Decision Document DD2016-114
Determination of the Safety of J.R. Simplot Company's Potato (Solanum tuberosum (L.)) Events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55

This Decision Document has been prepared to explain the regulatory decisions reached under Directive 94-08 (Dir94-08) - Assessment Criteria for Determining Environmental Safety of Plants with Novel Traits, its companion document The Biology of Solanum tuberosum (L.) (Potatoes) and Section 2.6 - Guidelines for the Assessment of Novel Feeds: Plant Sources, of Chapter 2 of the RG-1 Regulatory Guidance: Feed Registration Procedures and Labelling Standards.

The Canadian Food Inspection Agency (CFIA), specifically the Plant Biosafety Office of the Plant Health and Biosecurity Directorate, the Plant and Biotechnology Risk Assessment Unit of the Plant Health Science Directorate and the Animal Feed Division of the Animal Health Directorate, has evaluated information submitted by J.R. Simplot Company. This information is in regard to potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 with reduced levels of free amino acid asparagine, reduced levels of reducing sugars glucose and fructose and reduced levels of polyphenol oxidase. The CFIA has determined that these plants with novel traits (PNTs) do not present altered environmental risk nor, as a novel feed, do they present livestock feed safety concerns when compared to potato varieties currently grown and permitted to be used as livestock feed in Canada.

Potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 are subject to the same phytosanitary import requirements as unmodified potato varieties. Potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 must also meet the requirements of other jurisdictions, including but not limited to, the Food and Drugs Act and the Pest Control Products Act.

Please note that the livestock feed and environmental safety assessments of novel feeds and PNTs are critical steps in the potential commercialization of these plant types. Other requirements, such as the evaluation of food safety by Health Canada, have been addressed separately from this review.

(publié aussi en français)

March 18, 2016

This bulletin is published by the Canadian Food Inspection Agency. For further information, please contact the Plant Biosafety Office or the Animal Feed Division at:

1-800-442-2342
59 Camelot Drive, Ottawa
Ontario K1A 0Y9

On this page

1. Brief Identification of the Modified Plant
2. Background Information
3. Description of the Novel Traits
   1. Development Method
   2. Reduced Levels of Free Amino Acid Asparagine
   3. Reduced Levels of Reducing Sugars Glucose and Fructose
   4. Reduced levels of polyphenol oxidase
   5. Stable Integration into the Plant Genome
4. Criteria for the Environmental Assessment
   1. Potential for Potato Events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 to Become a Weed of Agriculture or be Invasive of Natural Habitats
   2. Potential for Gene Flow from Potato Events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 to Sexually Compatible Plants Whose Hybrid Offspring May Become More Weedy or More Invasive
   3. Potential for Potato Events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 to Become a Plant Pest
   4. Potential Impact of Potato Events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 and their Gene Products on Non-Target Organisms, including Humans
   5. Potential Impact of Potato Events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 on Biodiversity
5. Criteria for the Livestock Feed Assessment
   1. Potential Impact of Potato Events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 on Livestock Nutrition
   2. Potential Impact of Potato Events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 on Animal Health and Human Safety as it Relates to the Potential Transfer of Residues into Foods of Animal Origin and Worker/Bystander Exposure to the Feed
6. New Information Requirements
7. Regulatory Decision

I. Brief Identification of the Modified Plant

Designation of the Modified Plant:

Potato Events GEN1-F10 (OECD Unique Identifier SPS-ØØ-F1Ø-7), GEN1-E12 (OECD Unique Identifier SPS-ØØE12-8), GEN1-J3 (OECD Unique Identifier SPS-ØØØJ3-4) and GEN1-J55 (OECD Unique Identifier SPS-ØØJ55-2)

Applicant:

J.R. Simplot Company

Plant Species:

Potato (Solanum tuberosum (L.))

Novel Traits:

Reduced levels of free amino acid asparagine; reduced levels of reducing sugars glucose and fructose; reduced levels of polyphenol oxidase

Trait Introduction Method:

Agrobacterium-mediated transformation

Intended Use of the Modified Plant:

Intended Use of the Modified Plant: Potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 are intended to be grown for traditional potato human food and livestock feed uses. Potato Events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 are not intended to be grown outside the normal production area for potato in Canada.

II. Background Information

J.R. Simplot Company has developed four potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 that have:

• reduced levels of free amino acid asparagine, which decreases the potential for acrylamide formation during frying and baking;
• reduced levels of reducing sugars glucose and fructose, which improves the quality of tubers for processing and contributes to the decreased potential for acrylamide formation;
• reduced levels of polyphenol oxidase, which decreases the potential for black spot bruise.

