PGAS suppression cassette also contains a nptII gene encoding a neomycin phosphotransferase type II enzyme (NptII). This nptII gene is derived from the Escherichia coli Tn5 transposon ^{Footnote 1}. Expression of the nptII gene confers resistance to the antimicrobial kanamycin and acted as a selection marker during the transformation process to develop PG451. Like the PGAS suppression cassette, the history of safe use of the nptII gene was previously reviewed as part of the assessment of Arctic^® apple events GS784 and GD743.

The safety assessment performed by Food Directorate evaluators was conducted according to Health Canada's Guidance on the Pre-Market Assessment of Foods Derived from Retransformants, which are based upon the Department'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 PG451 was developed, how the composition and nutritional safety of this variety compare to its conventional comparator and related GM varieties, and what the potential is for this variety to present a toxic or allergenic concern. OSF has provided data to support that this apple variety is safe for use 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 Division 28 of Part B of the Food and Drug Regulations (Novel Foods). Foods derived from PG451 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."

Development of the Modified Plant

Arctic^® apple varieties (including PG451) are developed through Agrobacterium-mediated transformation of apple leaf tissue using the vector GEN-03. The same transformation and selection methods were previously reviewed in the assessment of Arctic^® apple events GD743 and GS784.

Characterization of the Modified Plant

Whole genome sequencing analysis of PG451 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 two T-DNA insertions and there were multiple GEN-03 fragments at each location. The two T-DNA insertions in PG451 also included one partial copy and one complete copy of the plasmid backbone, respectively, 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. PG451 was developed in 2011 and vegetatively propagated through multiple generations in tissue culture prior to entry into field trials in 2016. In 2021, the presence of the transgene in a propagation block of PG451 apples was confirmed using PCR, which was consistent for transgene stability in vegetatively propagated trees.

Product Information

PG451 differs from its conventional counterpart by the expression of chimeric suppression cassette (PGAS) comprising fragments of four apple polyphenol oxidase (PPO) genes (PPO2, GPO3, APO5 and pSR7), designed to suppress expression of the entire apple PPO gene family.

The desired characteristic of the GEN-03 vector T-DNA insertion is to trigger the plant's endogenous mechanism of RNA interference (RNAi) leading to suppression of polyphenol oxidase (PPO) enzyme expression and activity in the plant tissue, especially in the fruit tissues. The petitioner measures the amount of PPO suppression using an enzymatic assay, that is carried out in the same manner at multiple stages of plant growth/development. A comparison of PPO suppression in PG451 and the other Arctic^® apple events (i.e., NF872, GD743, and GS784) show that PG451 exhibits similar levels of PPO suppression in its mature fruit tissues compared to the previously approved Arctic^® apple events.

The only novel protein intentionally expressed in PG451 is the neomycin phosphotransferase type II enzyme (NptII) encoded by the nptII gene in the T-DNA sequence. Expression of NptII confers resistance to kanamycin in plants ^{Footnote 2}.

In the previous submissions for Arctic^® apple events GD743, and GS784, quantitative ELISA performed by Agdia (Elkhart, IN) had shown that NptII was not present at detectable levels in the mature fruit of GD743 and GS784. Agdia no longer offers quantitative NptII ELISA as a service, so OSF performed a semiquantitative NptII ELISA using a quantitative standard purchased from Agdia.

Mature apple fruit were harvested from a field trial in 2021. Six (6) apples of PG451 and six (6) apples of the parent variety (control) were randomly selected and stored at 4 °C until processing. The assay was performed according to the manufacturer's instructions. The levels of NptII protein in fruits of all samples tested were below the assay limit of quantitation (LOQ) which was 10 ng/g fresh weight of apple. This is consistent with quantitative ELISA results of the previously assessed events.

The pBIN19 vector, on which the GEN-03 vector is based, includes an inactivated tetracycline resistant determinant homologous to Pseudomonas aeruginosa plasmid RP4 (GenBank Accession X75761). The tetracycline resistance determinant is comprised of a regulatory gene (tetR) encoding a 26-kDa repressor protein and a structural gene (tetA) encoding a membrane protein (34 kDa) responsible for resistance. The two genes are adjacent and have divergent transcriptional polarity with their transcriptional signals located in the short intercistronic region ^{Footnote 3}. During the construction of pBIN19 the tetracycline resistance gene tetA was inactivated by the insertion of the T-DNA region ^{Footnote 4}. The tetR gene that remains in pBIN19 and GEN-03 is a repressor gene that normally suppresses expression of tetA and does not itself confer resistance to tetracycline.

