Decision Document DD2015-110
Determination of the Safety of Okanagan Specialty Fruits Inc.'s Apple (Malus domestica Borkh) Events GD743 and GS784

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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 biology document BIO2014-01 The Biology of Malus domestica Borkh (Apple) 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 Okanagan Specialty Fruits Inc. This information concerns the apple events GD743 and GS784, which display kanamycin resistance and reduced enzymatic browning. The CFIA has determined that this plant with a novel trait (PNT) does not present altered environmental risk nor, as a novel feed, does it present livestock feed safety concerns when compared to apple varieties currently grown and permitted to be used as livestock feed in Canada.

Taking into account these evaluations, unconfined release into the environment and use as livestock feed of apple events GD743 and GS784 are therefore authorized by the Plant Biosafety Office of the Plant Health and Biosecurity Directorate and the Animal Feed Division of the Animal Health Directorate, respectively, as of March 20, 2015. Any apple lines derived from apple events GD743 and GS784 may also be released into the environment and used as livestock feed, provided that:

  1. no inter-specific crosses are performed;
  2. the intended uses are similar;
  3. it is known, based on characterization, that these plants do not display any additional novel traits and are substantially equivalent to apple varieties that are currently grown and permitted to be used as livestock feed in Canada, in terms of their potential environmental impact and livestock feed safety; and
  4. the novel genes are expressed at levels similar to those of the authorized lines.

Apple events GD743 and GS784 are subject to the same phytosanitary import requirements as unmodified apple varieties. Apple events GD743 and GS784 are required to meet the requirements of other jurisdictions, including but not limited to, the Food & Drugs Act and the Pest Control Products Act.

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

March 20, 2015

This bulletin was created by the Canadian Food Inspection Agency. For further information, please contact the Plant Biosafety Office or the Animal Feed Division by visiting the contact page

Table of Contents

I. Brief Identification of the Modified Plant

Designation of the Modified Plant: Apple events GD743 (OECD unique identifier OKA-NBØØ1-8) and GS784 (OECD unique identifier OKA-NBØØ2-9)

Applicant: Okanagan Specialty Fruits Inc.

Plant Species: Apple (Malus domestica Borkh)

Novel Traits: Reduced enzymatic browning

Selectable Marker: Resistance to kanamycin

Trait Introduction Method: Agrobacterium-mediated transformation

Intended Use of the Modified Plant: Apple events GD743 and GS784 are intended to be grown for traditional apple human food and livestock feed uses. Apple events GD743 and GS784 are not intended to be grown outside the normal production area for apples in Canada.

II. Background Information

Okanagan Specialty Fruits Inc. has developed apple events that display reduced enzymatic browning and kanamycin resistance. Apple events Golden Delicious 743 (GD743) and Granny Smith 784 (GS784) were developed by Okanagan Specialty Fruits Inc. using recombinant deoxyribonucleic acid (rDNA) technology, resulting in the introduction of a suppression cassette, made up of gene sequences from 4 apple polyphenol oxidase (PPO) enzymes, as well as the Escherichia coli neomycin phosphotransferase type II (nptII) gene. The PPO suppression cassette was designed such that its transcription would result in suppression of the expression of the endogenous apple ppo genes through a process known as RNA interference (RNAi). PPO enzymes play a role in enzymatic browning of the flesh of apples following mechanical damage, such as slicing or bruising. As a result of the RNAi-mediated ppo gene suppression in GD743 and GS784, little to no PPO enzyme is produced in these events, as evidenced by greatly decreased levels of PPO enzyme activity. As a result, the flesh of apple events GD743 and GS784 does not brown but rather remains its original colour following mechanical damage. The NPTII protein encoded by the nptII gene provides resistance to the antibiotic kanamycin. This protein was introduced for use as a selectable marker during plant transformation and is not expressed at detectable levels in the mature apple fruit.