The potato events were developed by J.R. Simplot Company using recombinant deoxyribonucleic acid (DNA) technology to introduce two suppression cassettes into commercial potato varieties. Potato event GEN1-F10 is an introduction into the Ranger Russet variety, potato event GEN1-E12 is an introduction into the Russet Burbank variety and potato events GEN1-J3 and GEN1-J55 are introductions into the Atlantic variety. The suppression cassettes contain gene sequences from the endogenous potato asparagine synthetase-1 (Asn1), starch associated (R1) and phosphorylase-L (PhL) genes as well as the polyphenol oxidase-5 (Ppo5) gene from Solanum verrucosum. These suppression cassettes were designed such that their transcription would result in suppression of the expression of the homologous potato genes through a process known as ribonucleic acid interference (RNAi), which is mediated by small non-coding RNAs.

Acrylamide forms when free asparagine along with reducing sugars are heated, such as during frying and baking. The Asn1 gene encodes the asparagine synthetase-1 (ASN1) enzyme, which is involved in the synthesis of asparagine. Suppression of the Asn1 gene in potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 results in reduced levels of free amino acid asparagine and therefore a decreased potential for acrylamide formation during frying and baking.

The R1 and PhL genes encode the starch associated (R1) and phosphorylase-L (PHL) enzymes, respectively. These enzymes are involved in the conversion of starch to reducing sugars, and their suppression in potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 results in lowered levels of reducing sugars. Low levels of reducing sugars in tubers improve their quality since high levels of reducing sugars can result in excessive browning during frying and baking. In addition, low levels of reducing sugars also contribute to the decreased potential for acrylamide formation during frying and baking.

The Ppo5 gene encodes the polyphenol oxidase-5 (PPO5) enzyme. This enzyme is involved in the formation of black spot bruise following damage to tubers. Its suppression in potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 results in a reduced occurrence of black spot bruising.

J.R. Simplot Company has provided information on the identity of potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55, a detailed description of the transformation method; and information on insert copy number and intactness the role of the inserted sequences, and the levels of expression of the endogenous Asn1, R1, PhL and Ppo5 genes in the plant.

Potato events GEN1-F10 and GEN1-E12 were tested in the United States (US) at 3 locations in 2009, 4 locations in 2010 and 4 locations in 2011. Potato events GEN1-J3 and GEN1-J55 were tested in the US at 1 location in 2009, 6 locations in 2010 and 6 locations in 2011. Locations share similar environmental and agronomic conditions for potato production areas in Canada and were considered representative of major Canadian potato growing regions. Data from an additional 3 locations in the US were provided for potato events GEN1-J3 and GEN1-J55 that were not representative of major Canadian growing regions but were consistent with the results from the Canadian-equivalent locations. Unmodified control potato varieties, which share the same genetic backgrounds as potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55, were included in the field trials to act as comparators for potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55. Several reference potato varieties were also included in the field trials to establish a range of typical potato behaviour.

Agronomic characteristics of potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55, such as early emergence, final emergence, stems per plant, plant vigour, foliage colour, leaflet size, leaflet curl, flower colour, senescence, vine size and vine maturity, were compared to those of their respective unmodified control potato varieties and to the range established by the reference potato varieties.

Nutritional components of potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 tubers, such as protein, moisture, total fat, ash, crude fibre, total amino acids, free amino acids, fibre, vitamins, minerals, sugars and anti-nutrients, were compared with those of their respective unmodified control potato varieties and also to the nutrient ranges established from the reference potato varieties included in the field trials.

The Plant and Biotechnology Risk Assessment Unit of the Plant Health Science Directorate, CFIA, has reviewed the above information, in light of the assessment criteria for determining environmental safety of PNTs, as described in Directive 94-08 (Dir 94-08) - Assessment Criteria for Determining Environmental Safety of Plants with Novel Traits. The Plant and Biotechnology Risk Assessment Unit has considered:

• the potential for potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 to become a weed of agriculture or to be invasive of natural habitats;
• the potential for gene flow from potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 to sexually compatible plants whose hybrid offspring may become more weedy or more invasive;
• the potential for potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 to become a plant pest;
• the potential impact of potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 and their gene products on non-target organisms, including humans; and
• the potential impact of potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 on biodiversity.

The Animal Feed Division of the CFIA has also reviewed the above information with respect to the assessment criteria for determining the safety and efficacy of livestock feed, as described in Section 2.6 - Guidelines for the Assessment of Novel Feeds: Plant Sources, of Chapter 2 of the RG-1 Regulatory Guidance: Feed Registration Procedures and Labelling Standards.