As mentioned previously, while expression of the NptII enzyme confers resistance to kanamycin in plants, results of the semiquantitative ELISA assay show that NptII expression in PG451 is below the assay limit of quantitation (LOQ) (10 ng/g fresh weight).

The molecular characterization of PG451 demonstrates that the genetic changes introduced into PG451 result in the same mechanism(s) of action and characteristic(s) as the previously assessed Arctic^® apple events NF872, GD743, and GS784. Therefore, PG451 is confirmed to be a retransformant and a scientific rationale (and/or data) can be provided to support that the nutritional composition, toxicological safety, allergenic safety, chemical safety, and estimated dietary exposure of the retransformant are equivalent to the previously assessed GM plants.

Dietary exposure

It is expected that PG451 will be used in applications similar to conventional apple varieties. The petitioner does not anticipate a significant change in the food use of apples with the introduction of PG451.

Nutrition

Based on the molecular characterization of PG451, there is no hypothesis to suggest that the nutritional composition of PG451 compared to its parent variety would be different from the nutritional composition of the previously assessed Arctic^® apple events compared with their respective parent varieties. The petitioner provided compositional analysis data to further support this position.

OSF operates two field trial sites located in New York and Washington state, which represent the two largest apple-growing regions in the United States. Field trials are managed consistent with commercial apple cultivation methods, adhering to integrated pest management (IPM) and Good Agricultural Practices (GAP). By assuring this type of management, the data collected from the trials is both reproducible and can be extrapolated with confidence to a commercial setting.

The primary compositional analysis for PG451 was based on a side-by-side comparison with control fruit of the parent variety produced under the same growing conditions at the New York and Washington field trials. At each field location, mature fruits from PG451 and control trees were harvested and pooled using quality control and assurance practices consistent to those found within a commercial orchard setting. Harvested fruits were inspected, and culled fruit removed. Fruit for nutritional testing is randomly selected from the pool of harvested fruit and sent to testing facilities. All compositional analyses except phenolics were done by Element Materials Technology based in Portland, Oregon. Phenolics were determined by Eurofins Food Integrity & Innovation (Madison, Wisconsin).

The results of the compositional analysis show that there are no substantial differences between the fruit of PG451 and its parent variety in terms of proximates (i.e., fat, protein, moisture, ash, carbohydrates, and calories), sugar profile, and dietary fiber. Modifications to protocols at Elements and Europhins have made it so the vitamin C and phenolics levels in both PG451 and control fruit are below the current lower limit of detection (LLOD). Elements and Eurofins labs technicians at both labs have shared their observations with the petitioner that the phenolic levels of PG451 were noticed to be 5× to 10× higher in PG451 in comparison to the parent variety. This observation is in line with all previous PPO-suppressed Arctic^® apple events (i.e., NF872, GD743, and GS784).

Elements and Eurofins reported browning of control fruit samples, after grinding the apple samples and prior to measurement of phenolics and vitamin C, consistent with this observation (i.e., higher phenolic levels). In conventional apples, mechanical damage that results in loss of compartmentalization of PPO and its phenolic substrates would result in loss of phenolics and vitamin C. Arctic^® apples lack significant quantities of PPO and preserve phenolic and vitamin C levels under these conditions. It can be reasonably expected that the preservation of phenolics and vitamin C observed in Arctic^® apples would extend to other conditions where mechanical damage compromises cellular integrity leading to enzymatic browning, such as postharvest handling, slicing or juicing.

Nutritional composition of apple fruit can vary depending on several factors such as soil/environmental conditions, harvesting time, storage conditions, sample handling in the process of analysis. Nutritional composition data for PG451 was checked for its comparability to nutritional composition data of the other Arctic^® apple events (i.e., NF872, GD743, and GS784) previously assessed by Health Canada. Most of the nutritional components of PG451 are within previously reported limits of nutritional components in Arctic^® apple events (i.e., NF872, GD743, and GS784).

