Novel Food Information - Simplot Innate^® Potato Event Gen1-V11

Health Canada has notified J.R. Simplot Company that it has no objection to the food use of Simplot Innate® potato event Gen1-V11. The Department conducted a comprehensive assessment of this potato 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 J.R. Simplot Company and the evaluation by Heath Canada and contains no confidential business information.

1. Introduction

J.R. Simplot Company has developed a genetically modified potato event (Snowden variety) using recombinant DNA techniques. This potato event exhibits reduced levels of free asparagine in tubers, as well as lower levels of reducing sugars (i.e. glucose and fructose) in tubers. The reduction in asparagine alone significantly decreases acrylamide formation in tubers when subjected to various forms of thermal processing (e.g. baking, frying, etc.) however lower levels of reducing sugars further decreases acrylamide formation and limit heat-induced browning. This potato event also exhibits reduced expression of polyphenol oxidase 5 enzyme (Ppo5), resulting in a decreased incidence of black spot bruising in tubers.

These novel traits are achieved through the transcription of two inverted repeat sequences containing small fragments of DNA from four different endogenous genes (i.e. Asn1, Ppo5, PhL, and R1), which results in the reduced level of mRNA transcripts (and subsequently expressed proteins) for those same genes using the RNA interference (RNAi) pathway.

The novel traits exhibited by Simplot Innate® potato event Gen1-V11 are identical to traits previously assessed by Health Canada in the Simplot Innate® potato events Gen1-E12, Gen1-F10, Gen1-J3, and Gen1-J55 (Health Canada, 2016) and are achieved using identical genetic elements.

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 this potato event was developed; how the composition and nutritional quality of this event compared to non-modified potato varieties; and the potential for this potato event to be toxic or cause allergic reactions. J.R. Simplot Company has provided data that demonstrate that Simplot Innate® potato event Gen1-V11 is as safe and of the same nutritional quality as traditional potato 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 28). Simplot Innate® potato event Gen1-V11 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

(i) 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 Simplot Innate® potato event Gen1-V11 and the molecular biology data that characterize the genetic change, which results in reduced levels of free asparagine in tubers, lower levels of reducing sugars (i.e. glucose and fructose) in tubers, and reduced expression of polyphenol oxidase 5 enzyme (Ppo5). This phenotype was achieved by transformation of the conventional potato variety Snowden with a transgenic (T-DNA) expression cassette containing two inverted repeat sequences consisting of specific DNA fragments from four endogenous genes (i.e. Asn1, Ppo5, PhL, and R1). The target genes were chosen based upon their biochemical roles in asparagine production, accumulation of reducing sugars, or black spot bruising. The T-DNA insert in Simplot Innate® potato event Gen1-V11 contains two native promoters (i.e. pAgp and pGbss) that drive the transcription of two inverted repeat sequences, primarily in tuber tissue. Expression of these inverted repeat sequences results in the reduced level of mRNA (and subsequently expressed proteins) for those same genes using the RNA interference (RNAi) pathway.

The Asn1 gene encodes an asparagine synthetase 1 (Asn1) enzyme that catalyses the conversion of glutamine to asparagine by transferring the side-chain amine (NH2) from glutamine to aspartate to form asparagine. Asparagine is a substrate of the Maillard reaction which converts amino acids and reducing sugars to acrylamide during high-temperature processing. Reduction of Asn1 and asparagine levels in potato tubers results in a decreased potential for acrylamide formation in cooked potato food products.

The Ppo5 gene (as previously mentioned) encodes a polyphenol oxidase 5 (Ppo5) enzyme that catalyzes the conversion of o-diphenols to o-quinones. Reactive o-quinone molecules auto-polymerize to form melanins, which are responsible for the colouration of oxidized plant tissues. A loss of Ppo5 activity limits the colouration of oxidized tissues (thus reducing black spot bruising).

The PhL gene encodes an a-glucan phosphorylase, starch phosphorylase L (PhL) that degrades starch by phosphorolytic release of glucose-1-phosphate from glucan chains. A loss of PhL activity limits reducing sugar accumulation (further contributing to the lower potential for acrylamide formation).

The R1 gene encodes a starch-related R1 protein (a-glucan, water dikinase) that catalyzes the transfer of the g- and b-phosphates of ATP (through a phosphor-histidine intermediate) to α-glucan and water, resulting in phosphorylated starch. R1 is mainly responsible for phosphorylation at the C6 position. Phosphorylation affects the degree of crystalline packing within the starch granule and makes it more accessible to degradation. Thus, loss of R1 activity impairs starch degradation, which reduces accumulation of reducing sugars (further contributing to the lower acrylamide potential phenotype).