The Animal Feed Division has considered both intended and unintended effects and similarities and differences between potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55, and unmodified control potato varieties relative to the safety and efficacy of feed ingredients derived from potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55, for their intended purpose, including:

• the potential impact of potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 on livestock nutrition; and
• the potential impact of potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 on animal health and human safety, as it relates to the potential transfer of residues into foods of animal origin and worker/bystander exposure to the feed.

The Animal Feed Division has also considered whether feeds derived from potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55, meet the definitions and requirements of feeds as listed in Schedule IV of the Feeds Regulations.

III. Description of the Novel Traits

1. Development Method

Potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 were developed through Agrobacterium-mediated transformation of potato tissue and contain two suppression cassettes. The first suppression cassette contains sequences from the endogenous potato Asn1 gene and the Ppo5 gene from S. verrucosum, while the second suppression cassette contains sequences from the endogenous potato PhL and R1 genes. Transformed cells were identified using polymerase chain reaction (PCR). Potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 were selected as successful transformants for further development.

Transcription of suppression cassettes does not result in the production of novel proteins, therefore there is no risk of new toxins or allergens being introduced into potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55. Small noncoding RNAs are present in all plants and animals where they play central roles in endogenous gene regulation and response to exogenous DNA. The RNAs in Potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 are expected to function in a similar manner. Finally, the systemic absorption of exogenous RNAs in crops by livestock or human bystanders is considered to be unlikely based on numerous biological barriers.

2. Reduced Levels of Free Amino Acid Asparagine

Potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 were developed to have reduced levels of free amino acid asparagine by down-regulating the ASN1 enzyme. When potato tubers are fried or baked, the golden brown colour that forms is the result of the Maillard reaction, which occurs between free amino acids and reducing sugars. In some cases, particularly with asparagine, acrylamide can form as a by-product of this reaction. In potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55, transcription of the suppression cassette that includes the Asn1 gene sequence from potato results in suppression of the endogenous potato Asn1 gene through the RNAi process, resulting in reduced ASN1 enzyme levels. Reduced levels of the ASN1 enzyme result in reduced levels of free amino acid asparagine, which limits the potential for acrylamide formation during frying and baking.

The suppression cassettes in potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 contain promoters that are predominantly active in tubers and stolons. Northern blot analyses were used to compare the RNA levels of the endogenous Asn1 gene in tubers, leaves, stems and roots of potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 and their respective unmodified control potato varieties. The Northern blot data demonstrated a reduction in the level of Asn1 RNA compared to the unmodified control potato variety in the tubers and roots of all four events as well as in the stems of potato event GEN1-J3 and the leaves of potato event GEN1-F10.

For a more detailed discussion of the safety of RNAs produced by the suppression cassette containing the sequence from the Asn1 gene from potato, see Section V, part 2: Potential Impact of Potato Events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 on Animal Health and Human Safety as it Relates to the Potential Transfer of Residues into Foods of Animal Origin and Worker/Bystander Exposure to the Feed.

3. Reduced Levels of Reducing Sugars Glucose and Fructose

Potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 were developed to have reduced levels of reducing sugars by down-regulating the endogenous potato PhL and R1 genes. High levels of reducing sugars in potato tubers can lead to excessive browning from the Maillard reaction during frying or baking. In potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55, transcription of the suppression cassette that includes sequences from the PhL and R1 genes from potato results in suppression of the endogenous potato PhL and R1 genes through the RNAi process, resulting in reduced PHL and R1 enzyme levels. Reduced levels of these enzymes result in reduced conversion of starch to reducing sugars. This both improves the quality of potato tubers with respect to frying and baking and also contributes to limiting the potential for acrylamide formation.

The suppression cassettes in potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 contain promoters that are predominantly active in tubers and stolons. Northern blot analyses were used to compare the RNA levels of the endogenous PhL and R1 genes in tubers, leaves, stems and roots of potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 and their respective unmodified control potato varieties. The Northern blot data demonstrated a reduction in the level of PhL RNA compared to the unmodified control potato varieties in the tubers of potato events GEN1-E12, GEN1-J3 and GEN1-J55 but not GEN1-F10. No reduction in the level of PhL RNA was observed in roots, stems or leaves in potato events GEN1-E12, GEN1-F10, GEN1-J3 or GEN1-J55. The Northern blot data demonstrated a reduction in the level of R1 RNA compared to the unmodified control potato varieties in the tubers of all four events as well as in the roots of potato event GEN1-F10.

