Simplot Innate® Potato Events Gen1-E12, Gen1-F10, Gen1-J3, and Gen1-J55

Health Canada has notified J.R. Simplot Company that it has no objection to the food use of Simplot Innate^® Potato Events Gen1-E12, Gen1-F10, Gen1-J3, and Gen1-J55. The Department conducted a comprehensive assessment of these potato events 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 four genetically modified potato events (i.e. 1 Russet Burbank variety, 1 Ranger Russet variety, and 2 Atlantic varieties, respectively) using recombinant DNA techniques. These potato events exhibit 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 decrease acrylamide formation and limit heat-induced browning. These potato events also exhibit 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 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 these potato events were developed; how the composition and nutritional quality of these events compared to non-modified potato varieties; and the potential for these potato events to be toxic or cause allergic reactions. J.R. Simplot Company has provided data that demonstrate that Simplot Innate^® Potato Events Gen1-E12, Gen1-F10, Gen1-J3, and Gen1-J55 are 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 Events Gen1-E12, Gen1-F10, Gen1-J3, and Gen1-J55 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

(i) the plant, animal or microorganism exhibits characteristics that were not previously observed in that plant, animal or microorganism.

2. Development of the Modified Plants

The petitioner has provided information describing the methods used to develop Simplot Innate^® Potato Events Gen1-E12, Gen1-F10, Gen1-J3, and Gen1-J55 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 varieties Russet Burbank (for event Gen1-E12), Ranger Russet (for event Gen1-F10), and Atlantic (for events Gen1-J3 and Gen1-J55) 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 each of the Simplot Innate^® potato events 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 α-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 (α-glucan, water dikinase) that catalyzes the transfer of the γ- and β-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) (1^st 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) (1^st 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) (2^nd 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) (2^nd 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 Events Gen1-E12, Gen1-F10, Gen1-J3, and Gen1-J55 were 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 and DNA sequencing of Simplot Innate^® Potato Events Gen1-E12, Gen1-F10, Gen1-J3, and Gen1-J55 demonstrated the presence of the T-DNA expression cassette in each transformed event.

For events Gen1-E12 (Russet Burbank) and Gen1-F10 (Ranger Russet), Southern blot analysis confirmed that their respective genomes contain a single complete copy of the T-DNA expression cassette (from the Left Border sequence to the Right Border sequence).

For event Gen1-J3, Southern blot analysis coupled with direct sequence information revealed a complex rearrangement of the T-DNA expression cassette which is described as three sections (i.e. left, middle, and right) inserted at a single locus within the host genome. The left section of the insert consists of an Asn1/Ppo5 inverted repeat sequence in the reverse orientation and includes a truncated pAgp oriented to the flanking genome region. The middle section of the insert represents a complete copy of the T-DNA expression cassette except for the deletion of the T-DNA Left Border sequence, originally adjacent to the pAgp. The right section of the insert consists of a partial PhL/R1 inverted repeat sequence at the junction site that was adjacent to the long form of the pGbss. Overall, the single insertion site in the event Gen1-J3 genome contains a single complete copy of the T-DNA expression cassette, plus an additional Asn1/Ppo5 inverted repeat sequence and a partial PhL/R1 inverted repeat sequence. The petitioner addressed the potential for these partial sequences in the left and right sections of the insert to result in the expression of a novel protein product (e.g. by polymerase read-through into the flanking genomic sequence). While there is a pAgp element in the reverse orientation in the left section of the insert, it is a small, non-functional version of the pAgp promoter and would not initiate transcription. Similarly, there is another pGbss element in the right section of the insert however Open Reading Frame (ORF) analysis demonstrated that no potential ORFs span the junction sequence between the insert DNA and flanking genomic DNA. Thus, it is highly unlikely that any unintended expressed product would result from these partial insert sequences in the left and right sections of the Gen1-J3 insert.

For event Gen1-J55, Southern blot analysis coupled with direct sequence information revealed another rearrangement of the T-DNA expression cassette inserted at a single locus within the host genome. The insert contains one complete copy of the T-DNA expression cassette, coupled to a truncated copy of the cassette in the reverse orientation that lacks the PhL/R1 inverted repeat sequence (i.e. partial sequence of the 2^nd copy of the pAgp meets at a junction with the T-DNA Right Border sequence). The only expressed product resulting from the truncated copy of the cassette would be another copy of the Asn1/Ppo5 dsRNA molecule due to the converging nature of the intact pAgp and pGbss promoters.