The notable variations observed for PG451 (i.e., potassium, vitamin C, and phenolics) can be explained and attributed to factors other than inherent fruit composition. Health Canada in its previous assessments for Arctic^® apple events NF872, GD743, and GS784, had also observed variation in potassium and vitamin C of tested fruit, attributed to external factors. Based on all the information provided, the petitioner believes that nutritional composition of PG451 is comparable to the previously assessed Arctic^® apple events.

Potassium appears to be higher (on average) in PG451 compared to previously assessed varieties, however when the data for potassium is broken down by sample and origin rather than being an average value, it becomes clear that within each field trial, PG451 and its parent variety have very similar levels of potassium and the discrepancy is rather between the two field trials.

There are multiple factors that occurred during the 2021 season and subsequent fruit harvest that likely led to this increase in potassium. No calcium was added to the soil in Washington in 2021 which has a competitive uptake with potassium by plants ^{Footnote 5}. Less calcium to inhibit potassium uptake meant more potassium in the plant. The 2021 growing season had a significant heat dome event in Washington that produced higher temperatures than normal and increases in soil temperature have been noted to encourage the uptake of potassium by the apple trees. Finally, the fruit was harvested quite late in the season (mid-late October). This fruit was over-mature having been on the tree longer leading to increased levels of potassium to be fixed in the fruit.

In conclusion, the compositional analysis data provided further supports that the nutritional composition of PG451 is comparable to its parent variety and the previously assessed Arctic^® apple events.

Chemistry

Chemical contaminant residue data have not been provided as part of the novel food submission for PG451, nor have any unique contaminant considerations been identified. All types of apple juice and unfermented apple cider sold in Canada must comply with Health Canada's maximum level (ML) of 50 ppb patulin set out in the List of Contaminants and Other Adulterating Substances. As well, Health Canada has established MLs of 0.01 ppm inorganic arsenic (sum of arsenite (As III) and arsenate (As V)) and 0.05 ppm lead in fruit juice and fruit nectar set out in the above List.

As with any food or food ingredient sold in Canada, it is the responsibility of the food manufacturer to ensure that its use does not result in a violation of Sections 4(1)(a) and (d) of the Food and Drugs Act, which state that no person shall sell an article of food that has in or on it any poisonous or harmful substance or is adulterated. If an elevated concentration of any chemical contaminant is found in any type of food, the Bureau of Chemical Safety (BCS) may conduct a human health risk assessment to determine if there is a potential safety concern and whether risk management measures are required.

Toxicology

Data pertaining to the toxicological safety of the genetic elements present in the GEN-03 vector were provided to Health Canada in the previously assessed Arctic^® apple event NF872.

Based on the molecular characterization of PG451, there is no hypothesis to suggest that PG451 would express the NptII protein differently compared to the previously assessed Arctic^® apple events (i.e., NF872, GD743, and GS784). Furthermore, the bioinformatic analysis of the putative ORFs created from the unique T-DNA insertions sites created in PG451 identified no homology to known toxins and are not expected to result in the expression of a protein with similarity to a known toxin relevant to human health.

As mentioned above, there is no evidence that the NptII protein is toxic in animals, bioinformatic assessment has not identified homology to known toxins, and animal feeding studies have failed to establish deleterious effects of NptII (previously reviewed in the assessment of Arctic^® apple event NF872).

In conclusion, the toxicological safety of PG451 is equivalent to its parent variety and the previously assessed Arctic^® apple events.

Allergenicity

Data pertaining to the allergenic safety of the genetic elements present in the GEN-03 vector were provided to Health Canada in the previously assessed Arctic^® apple event NF872.

Based on the molecular characterization of PG451, there is no hypothesis to suggest that PG451 would express the NptII protein differently compared to the previously assessed Arctic^® apple events (i.e., NF872, GD743, and GS784). Furthermore, the bioinformatic analysis of the putative ORFs created from the unique T-DNA insertions sites created in PG451 would not result in the expression of a protein with similarity to a known allergen.

In conclusion, the allergenic safety of PG451 is equivalent to its parent variety and the previously assessed Arctic^® apple events.

Conclusion

Health Canada's review of the information presented in support of the use of PG451 does not raise concerns related to food safety.

Health Canada's opinion refers only to the food use of PG451. Issues related to its use as animal feed have been addressed separately through existing regulatory processes in the Canadian Food Inspection Agency.

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