Due to the nature of the inverted repeat sequences, their transcripts form double-stranded RNA (dsRNA) molecules through complementary binding. The dsRNA molecules act as a precursor for the plant's own RNAi post-transcriptional regulatory pathway. A cellular RNase III enzyme (i.e. Dicer) recognizes and processes these precursor dsRNA molecules into small, 21-bp duplexes consisting of two individual strands (denoted as 'guide' and 'passenger', respectively) termed small interfering RNA (siRNA). The siRNA duplexes are subsequently bound by the RNA Induced Silencing Complex (RISC), which selectively degrades the 'passenger' strand of each duplex. The 'guide' strand (still bound to the Complex) serves to activate RISC and turn it into a silencing complex. In plants, the activated RISC binds to any messenger RNA (mRNA) that has complete complementary sequence to the bound 'guide' strand, and destroys the bound mRNA by enzymatic cleavage. The cleavage of the target mRNA can result in reduced expression of the associated protein (although this reduction in expression can vary for individual siRNA).

The T-DNA expression cassette contains the following genetic elements: the promoter for an ADP glucose pyrophosphorylase gene (pAgp) (1st copy), one of two convergent promoters that drive the expression of an inverted repeat sequence containing fragments of the Asn1 and Ppo5 genes, derived from Solanum tuberosum (potato) var. Ranger Russet; the inverted repeat sequence containing fragments of the Asn1 and Ppo5 genes, the promoter for the granule-bound starch synthase (pGbss) gene (in the reverse orientation) (1st copy), the second of two convergent promoters that drive the expression of the inverted repeat sequence containing fragments of the Asn1 and Ppo5 genes, derived from S. tuberosum var. Ranger Russet; the promoter for an ADP glucose pyrophosphorylase gene (pAgp) (2nd copy), one of two convergent promoters that drive the expression of an inverted repeat sequence containing fragments of the PhL and R1 genes, derived from S. tuberosum var. Ranger Russet; the inverted repeat sequence containing fragments of the PhL and R1 genes, and the promoter for the granule-bound starch synthase (pGbss) gene (in the reverse orientation) (2nd copy), the second of two convergent promoters that drive the expression of the inverted repeat sequence containing fragments of the PhL and R1 genes, derived from S. tuberosum var. Ranger Russet.

Simplot Innate® potato event Gen1-V11 was developed using Agrobacterium-mediated transformation of 4-week old potato internode segments (of 4 to 6 mm) using the transformation plasmid pSIM1278 (containing the T-DNA expression cassette).

3. Characterization of the Modified Plants

Southern blot analysis of Simplot Innate® potato event Gen1-V11 demonstrated the presence of the T-DNA expression cassette in the transformed event. The analysis confirmed that Simplot Innate® potato event Gen1-V11 contains a single complete copy of the T-DNA expression cassette (from the Left Border sequence to the Right Border sequence). Further sequence analyses revealed that the single insert has a 14-bp deletion of the Left Border sequence and a 3-bp deletion of the Right Border sequence.

Furthermore, Southern blot analysis confirmed an absence of the pSIM1278 plasmid backbone sequence in the recipient genome.

The potato is a vegetatively propagated crop and reproduction does not involve meiosis. Thus the T-DNA insertion within Simplot Innate® potato event Gen1-V11 was expected to be genetically stable. Nevertheless, stability of the T-DNA insert was confirmed over multiple propagations by Southern blot analysis.

4. Product Information

Simplot Innate® potato event Gen1-V11 differs from its traditional counterpart by the addition of two inverted repeat sequences containing sequence fragments of 4 endogenous genes (i.e. Asn1, Ppo5, PhL, and R1), and the converging promoters (i.e. pAgp and pGbss) that drive their expression. Expression of these inverted repeat sequences results in the reduced level of mRNA (and subsequently expressed proteins) for those same genes using the RNA interference (RNAi) pathway of the host plant. Reduction in the protein expression of Asn1, Ppo5, PhL, and R1 (for individual functions see Section 2: Development of the Modified Plants) results in reduced levels of asparagine and reducing sugars (i.e. glucose and fructose) in tubers as well as lower levels of the Ppo5 enzyme. The reduction in asparagine alone significantly decreases acrylamide formation in tubers when subjected to various forms of thermal processing (e.g. baking, frying, etc.) however lower levels of reducing sugars further decreases acrylamide formation and limits heat-induced browning. Reducing levels of the Ppo5 enzyme results in a decreased occurrence of black spot bruising in tubers.

The host organism, Solanum tuberosum (potato), is produced in all provinces and territories of Canada and is an integral part of the world's food supply. Many varieties of this species have a long history of safe use as food. Simplot Innate® potato event Gen1-V11 contains genomic DNA sequences derived from conventional potato and a wild potato species (Solanum verrucosum). According to the Organization for Economic Cooperation and Development (OECD), S. verrucosum is an acceptable source of genetic material used to improve potato varieties and has a history of safe food use. Thus, the use of genetic material from both sources in Simplot Innate® potato event Gen1-V11 would not be expected to pose a safety concern.