For each of the transformed events, Southern blot analysis confirmed an absence of the pSIM1278 plasmid backbone sequence in each host genome.

The potato is a vegetatively propagated crop and reproduction does not involve meiosis. Thus the T-DNA insertions within potato events Gen1-E12, Gen1-F10, Gen1-J3, and Gen1-J55 were expected to be genetically stable. Nevertheless, stability of each T-DNA insert was confirmed over 4 generations of each event by Southern blot analysis.

4. Product Information

Simplot Innate^® Potato Events Gen1-E12, Gen1-F10, Gen1-J3, and Gen1-J55 differ from their traditional counterparts 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 events contain 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 events would not be expected to pose a safety concern.

The T-DNA present in Simplot Innate^® potato events do 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 events to changes at the molecular level. RNA was isolated from various tissues of each event and their respective untransformed counterparts 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 proteins were mostly reduced in events Gen1-F10, Gen1-E12, Gen1-J3, and Gen1-J55. Reduced transcript levels for the Asn1 and Ppo5 genes were observed in tuber tissue for all four events and are associated with the reduced concentration of asparagine in these tissues (and thus low-acrylamide potential) and black spot bruise tolerance, respectively. Reduced transcript levels for the PhL gene were expected in the tuber tissues of all events and partial reduction was observed in all events except Gen1-F10. As previously mentioned, reduced concentrations of PhL protein is linked with reduced concentrations of glucose and fructose. Transcripts for the R1 gene were partially reduced in the tubers of all events to help limit the degradation of starch into sugars. As noted by the petitioner, the lack of reduction of PhL transcripts in event Gen1-F10 tissues along with only slight reductions of R1 transcripts in all the events, show that 'silencing' of the PhL and R1 genes was not as pronounced as expected, yet the intended phenotypes were observed for all the events.

5. Dietary Exposure

It is expected that Simplot Innate^® Potato Events Gen1-E12, Gen1-F10, Gen1-J3, and Gen1-J55 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 these transformed events.

6. Nutrition

Simplot Innate^® potato events (i.e. Gen1-E12, Gen1-F10, Gen1-J3, and Gen1-J55) were grown in confined field trials of randomized complete block design with 3, 4, or 5 replicates per line over 3 years, with parental lines and other cultivated varieties of potato as controls, to identify any biologically relevant differences in the nutritional and anti-nutritional compounds which could result in an increased risk to humans when used for food, to animals when used as feed, or other non-target organisms.

For statistical analysis, all attributes for the Russet Burbank, Ranger Russet, and Atlantic varieties were analyzed by combining data from multiple test years and locations using a linear mixed effect model.

The petitioner's compositional assessment of the four potato events included the amounts of: proximates (i.e. moisture, protein, total fat, ash, crude fibre, carbohydrate; calories; vitamins B3, B6, and C; minerals (i.e. copper, magnesium, and potassium)); glycoalkaloids; free amino acids; total amino acids; and reducing sugars for tubers collected from events grown in potato-growing areas of the United States.

The petitioner provided summaries and detailed information on comparisons of the nutrient composition of tubers from the transformed 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 their 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 and magnesium in addition to the recommended potassium. Comparative analyses were further facilitated by the petitioner's provision of combined literature range values and their own experimental tolerance intervals for several commercial and proprietary varieties of potatoes grown under identical conditions to the transformed events and their corresponding controls.

Statistically significant differences were observed in the following analytes: increased vitamin B3 and C (event Gen1-F10), and decreased vitamin B6 (events Gen1-J3 and Gen1-J55); total amino acids: decreased aspartate and asparagine (events Gen1-F10 and Gen1-E12), and increased glutamate and glutamine (events Gen1-F10 and Gen1-E12); free amino acids: decreased asparagine (events Gen1-F10, Gen1-J3, and Gen1-J55), and increased glutamine (event Gen1-F10); and reducing sugars (one month after harvest): decreased glucose (all events), and decreased sucrose (event Gen1-E12). However, the levels of analytes in the transformed events fell within the ranges of the levels of those analytes in conventional potatoes that have been reported in the scientific literature including the OECD (2002) guide for new varieties of potatoes.