For a more detailed discussion of the safety of RNAs produced by the suppression cassettes containing the sequences from the PhL and R1 genes from potato, see Section V, part 2: Potential Impact of Potato Events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 on Animal Health and Human Safety as it Relates to the Potential Transfer of Residues into Foods of Animal Origin and Worker/Bystander Exposure to the Feed.

4. Reduced Levels of Polyphenol Oxidase

Potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 were developed to have reduced levels of the PPO5 enzyme by down-regulating the endogenous potato Ppo5 gene. PPOs are a diverse group of enzymes that are responsible for browning following damage to plant cells, such as through cutting or bruising. In potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55, transcription of the suppression cassette that includes the Ppo5 gene sequence from S. verrucosum results in the suppression of the homologous Ppo5 gene in potato through the RNAi process, resulting in reduced PPO5 enzyme levels. Thus when tubers from potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 are subjected to mechanical damage, the tuber is less likely to develop black spot bruising.

The suppression cassettes in potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 contain promoters that are predominantly active in tubers and stolons. Northern blot analyses were used to compare the RNA levels of the endogenous Ppo5 gene in tubers, leaves, stems and roots of potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 and their respective unmodified control potato varieties. The Northern blot data demonstrated a reduction in the level of Ppo5 RNA compared to the unmodified control potato varieties in the tubers and stems of all four events as well as in the roots of potato events GEN1-F10 and GEN1-E12. Ppo5 RNA was undetectable in leaves of all four events and their respective unmodified control potato varieties.

For a more detailed discussion of the safety of RNAs produced by the suppression cassette containing the sequence from the Ppo5 gene from potato, see Section V, part 2: Potential Impact of Potato Events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 on Animal Health and Human Safety as it Relates to the Potential Transfer of Residues into Foods of Animal Origin and Worker/Bystander Exposure to the Feed.

5. Stable Integration into the Plant Genome

a. Potato Event GEN1-F10

Molecular characterization by Southern blot analysis demonstrated that potato event GEN1-F10 contains a single insertion at a single site in the potato genome with one intact copy of the two suppression cassettes. No additional elements, including intact or partial DNA fragments of the suppression cassettes or backbone sequences from the plasmid vector, linked or unlinked to the intact insert, were detected in potato event GEN1-F10.

The stability of the insert within potato event GEN1-F10 was verified by Southern blot analysis over four generations of vegetative propagation.

b. Potato Event GEN1-E12

Molecular characterization by Southern blot analysis demonstrated that potato event GEN1-E12 contains a single insertion at a single site in the potato genome with one intact copy of the two suppression cassettes. No additional elements, including intact or partial DNA fragments of the suppression cassettes or backbone sequences from the plasmid vector, linked or unlinked to the intact insert, were detected in potato event GEN1-E12.

The stability of the insert within potato event GEN1-E12 was verified by Southern blot analysis over four generations of vegetative propagation.

c. Potato Event GEN1-J3

Molecular characterization by Southern blot analysis demonstrated that potato event GEN1-J3 contains three copies of the suppression cassettes inserted at a single site in the potato genome. There is one nearly intact copy of the two suppression cassettes and two partial copies. These partial copies are not expected to result in the formation of functional genes that would express novel proteins in potato event GEN1-J3. No backbone sequences from the plasmid vector, linked or unlinked to the intact insert, were detected in potato event GEN1-J3.

The stability of the insert within potato event GEN1-J3 was verified by Southern blot analysis over four generations of vegetative propagation.

d. Potato Event GEN1-J55

Molecular characterization by Southern blot analysis demonstrated that potato event GEN1-J55 contains one intact and one partial copy of the suppression cassettes inserted at a single site in the potato genome. The partial copy is not expected to result in the formation of a functional gene that would express a novel protein in potato event GEN1-J55. No backbone sequences from the plasmid vector, linked or unlinked to the intact insert, were detected in potato event GEN1-J55.

The stability of the insert within potato event GEN1-J55 was verified by Southern blot analysis over four generations of vegetative propagation.

IV. Criteria for the Environmental Assessment

1. Potential for Potato Events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 to Become a Weed of Agriculture or be Invasive of Natural Habitats

The biology of potato, described in the CFIA biology document The Biology of Solanum tuberosum (L.) (Potatoes), states that unmodified plants of this species are not invasive of unmanaged habitats in Canada. Although limited distribution is possible through dispersal of tubers, volunteers will not persist in cultivated habitats in the presence of normal agronomic practices or compete in uncultivated habitats. According to the information provided by J.R. Simplot Company, potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 were determined not to be significantly different from unmodified control potato varieties in this respect.