The T-DNA present in Simplot Innate® potato event Gen1-V11 does not result in the expression of a novel protein(s), but rather the expression of dsRNA molecules that are further processed by the endogenous enzyme Dicer into small siRNAs that will subsequently activate the RNAi pathway, resulting in reduced levels of the mRNA transcripts for four endogenous proteins (i.e. Asn1, Ppo5, PhL, and R1). As such, mRNA transcript levels (rather than protein concentrations) were evaluated to link the new phenotypic traits in Simplot Innate® potato event Gen1-V11 to changes at the molecular level. RNA was isolated from various tissues of Simplot Innate® potato event Gen1-V11 and its untransformed counterpart and used to carry out Northern blot analyses.

Based on the Northern blot analyses, it was demonstrated that mRNA transcript levels for the four target genes were mostly reduced in tubers, the primary target tissue. This was expected as the promoters (i.e. pAgp and pGbss) express primarily in tubers. Reduced transcript levels in the other tissues were observed for Asn1 in leaves and stems. This pattern of RNAi-based down-regulation is similar to what was observed for previously assessed Simplot Innate® potato events Gen1-E12, Gen1-F10, Gen1-J3, and Gen1-J55 (Health Canada, 2016).

5. Dietary Exposure

It is expected that Simplot Innate® potato event Gen1-V11 will be used in applications similar to conventional potato varieties. The petitioner does not anticipate a significant change in the food use of potatoes with the introduction of this transformed event.

6. Nutrition

Simplot Innate® potato event Gen1-V11 potatoes were grown in field trials of randomized complete block design with 3 or 4 replicates per line in 2012 at 2 sites (i.e. St. Johns, Florida;; Montcalm, Michigan) and in 2013 at 4 sites (Montcalm, Michigan; Walworth, Wisconsin; Grant, Washington; Berks, Pennsylvania), with the parental control, Snowden, and other reference varieties to identify any biologically relevant differences in the nutritional and anti-nutritional compounds.

The nutritional and compositional analytes measured in the Simplot Innate® potato event Gen1-V11 potato, non-transgenic Snowden control and reference varieties were:  proximates (i.e., moisture, protein, total fat, ash, crude fibre, carbohydrate, and calories), vitamins (i.e. B3, B6 and C), minerals (i.e., copper, magnesium, and potassium); total amino acids; free amino acids (i.e. asparagine, aspartic acid, glutamine, and glutamic acid); and reducing sugars.

The petitioner provided summaries and detailed information on comparisons on the nutrient composition of tubers from the Simplot Innate® potato event Gen1-V11 and the control potatoes as per the recommendations of the 2002 Organization for Economic Co-Operation and Development (OECD) Consensus Document on Compositional Considerations for New Varieties of Potatoes.  In addition to protein content, the petitioner provided the total and free amino acid concentrations due to its relevance to the transformed events. In addition to the OECD recommended vitamin C analysis, the petitioner provided data on vitamin B3 and B6, as well as the content of copper, magnesium, and potassium.

Statistical differences were observed in the following analytes in Simplot Innate® potato event Gen1-V11 potatoes compared to controls: Vitamin C (higher); total amino acids:  aspartic acid and asparagine (42% lower in Gen1-V11 potatoes compared to the control), and higher levels of glutamic acid and glutamine, alanine, arginine, glycine, isoleucine, leucine, serine, threonine, tyrosine and valine; free amino acids: asparagine (74% lower in Gen1-V11 potatoes compared to the control) and glutamine (higher). However, the levels of analytes in the Simplot Innate® potatoes fell within the ranges of the levels of those analytes in the reference varieties and/or conventional potatoes that have been reported in the scientific literature including the OECD (2002) guide for new varieties of potatoes.

The intended effects on levels of nutrients in the Simplot Innate® potato event Gen1-V11 potatoes are reductions in the content of asparagine and the reducing sugars glucose and fructose, in order to reduce acrylamide formation during high-temperature processing (e.g., the production of fries and chips). The level of asparagine in the Simplot Innate® potato event Gen1-V11 potatoes was reduced as intended. The asparagine levels in both Simplot Innate® potato event Gen1-V11 potatoes and the control are, however, not outside ranges found in reference varieties. The levels of reducing sugars in the Simplot Innate® potato event Gen1-V11 potatoes however were not significantly different from the control.