The results for vitamin content showed that the vitamin B3, B6, and C values for the events and their controls all fell within the tolerance intervals and combined literature ranges provided by the petitioner and the range of vitamin C mentioned in the OECD document. Mineral nutrient composition results were also within tolerance intervals and combined literature ranges as well as the potassium range of the OECD document.

The intended effects on levels of nutrients in the Simplot Innate^® potato events Gen1-E12, Gen1-F10, Gen1-J3, and Gen1-J55 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 levels of these analytes in the transformed events are within the ranges found in conventional potatoes and do not present an increased nutritional risk. Another intended effect is a reduction in oxidized phenols comprising the precipitated dark pigments of black spot bruises. This change does not appear to have adversely affected nutrient levels based on compositional analyses of the events, as all analytes were within the ranges found in conventional potatoes.

The intended changes in analytes are accompanied by certain predictable changes in other analytes. 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 glutamine to asparagine. Reducing the expression of the PhL gene coding for starch phosphorylase enzyme and the R1 gene coding for the starch-associated α-glucan water dikinase enzyme reduced the breakdown of starch into glucose and fructose as intended, and also reduced the production of sucrose which is a disaccharide of glucose and fructose. Since these nutrients are also at levels in the transformed events that are within ranges found in conventional potato varieties, there does not appear to be any increased nutritional risk from these consequential changes.

Reducing the expression the polyphenol oxidase-5 enzyme, for the purpose of reducing the oxidation of polyphenols to precipitated dark pigments, may also have unintended effects. Information available in the scientific literature 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 Events Gen1-E12, Gen1-F10, Gen1-J3, and Gen1-J55 tubers have similar compositions compared to their non-transgenic controls and therefore would not pose an increased nutritional risk to consumers.

7. Chemistry/Toxicology

The petitioner stated that Simplot Innate^® potatoes do not produce any novel proteins as all the RNA products that will be 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 these transformed events.

The petitioner stated that siRNA generated by the potato events would not be bioavailable, and that most of the siRNAs would be degraded during cooking or digested when consumed. They also stated that siRNAs may be prevented from reaching target tissues due to the numerous biological barriers that exist in the human body. Although plausible, no data were provided to support these statements.

The petitioner demonstrated that all potential siRNA sequences derived from the T-DNA inserts in the transformed events do not share perfect sequence complementarity with human mRNA transcripts (RefSeq: NCBI Reference Sequence RNA database; 163,241 sequences) or protein-coding sequences in the human genome (i.e. exons; human genome published by the University of California, Santa Cruz). These data support the statement that the siRNAs are not expected to have an off-target effect in humans (e.g. reduce levels of human mRNAs), and therefore would not be expected to pose a hazard.

The petitioner provided an estimated exposure to Simplot Innate^® potato siRNAs that was calculated using data from published literature. The petitioner and PTAS calculated levels of siRNA exposure in the greatest consumers (i.e. 95^th percentile), based on very conservative assumptions. For example, it was assumed that all siRNAs produced by Simplot Innate^® 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. On this basis, the Simplot Innate^® potato events would not be expected to pose a hazard.

Glycoalkaloids, endogenous toxins naturally present in potatoes, were present in Simplot Innate^® potato events at levels consistent with conventional potato varieties (i.e. <200 mg/kg fresh tuber) and therefore these levels were 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 these transformed potato events.

Based on the available data regarding the toxicological and allergenic potential of Simplot Innate^® potato events Gen1-E12, Gen1-F10, Gen1-J3, and Gen1-J55, 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 events Gen1-E12, Gen1-F10, Gen1-J3, and Gen1-J55 does not raise concerns related to food safety. Health Canada is of the opinion that food derived from these transformed events 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 events Gen1-E12, Gen1-F10, Gen1-J3, and Gen1-J55. Issues related to its use as animal feed have been addressed separately through existing regulatory processes in the Canadian Food Inspection Agency (CFIA). The CFIA evaluated information provided on the environmental, animal, and human health safety of Simplot Innate^® potato events Gen1-E12, Gen1-F10, Gen1-J3, and Gen1-J55 with the intended use in animal feed.  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 Simplot Innate^® potato events Gen1-E12, Gen1-F10, Gen1-J3, and Gen1-J55 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.

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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
novelfoods-alimentsnouveaux@hc-sc.gc.ca