The CFIA evaluated data submitted by J.R. Simplot Company on the reproductive biology and life history traits of potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55. As previously mentioned, potato events GEN1-E12 and GEN1-F10 were tested in the US at 3 locations in 2009, 4 locations in 2010 and 4 locations in 2011. Potato events GEN1-J3 and GEN1-J55 were tested in the US at 1 location in 2009, 6 locations in 2010 and 6 locations in 2011. These locations share similar environmental and agronomic conditions for potato production areas in Canada and were considered representative of major Canadian potato growing regions. Data from an additional 3 locations in the US were provided for potato events GEN1-J3 and GEN1-J55 that were not representative of major Canadian growing regions but were consistent with the results from the Canadian-equivalent locations. During the field trials, potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 were compared to the unmodified control potato varieties (Russet Burbank, Ranger Russet and Atlantic, respectively). Reference potato varieties were also included in these trials to establish ranges of comparative values that are representative of currently grown potato varieties. Phenotypic and agronomic traits were evaluated, covering a broad range of characteristics that encompass the entire life cycle of the potato plant. The traits included early emergence, final emergence, stems per plant, plant vigour, foliage colour, leaflet size, leaflet curl, flower colour, senescence, vine size and vine maturity. Although instances of statistically significant differences were observed between potato events GEN-F10, GEN1-E12, GEN1-J3 and GEN1-J55 and their respective unmodified control potato varieties for some agronomic traits in the individual-site analyses, there was no consistent trend in the data across years and locations that would indicate the differences were due to the genetic modification, and the values for potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 were within the reference range established for the reference potato varieties included in the same field trials. Therefore, these instances of statistically significant differences are not considered biologically meaningful and the data support a conclusion of phenotypic and agronomic equivalence to currently grown potato varieties. In addition, tuber yield and grading characteristics were also evaluated in the field at the same locations as the agronomic characteristic studies. Some of the events showed reduced yield along with greater numbers of smaller potatoes and fewer larger potatoes. However, these observations, if they persist with commercial production, would not contribute to weediness or invasiveness potential in Canada compared to currently grown potato varieties.

Potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 were exposed to frost, hail, heat, herbicide and wind in the field during the agronomic characteristic studies. No trend in increased or decreased susceptibility to these abiotic stressors was observed in potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 and their respective unmodified control potato varieties.

The susceptibility of potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 to various potato pests and pathogens was evaluated in the field at the same locations as the agronomic characteristic studies (further detail provided below in Section 3: Potential for Potato Events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 to Become a Plant Pest). No trend in increased or decreased susceptibility to pests or pathogens was observed in potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 compared to their respective unmodified control potato varieties.

No competitive advantage was conferred to potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55, as the reproductive characteristics, growth characteristics and tolerance to pests and pathogens of potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 were comparable to those of their respective unmodified control potato varieties.

The novel traits have no intended or observed effects on weediness or invasiveness. The CFIA has therefore concluded that potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 have no altered weediness or invasiveness potential in Canada compared to currently grown potato varieties.

2. Potential for Gene Flow from Potato Events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 to Sexually Compatible Plants Whose Hybrid Offspring May Become More Weedy or More Invasive

The biology of potato, as described in the CFIA biology document The Biology of Solanum tuberosum (L.) (Potatoes), indicates that there are no sexually compatible plants in Canada that can hybridize with potato.

The novel traits introduced into potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 (reduced levels of free amino acid asparagine; reduced levels of reducing glucose and fructose; and reduced levels of PPO) are unrelated to sexual compatibility.

The CFIA has therefore concluded that gene flow from potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 to sexually compatible plants is not possible in Canada.

3. Potential for Potato Events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 to Become a Plant Pest

Potato is not considered a plant pest in Canada. Two of the novel traits introduced in potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 (reduced levels of free amino acid asparagine and reduced levels of reducing glucose and fructose) are unrelated to plant pest potential (i.e., the potential for the plant to harbour new or increased populations of pathogens or pests). The third trait, reduced levels of PPO, may be related to plant pest potential. The physiological role of PPO enzymes in potato and other plants is not well established, but there is evidence that some PPO enzymes may play a role in plant defense against pests and pathogens. Furthermore, some studies have shown that reduction of PPO enzyme activity can increase resistance to some pathogens in potato. As such, the susceptibility of potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 to various potato pests and pathogens was evaluated in the field at the same locations as the agronomic characteristic studies in 2011. The stressors observed included aphids, Colorado potato beetles, flea beetles, loopers, potato leafhoppers, seed corn maggots, black leg, Botrytis sp., early blight, late blight, Rizoctonia sp., Verticillium sp., virus and white mold. The evaluations of potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 did not show any increased or decreased susceptibility to these potato pests or pathogens compared to their respective unmodified control potato varieties.