Okanagan Specialty Fruits Inc. has provided information on the identity of apple events GD743 and GS784; a detailed description of the transformation method; and information on insert copy number and intactness, levels of nptII gene expression and PPO enzyme activity in the plants and the role of the inserted genes and regulatory sequences. The NPTII protein was identified and characterized. Information was provided for the evaluation of the potential toxicity of the novel NPTII protein to livestock and non-target organisms and its potential allergenicity to humans and to livestock.

Apple events GD743 and GS784 were planted in Washington and New York states in the United States (US) in 2003, 2004, 2005 and 2008. Agronomic data was collected from these trials between 2004 and 2011. The locations of these trials share similar environmental and agronomic conditions to apple production areas in Canada and were considered representative of major Canadian apple growing regions. Unmodified control Golden Delicious and Granny Smith apple varieties, which share the same genetic background as apple events GD743 and GS784, respectively, but have not been modified, were included in the trials to act as comparators for apple events GD743 and GS784.

Agronomic characteristics of apple events GD743 and GS784, such as tree height, trunk cross-sectional area, date of first bloom, duration of bloom, flower cluster number, date of fruit maturity and fruit number at harvest, were compared to those of their respective unmodified control apple varieties.

Nutritional components of apple events GD743 and GS784 fruit, such as moisture, crude protein, carbohydrates, calories, crude fat, ash, dietary fibre, sugar, potassium, vitamin C and total phenolics, were compared to those of their respective unmodified control apple varieties and to values found in published data for apple provided by the United States Department of Agriculture's (USDA's) National Nutrient Database for Standard References 2009, (USDA, 2009) and other scientific published literature values.

The Plant and Biotechnology Risk Assessment (PBRA) 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 Directive 94-08 (Dir94-08) - Assessment Criteria for Determining Environmental Safety of Plants with Novel Traits. The PBRA Unit has considered:

  • the potential for apple events GD743 and GS784 to become a weed of agriculture or to be invasive of natural habitats;
  • the potential for gene flow from apple events GD743 and GS784 to sexually compatible plants whose hybrid offspring may become more weedy or more invasive;
  • the potential for apple events GD743 and GS784 to become a plant pest;
  • the potential impact of apple events GD743 and GS784 and their gene products on non-target organisms, including humans; and
  • the potential impact of apple events GD743 and GS784 on biodiversity.

The Animal Feed Division (AFD) 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 AFD has considered both intended and unintended effects and similarities and differences between apple events GD743 and GS784 and unmodified apple varieties relative to the safety and efficacy of feed ingredients derived from apple events GD743 and GS784 for their intended purpose, including:

  • the potential impact of apple events GD743 and GS784 on livestock nutrition; and
  • the potential impact of apple events GD743 and GS784 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 AFD has also considered whether feeds derived from apple events GD743 and GS784 meet the definitions and requirements of feeds as listed in Schedule IV of the Feeds Regulations.

Okanagan Specialty Fruits Inc. has provided the CFIA with a method for the detection and identification of apple events GD743 and GS784.

III. Description of the Novel Traits

1. Development Method

Apple events GD743 and GS784 were developed through Agrobacterium-mediated transformation of cells from Golden Delicious and Granny Smith varieties, respectively, and contain a PPO suppression cassette, made up of sequences from 4 endogenous apple ppo genes, and the E. coli nptII gene, which confers resistance to kanamycin. Transformed cells were selected on the basis of resistance to kanamycin and regenerated to produce plants. Apple events GD743 and GS784 were identified as successful transformants based on molecular analyses and phenotypic screening for low PPO enzyme activity and were thus chosen for further development.

2. Reduced Enzymatic Browning

PPOs are a diverse group of enzymes that are able to insert oxygen in a position ortho- to an existing hydroxyl group in an aromatic ring to create a diphenol, followed by the oxidation of the diphenol to the corresponding quinone. The resulting compounds are very unstable and are easily oxidized to melanins, resulting in brown pigment. In apples, PPO enzymes are located in the plastids of plant cells while their phenolic substrates are present in the vacuoles of plants cells. Thus, enzymatic browning by PPO enzymes only occurs following damage to the plant cells, such as through cutting or bruising. In apple events GD743 and GS784, transcription of the PPO suppression cassette results in suppression of the expression of endogenous apple ppo genes, likely through the RNAi process, resulting in reduced PPO enzyme levels. Thus, when fruit from apple events GD743 and GS784 are subjected to mechanical damage, the flesh does not brown, but remains its original colour.