The intended changes in analytes are accompanied by certain predictable changes in other amino acids. An increase in glutamine is a direct result of a reduction in the expression of the Asn1 gene that codes for the asparagine synthetase enzyme that converts aspartate to asparagine. Concomitantly, due to the lower asparagine levels, there were small but significantly higher levels of some amino acids (listed above) when considered as a percent of total amino acids, with glutamine being the highest.  Since these nutrient levels in the Simplot Innate® potato event Gen1-V11 potatoes are within ranges found in reference varieties, there is no nutritional safety concern.

Reducing the expression of the Ppo5 gene that codes for the polyphenol oxidase-5 enzyme, for the purpose of reducing the oxidation of polyphenols to precipitated dark pigments, may also have unintended effects. Literature information suggests that reduced polyphenol oxidase activity could reduce degradation of antioxidant polyphenols and reduce production of potentially toxic plant defense compounds such as quinones. Thus, these unmeasured but plausible unintended effects are unlikely to pose any increased nutritional risk and could be beneficial.

The petitioner has sufficiently demonstrated that Simplot Innate® potato event Gen1-V11 tubers have similar compositions compared to its non-transgenic control and therefore would not pose an increased nutritional risk to consumers.

7. Chemistry/Toxicology

Simplot Innate® potato event Gen1-V11 contains the same genetic insert as previously approved Simplot Innate® potato events Gen1-E12, Gen1-F10, Gen1-J3 and Gen1-J55 which were found to be as safe as conventional potatoes.

The Simplot Innate® potato event Gen1-V11 potato contains genomic DNA sequences derived from conventional potato (Solanum tuberosum) and wild potato (Solanum verrucosum).  According to the Organisation for Economic Co-operation and Development (OECD, 1997), S. verrucosum is an acceptable source of genetic material used to improve potato varieties and has a history of safe food use.  Thus, the use of genetic sequences from both sources in Simplot Innate® potato event Gen1-V11 potatoes would not be expected to pose a toxicological concern.

The petitioner stated that Simplot Innate® potato event Gen1-V11 potatoes do not produce any novel proteins as all the RNA products that are transcribed from the transgenes will be processed into siRNA. In the absence of any exposure to novel proteins, there would be no additional hazard from novel toxins or allergens in this transformed event.

The petitioner demonstrated that all potential siRNA sequences derived from the DNA inserts do not share perfect sequence complementarity with human transcripts (RefSeq; NCBI Reference Sequence RNA database; 163 241 sequences) or protein-coding sequences in the human genome (exons; human genome published by the University of California Santa Cruz).  These data support the argument that the siRNAs are not expected to have an off-target effect, such as silencing of human mRNAs, and therefore would not be expected to create a hazard.

The petitioner provided an exposure estimate for Simplot Innate® potato event Gen1-V11 potato siRNA that was calculated using data from published literature. The petitioner and Health Canada calculated levels of siRNA exposure in the greatest consumers, based on very conservative assumptions.  For example, it was assumed that all siRNAs produced by Simplot Innate® potato event Gen1-V11 potatoes would become bioavailable and reach target tissues, when consumed.  In both cases, the estimates of exposure to siRNAs were below the biologically relevant concentrations that were previously reported in the published literature.  However, it was acknowledged that much of the siRNAs would likely not be bioavailable, since they would probably be degraded during cooking or digestion and would be largely prevented from reaching target tissues due to numerous biological barriers that exist in the human body. On this basis, the Simplot Innate® potato event Gen1-V11 potato would not be expected to pose a hazard.

The level of glycoalkaloids, an endogenous toxin naturally present in potatoes, was present in Simplot Innate® potato event Gen1-V11 potatoes at levels consistent with conventional potatoes and therefore, the level was not considered to pose a health concern.

Additionally, there is no mechanistic reason to suggest that the level of patatin (an endogenous allergen found in conventional potato varieties) would be elevated in this transformed potato event.

Based on the available data regarding the toxicological and allergenic potential of Simplot Innate® potato event Gen1-V11, these potatoes are considered to be as safe as conventional potato varieties currently available in the Canadian marketplace from the perspective of toxicological and allergenic risk.

Conclusion:

Health Canada's review of the information presented in support of the food use of Simplot Innate® potato event Gen1-V11 does not raise concerns related to food safety. Health Canada is of the opinion that food derived from this transformed event to be as safe and nutritious as food from current commercial potato varieties.

Health Canada's opinion deals only with the food use of Simplot Innate® potato event Gen1-V11. Issues related to its use as animal feed have been addressed separately through existing regulatory processes in the Canadian Food Inspection Agency (CFIA). As Simplot Innate® potato event Gen1-V11 contains the identical construct and exhibits the same phenotype as previously assessed for Simplot Innate® potato events Gen1-E12, Gen1-F10, Gen1-J3, and Gen1-J55, this event meets the definition of a retransformation event according to the CFIA's retransformation/remutation policies.