In addition, J.R. Simplot Company quantitatively assessed the response of potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 to late blight and bacterial soft rot by intentionally infecting tubers or plants of potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 and their respective unmodified control potato varieties. Tubers from the 2011 growing season were used to test late blight susceptibility. Potato event GEN1-E12 was found to be less susceptible to late blight than its unmodified control potato variety. However, potato event GEN1-J3 was found to be more susceptible to late blight than its unmodified control potato variety. Potato events GEN1-F10 and GEN1-J55 events were found to be as susceptible to late blight as their respective unmodified control potato varieties. Late blight susceptibility was also evaluated using potato plants from the 2011 growing season. A significant difference was observed for potato event GEN1-F10 only, which was found to be less susceptible to late blight than its unmodified control potato variety. Tubers from the 2009 and 2011 growing seasons were used to test susceptibility to bacterial soft rot. Potato events GEN1-J3 and GEN1-J55 were less susceptible to soft rot than their unmodified control potato variety. Potato events GEN1-E12 and GEN1-F10 were found to be as susceptible to soft rot as their respective unmodified control potato varieties. These observations showed no consistent trend in the response of potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 to late blight or bacterial soft rot that would indicate that any difference in response to late blight or bacterial soft rot compared to their respective unmodified control potato varieties was associated with reduced PPO5 levels.

The CFIA has therefore concluded that potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 do not display any altered plant pest potential compared to currently grown potato varieties.

4. Potential Impact of Potato Events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 and their Gene Products on Non-Target Organisms, including Humans

The novel traits introduced into potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 (reduced levels of free amino acid asparagine; reduced levels of reducing glucose and fructose; and reduced levels of polyphenol oxidase) are unrelated to a potential impact on non-target organisms.

The suppression cassettes introduced into potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 do not result in the expression of a novel protein, therefore, there is no risk of new toxins or allergens being introduced into potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55. Small non-coding RNAs are present in all plants and animals where they play central roles in endogenous gene regulation and response to exogenous DNA. The suppression cassettes were not designed to mediate gene suppression in any organism interacting with potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55, and based on limited sequence conservation of the targeted genes outside of the plant kingdom, this is unlikely to occur unintentionally. Therefore, no negative impacts resulting from exposure of organisms to RNAs expressed from the suppression cassettes in potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 are expected.

Composition analyses showed that there were some statistically significant differences in key nutrients between potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 and their respective unmodified control potato varieties, including levels of asparagine and reducing sugars (glucose and fructose), as expected; however, all mean values, including levels of asparagine, glucose and fructose, were within the range established from the reference potato varieties and therefore the differences were considered to be not biologically significant (see Section V, part 1: Potential Impact of Potato Events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 on Livestock Nutrition). In addition, the glycoalkaloid levels in potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 were found to be similar to those of their respective unmodified control potato varieties. Therefore, it is unlikely that the introduction of the novel traits may have caused unintended changes to the composition of potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 tissues that would negatively impact organisms interacting with potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55.

Field evaluations of potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 did not show any increased resistance to potato pests or pathogens compared to their respective unmodified control potato varieties (see Section IV, part 3: Potential for Potato Events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 to Become a Plant Pest).

Collectively, these information elements indicate that the interactions between potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 and the populations of animals and microorganisms interacting with potato crops will be similar compared to currently grown potato varieties.

The CFIA has therefore determined that the unconfined environmental release of potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 in Canada will not result in altered impacts on non-target organisms, including humans, compared to currently grown potato varieties.

5. Potential Impact of Potato Events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 on Biodiversity

Potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 express no novel phenotypic characteristics that would extend their range beyond the current geographic range of potato production in Canada. Since potato has no sexually compatible relatives with which it can outcross in Canada, there will be no transfer of the novel traits to other species in unmanaged environments. Potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 are unlikely to cause adverse effects on non-target organisms and do not display increased weediness, invasiveness or plant pest potential. It is therefore unlikely that potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 will have any direct effects on biodiversity, in comparison to the effects that would be expected from the cultivation of potato varieties that are currently grown in Canada.

The CFIA has concluded that the introduced DNA sequences and their corresponding novel traits do not confer to potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 any characteristic that would result in unintended environmental effects following unconfined release. The CFIA has therefore concluded that the potential impact on biodiversity of potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 is unlikely to be different from that of the potato varieties that are currently grown in Canada.