Since the reduced enzymatic browning trait introduced into apple events GD743 and GS784 is the result of suppression of the expression of endogenous ppo genes, no new proteins are produced in these events to generate this trait. Transcription of the PPO suppression cassette in apple events GD743 and GS784 is driven by a constitutive promoter. To confirm that levels of endogenous PPO enzymes were reduced in apple events GD743 and GS784, PPO enzyme activity in apple events GD743 and GS784 was compared to that of their respective unmodified control apple varieties. Tissue samples were collected from plants from a field trial in the US. For apple event GD743, the relative levels of PPO enzyme activity, as compared to its unmodified control apple variety, were as follows: PPO enzyme activity was 82% lower in mature leaves and 91% lower in mature fruit. For apple event GS784, the relative levels of PPO enzyme activity, as compared to its unmodified control apple variety, were as follows: PPO enzyme activity was 76% lower in mature leaves and 90% lower in mature fruit. A significant reduction in the change in colour of the apple flesh following bruising was also observed in apple events GD743 and GS784 compared to their respective unmodified control apple varieties. These phenotypic changes indicate that the PPO suppression cassette is transcribed in apple events GD743 and GS784 and results in the expected reduction of endogenous PPO enzyme activity.

Expression of the PPO suppression cassette does not result in production of novel proteins, therefore there is no risk of new protein toxins or allergens being introduced into apple events GD743 and GS784. Furthermore, small noncoding RNAs (ncRNAs), such as those present in apple events GD743 and GS784, are present in all plants and animals where they play central roles in endogenous gene regulation and response to exogenous DNA. The ncRNAs in apple events GD743 and GS784 are expected to function in a similar manner. Finally, the systemic absorption of exogenous ncRNAs in crops by livestock or human bystanders is considered to be unlikely based on information in the scientific literature.

3. Resistance to Kanamycin

The antibiotic kanamycin binds to bacterial ribosomes, disrupting normal protein synthesis and killing cells exposed to the antibiotic. Apple events GD743 and GS784 were developed to be tolerant to kanamycin by incorporation of the E. coli nptII gene. The nptII gene encodes the NPTII protein, which phosphorylates kanamycin, rendering it inactive. Introduction of the nptII gene into apple events GD743 and GS784 confers resistance to kanamycin. This resistance was used to select for transformed cells during the initial development of apple events GD743 and GS784.

The nptII genes in apple events GD743 and GS784 are linked to a weak constitutive promoter. Apple fruit and leaf tissue samples were collected from apple events GD743 and GS784 in field trials in New York and Washington states in the US and tested by enzyme-linked immunosorbant assay (ELISA). Apple fruit and leaf tissue samples from their respective unmodified control apple varieties were also tested. Average NPTII protein concentrations, expressed in micro-grams NPTII protein per gram dry weight tissue (µg/g dwt), were 5.0 µg/g dwt in leaves and 0.1 µg/g dwt in fruit for apple event GD743 and 3.8 µg/g dwt in leaves and 0.1 µg/g dwt in fruit for apple event GS783. NPTII protein concentrations in both of the unmodified control apple varieties were 0.1 µg/g dwt in both fruit and leaves. Therefore, fruit of apple events GD743 and GS784 did not contain NPTII protein at concentrations above those of the background readings for the ELISA.

Apple events GD743 and GS784 did not express the NPTII protein in sufficient quantities for assessment of environmental and livestock feed safety. However, the NPTII protein was previously expressed in an E. coli production system and characterized in published safety studies by Fuchs et al. (1993a, 1993bFootnote 1). Equivalency was demonstrated between the NPTII protein produced in apple events GD743 and GS784 and the E. coli-produced NPTII protein previously characterized by Fuchs et al. (1993a, 1993b) on the basis of identical amino acid sequences and recognition by anti-NPTII antiserum. Demonstration of equivalence between the NPTII protein produced in E. coli and the NPTII protein produced in apple events GD743 and GS784 allowed the use of the published studies by Fuchs et al. (1993a, 1993b) to support the safety of the NPTII protein produced in apple events GD743 and GS784.

The potential allergenicity and toxicity of the NPTII protein to livestock and non-target organisms were evaluated. The weight of evidence indicates that the NPTII protein is unlikely to be allergenic, based on the following information. The source of the nptII gene, E. coli, is not commonly associated with allergenicity. The NPTII protein amino acid sequence lacks relevant similarities to known allergens. Finally, unlike many allergens, the E. coli-produced NPTII protein was shown experimentally to be rapidly degraded in simulated gastric fluid and simulated intestinal fluid. It was also concluded that the NPTII protein is unlikely to be toxic to livestock and non-target organisms because it lacks a mode of action to suggest that it is intrinsically toxic to livestock or non-target organisms. The NPTII protein amino acid sequence lacks relevant similarities to known toxins. Furthermore, no adverse effects were observed in the studies by Fuchs et al. (1993a, 1993b) when E. coli NPTII protein was ingested by mice at doses of approximately 5000 mg/kg bwt. For a more detailed discussion of the potential allergenicity and toxicity of the NPTII protein, see Section V, part 2: Potential Impact of Apple events GD743 and GS784 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. Stable Integration into the Plant Genome

Molecular characterization by Southern blot analysis and DNA sequencing demonstrated that apple event GD743 contains 2 intact copies and 1 imperfect inverted repeat of the chimeric PPO transgene and nptII gene cassette. Molecular characterization by Southern blot analysis and DNA sequencing demonstrated that apple event GS784 contains 4 intact copies and 1 imperfect inverted repeat of the gene cassette. Two insertions, containing fragments of the plasmid backbone, were also identified in apple event GS784.

The stability of the DNA insert within apple events GD743 and GS784 was verified by testing leaf samples from 14 trees each of apple events GD743 and GS784 by a polymerase chain reaction (PCR) and an ELISA specific for the nptII gene and the NPTII protein, respectively. The PCR and ELISA results indicated that the DNA inserts in apple events GD743 and GS784 were stable and that the NPTII protein was stably expressed in apple leaves.

IV. Criteria for the Environmental Assessment

1. Potential for Apple Events GD743 and GS784 to Become a Weed of Agriculture or be Invasive of Natural Habitats

The biology of apple, as described in the CFIA biology document BIO2014-01 The Biology of Malus domestica Borkh (Apple), is such that unmodified plants of this species are not weeds of managed habitats or invasive of unmanaged habitats in Canada. Cultivated apple is not weedy, as volunteer seedlings originating from seeds in apple orchards are very rare due to typical orchard management practices, including herbicide treatments of the tree rows and mowing of the alleys between rows. Although apple seedlings and trees can survive and persist outside of cultivation, M. domestica is not regarded as an invasive species in Canada or elsewhere. According to the information provided by Okanagan Specialty Fruits Inc., apple events GD743 and GS784 were determined not to be significantly different from unmodified apple varieties in this respect.

The CFIA evaluated data submitted by Okanagan Specialty Fruits Inc. on the reproductive biology and life history traits of apple events GD743 and GS784. As previously mentioned, these events were tested at trial sites in New York and Washington states. It was determined that the New York and Washington state locations share similar environmental and agronomic conditions to Ontario and British Columbia, respectively, and were considered to be representative of the major Canadian apple growing regions. The trees were planted in 2003, 2004, 2005 and 2008, and agronomic data was collected from these trials between 2004 and 2011. The trees were grown under typical highly-managed conditions for commercial orchards. During the field trials, apple events GD743 and GS784 were compared to their respective unmodified control apple varieties. Phenotypic and agronomic traits were evaluated, including tree height, trunk cross-sectional area, date of first bloom, duration of bloom, flower cluster number, date of fruit maturity and fruit number at harvest. The results showed no biologically meaningful differences between apple events GD743 and GS784 and their respective unmodified control apple varieties and support a conclusion of phenotypic and agronomic equivalence to currently grown apple varieties.