V. Criteria for the Livestock Feed Assessment

1. Potential Impact of Potato Events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 on Livestock Nutrition

Nutritional and Anti-Nutrient Composition

The compositional equivalence of potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 were compared to their unmodified control potato varieties and 9 reference potato varieties from 11-15 replicated sites in the US over a 3 year period (2009-2011). Potato tuber samples (with skin) from potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 and their unmodified control potato varieties were randomly selected from each replicated block at each site. Samples were freeze-dried, ground and stored (−80°C) until compositional analysis was conducted for protein, moisture, total fat, ash, crude fibre, carbohydrates, calories, vitamins (C, B₃ and B₆), minerals (copper, magnesium and potassium), total amino acids, free amino acids, reducing sugars (fructose and glucose), sucrose and antinutrient (glycoalkaloids), as recommended by the OECD consensus document for new varieties of potatoes [OECD, 2002 - PDF (208 kb)]. In addition, the decreased acrylamide trait in processed products from potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 was evaluated during storage. Samples of potato events GEN1-F10, GEN1-E12 and their respective unmodified control potato varieties were processed into french fries, while potato events GEN1-J3, GEN1-J55 and their unmodified control potato varieties were processed into potato chips. Composition data was analysed statistically using analysis of variance and statistically significant differences among potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 and their respective unmodified control potato varieties were identified (P≤0.05). The biological relevance of any statistically significant differences between potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 and their respective unmodified control potato varieties were compared within the range of the values of the reference potato varieties grown in the trials and/or in the published scientific literature.

No statistically significant differences were observed between potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 and their respective unmodified control potato varieties for protein, moisture, total fat, ash, crude fibre, carbohydrates and calories measured. Statistically significant differences were observed between potato event GEN1-F10 and its unmodified control potato variety for vitamins B₃ and C, as well as between potato events GEN1-J3 and GEN1-J55 and their unmodified control potato variety for vitamin B₆. All means were however within the range of the values of the reference potato varieties and in the published scientific literature. No statistically significant differences were observed between potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 and their respective unmodified control potato varieties for total amino acids measured. The glycoalkaloid levels in potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 ranged from 5.7-7.0 mg/100 g FW and were not statistically significantly different from the levels of their respective unmodified control potato varieties (6.3- 7.4 mg/100 g FW). All glycoalkaloids in potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 were within the range of the values of the reference potato varieties as well as the safety limit of 20 mg/100 g FW for potato tubers (OECD 2002). Free asparagine was statistically significantly lower in potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 compared to their respective unmodified control potato varieties, but the means were within the range of the values of the reference potato varieties and in the published scientific literature. On the other hand, statistically significantly higher free glutamine was observed in potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 compared to their respective unmodified control potato varieties; however the means were within the range of the values of the reference control potato varieties and in the published scientific literature. As expected, an increase in free glutamine was observed as a direct result of silencing the gene that codes for the asparagine synthase enzyme that converts glutamine to asparagine. Free aspartic and glutamic acids were significantly higher in potato event GEN1-F10 compared to its unmodified control potato variety, but the means of potato event GEN1-F10 were within the range of the values of the reference potato varieties and in the published scientific literature. Free valine was statistically significantly lower in potato events GEN1-J3 and GEN1-J55 than their unmodified control potato varieties, however both means were within the range of the values of the reference potato varieties and therefore the differences were not considered biological relevant from a feed safety perspective. Statistically significantly lower levels of reducing sugars (fructose and glucose) was observed at the time of harvest (potato events GEN1-J3 and GEN1-J55) and/or after one month of storage (potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55), when compared to their respective unmodified control potato varieties. After 2-5 months of storage, the levels of reducing sugars in the four potato events were lower than their respective unmodified control potato varieties. All means were within the range of values of the reference potato varieties and in the published scientific literature. The levels of acrylamide were statistically significantly lower in all four processed potato events when compared to their respective unmodified control potato varieties during storage. On average the acrylamide levels in potato events GEN1-E12 and GEN1-F10 french fries and potato events GEN1-J3 and GEN1-J55 potato chips were statistically significantly lower (60-72%) than their respective unmodified control potato varieties during the storage period (2-7 months). All mean levels of the four potato events were however within the range of the values of the reference potato varieties.

Conclusion

The evidence provided by J.R. Simplot Company supports the conclusion that the nutritional composition of potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 are similar to their respective unmodified control potato varieties, except for decreased free asparagine, increased free glutamine and decreased acrylamide in processed products in storage. Feed ingredients derived from potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 are considered to meet present ingredient definitions for potatoes, since the levels of free asparagine, glutamine and acrylamide are within the range of the values established for the reference potato varieties.