The susceptibility of apple events GD743 and GS784 to various apple pests and pathogens was evaluated in the field at the same locations as the agronomic characteristics studies. Further detail provided below in Section IV, part 3: Potential for Apple Events GD743 and GS784 to Become a Plant Pest. No trend in increased or decreased susceptibility to pests and pathogens was observed in apple events GD743 and GS784 compared to their respective unmodified control apple varieties.

No competitive advantage was conferred to apple events GD743 and GS784, as the reproductive characteristics, growth characteristics and tolerance to pests and pathogens of apple events GD743 and GS784 were comparable to those of their respective unmodified control apple varieties.

The novel traits have no intended or observed effects on weediness or invasiveness. The CFIA has therefore concluded that apple events GD743 and GS784 have no altered weediness or invasiveness potential in Canada compared to currently grown apple varieties.

2. Potential for Gene Flow from Apple Events GD743 and GS784 to Sexually Compatible Plants Whose Hybrid Offspring May Become More Weedy or More Invasive

As described in the CFIA biology document BIO2014-01 The Biology of Malus domestica Borkh (Apple), there are four Malus species present in Canada in addition to M. domestica. Two of these are native to North America: the sweet crabapple (M. coronaria) and the Oregon or Pacific crabapple (M. fusca). The other two are introduced: the Siberian crabapple (M. baccata) and the plum- or pear-leaved crabapple (M. prunifolia). In Canada, M. coronaria is present only in Southern Ontario, while M. fusca occurs in coastal British Columbia. As for the two introduced species, M. baccata is reported from Ontario, Quebec, New Brunswick, Nova Scotia and possibly Newfoundland and Labrador, while M. prunifolia is only reported from New Brunswick and Nova Scotia. All four crabapple species are sexually compatible with M. domestica. M. domestica flowers are predominantly insect-pollinated, mainly by bees when grown as a commercial crop. Natural hybridization between cultivated apple (M. domestica) and crabapple species is possible when synchronous flowering and close proximity of the two species occur. However, the extent of gene introgression from M. domestica into crabapple species in Canada appears to be limited. Natural hybridization (i.e. outside of breeding programs) between M. domestica and species outside of the genus Malus has not been reported.

The resistance to kanamycin and reduced enzymatic browning traits introduced into apple events GD743 and GS784 are not related to increased weediness or invasiveness potential. If individuals arose through interspecific hybridization between apple events GD743 or GS784 and crabapple species, the novel traits would confer no competitive advantage to these plants.

This information led the CFIA to conclude that gene flow from apple events GD743 and GS784 to related species in Canada is possible, but would not result in increased weediness or invasiveness of the resulting progeny.

3. Potential for Apple Events GD743 and GS784 to Become a Plant Pest

Cultivated apple is not considered a plant pest in Canada and the resistance to kanamycin trait introduced into apple events GD743 and GS784 is unrelated to plant pest potential (i.e. the potential for the plant to harbour new or increased populations of pests or pathogens).

The physiological role of PPO enzymes in apple 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 alter pest and pathogen susceptibility, although this has not been demonstrated in apple. As such, the susceptibility of apple events GD743 and GS784 to various apple pests and pathogens was evaluated in the field from 2004 to 2011 at the same locations as the agronomic characteristics studies. The pests and pathogens observed included green apple aphid, woolly aphid, Japanese beetle, tentiform leaf miner, campylomma bug, powdery mildew and fireblight. Although instances of statistically significant differences were observed between apple events GD743 and GS784 and their respective unmodified control apple varieties for some pests and pathogens, there was no consistent trend in the data across years that would indicate the differences were due to the genetic modification. Thus, the evaluations of apple events GD743 and GS784 did not show any increased or decreased susceptibility to pests or pathogens compared to their respective unmodified control apple varieties.

The CFIA has therefore concluded that apple events GD743 and GS784 do not display any altered plant pest potential compared to currently grown apple varieties in Canada.