2. Potential Impact of Potato Events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 on Animal Health and Human Safety as it Relates to the Potential Transfer of Residues into Foods of Animal Origin and Worker/Bystander Exposure to the Feed

Potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 display reduced levels of free amino acid asparagine, reduced levels of reducing sugars glucose and fructose as well as reduced levels of polyphenol oxidase. This is due to the suppression of four genes, Asn1, R1, PhL and Ppo5 through RNAi. The assessment of potato events GEN1-F10, GEN1-E12, GEN1-J3, and GEN1-J55 evaluated the impact of the following potential hazards relative to the safety of feed ingredients derived from this event:

• RNAi-mediated suppression of Asn1, R1, PhL and the Ppo5 genes.

RNAi-Mediated Suppression of Asn1, R1, PhL and the Ppo5 genes

Expression of the Asn1, R1, PhL and the Ppo5 suppression cassettes do not result in the production of novel proteins, therefore, it is unlikely that new toxins or allergens would be introduced into potato events GEN1-F10, GEN1-E12, GEN1-J3, and GEN1-J55. Thus, the livestock feed safety assessment of potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 focused on the safety of the reduced abundance of ASN1, R1, PHL and PPO5 enzymes, and the RNAs. Reduction of ASN1, R1, PHL and PPO5 enzyme levels in potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 were not considered to present a risk to human or animal health or the environment as they are not associated with a toxic mode of action. There was no reason to believe that reduction of ASN1, R1, PHL and PPO5 enzyme levels would impact endogenous potato toxin or allergen levels. Small noncoding RNAs present in potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 were not considered to present a risk to human or animal health or the environment because bioinformatic analyses demonstrated that the DNA sequences inserted into potato events GEN1-F10, GEN1-E12, GEN1-J3, and GEN1-J55 did not have significant similarity to protein-coding genomic regions of livestock animals or humans thus were not expected to affect livestock or human bystander protein expression. The Asn1, R1 and PhL genes are derived from S. tuberosum and as such they have a history of consumption. In addition, RNAs play central roles in endogenous gene regulation and response to exogenous DNA in plants and animals. The RNAs in Potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 are expected to function in a similar manner. Finally, the systemic absorption of exogenous RNAs in crops by livestock or human bystanders is considered to be unlikely based on numerous biological barriers.

Conclusions

It was concluded, based on the evidence provided by J.R. Simplot Company, that the novel reduced levels of free amino acid asparagine, reduced levels of reducing sugars glucose and fructose, and reduced levels of polyphenol oxidase traits will not confer to potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 any characteristic that would raise concerns regarding the safety of potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55. Feed ingredients derived from potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 are considered to meet present ingredient definitions for potato.

VI. New Information Requirements

If at any time, J.R. Simplot Company becomes aware of any new information regarding risk to the environment, livestock or human health, which could result from release or livestock feed use of potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 or lines derived from it, J.R. Simplot Company is required to immediately provide such information to the CFIA. On the basis of such new information, the CFIA will re-evaluate the potential impact of potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 on the environment, livestock and human health and may re-evaluate its decision with respect to the livestock feed use and environmental release authorizations of potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55.

VII. Regulatory Decision

Based on the review of the data and information submitted by J.R. Simplot Company and input from other relevant scientific sources, the Plant and Biotechnology Risk Assessment Unit of the Plant Health Science Directorate, CFIA, has concluded that the unconfined environmental release of potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 does not present altered environmental risk when compared to potato varieties that are currently grown in Canada.

Based on the review of the data and information submitted by J.R. Simplot Company and input from other relevant scientific sources, the Animal Feed Division of the Animal Health Directorate, CFIA, has concluded that the novel reduced levels of free amino acid asparagine, reduced levels of reducing sugars glucose and fructose and reduced levels of polyphenol oxidase traits will not confer to potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 any characteristic that would raise any concerns regarding the safety or nutritional composition of potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55. Potato and its by-products are currently listed in IV of the Feeds Regulations and are, therefore approved for use in livestock feeds in Canada. Potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 have been found to be as safe as and as nutritious as currently and historically grown potato varieties. Potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 and their products are considered to meet present ingredient definitions and are approved for use as livestock feed ingredients in Canada.

Potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 are subject to the same phytosanitary import requirements as unmodified potato varieties. Potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55 must also meet the requirements of other jurisdictions, including but not limited to, the Food and Drugs Act and the Pest Control Products Act.

Please refer to Health Canada's Decisions on Novel Foods for a description of the food safety assessment of potato events GEN1-F10, GEN1-E12, GEN1-J3 and GEN1-J55.