4. Potential Impact of Apple Events GD743 and GS784 and their gene products on Non-Target Organisms, Including Humans

The resistance to kanamycin and reduced enzymatic browning traits introduced into apple events GD743 and GS784 are unrelated to a potential impact on non-target organisms.

The PPO suppression cassette introduced into apple events GD743 and GS784 does not result in the expression of a novel protein; therefore, there is no risk of new allergens being introduced into apple events GD743 and GS784. The safety assessment of apple events GD743 and GS784 focused on ncRNAs and the suppression of the expression of endogenous PPO enzyme levels.

The production of ncRNA for naturally occurring endogenous RNAi is common in plants and animals, and the PPO suppression cassette in apple events GD743 and GS784 was specifically designed to target apple. Therefore, the apple PPO suppression cassette in apple events GD743 and GS784 is not expected to mediate the inhibition of gene expression in organisms interacting with apple events GD743 and GS784. Based on the evidence provided by Okanagan Specialty Fruits Inc., no negative impacts resulting from exposure of organisms to ncRNA expressed from the PPO suppression cassette in apple events GD743 and GS784 are expected.

Detailed characterization of the NPTII protein expressed in apple events GD743 and GS784 led to the conclusion that this protein does not display any characteristics of a potential toxin or allergen (see Section V, part 2: Potential Impact of Apple Events GD743 and GS784 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). Therefore, no negative impacts resulting from exposure of organisms to the NPTII protein expressed in apple events GD743 and GS784 are expected.

Composition analyses showed that the levels of key nutrients in fruit from apple events GD743 and GS784 are comparable to those in conventional apple varieties (see Section V, part 1: Potential Impact of Apple Events GD743 and GS784 on Livestock Nutrition). Therefore, it is very unlikely that the introduction of the novel traits may have caused unintended changes to the composition of apple events GD743 and GS784 tissues that would negatively impact organisms interacting with apple events GD743 and GS784. In addition, it is unlikely that the novel traits would impact endogenous apple toxin or allergen levels in a manner that would present a risk to organisms interacting with apple events GD743 or GS784.

Field evaluations of apple events GD743 and GS784 did not show any increased resistance to apple pests or pathogens compared to their respective unmodified control apple varieties (see Section IV, part 3: Potential for Apple Events GD743 and GS784 to Become a Plant Pest).

As the novel traits are unrelated to a potential impact on non-target organisms and apple events GD743 and GS784 are equivalent to their respective unmodified apple varieties in terms of their floral traits (see Section IV, part 1: Potential for Apple Events GD743 and GS784 to Become a Weed of Agriculture or be Invasive of Natural Habitats), apple events GD743 and GS784 are not expected to negatively impact pollinators of apple, including honeybees. In addition, no differences in pollination, honeybee health or honeybee mortality were observed during the field trials of apple events GD743 and GS784.

Collectively, these information elements indicate that the interactions between apple events GD743 and GS784 and the populations of animals and microorganisms interacting with apple crops will be similar compared to currently grown apple varieties.

The CFIA has therefore determined that the unconfined environmental release of apple events GD743 and GS784 in Canada will not result in altered impacts on non-target organisms, including humans, compared to currently grown apple varieties.

5. Potential Impact of Apple Events GD743 and GS784 on Biodiversity

Apple events GD743 and GS784 express no novel phenotypic characteristics that would extend their range beyond the current geographic range of apple production in Canada. Apple events GD743 and GS784 are unlikely to cause adverse effects on non-target organisms and do not display increased weediness, invasiveness or plant pest potential. Cultivated apple M. domestica can outcross to crabapples under natural conditions in Canada. However, the consequences of the transfer of the reduced enzymatic browning and resistance to kanamycin traits to crabapples are minimal as these traits do not confer any selective advantage and are unlikely to negatively impact organisms interacting with the cultivated apple. Growing apple events GD743 and GS784 will not necessitate changes to current orchard management practices. It is therefore unlikely that apple events GD743 and GS784 will have any direct or indirect effects on biodiversity, in comparison to the effects that would be expected from the cultivation of apple varieties that are currently grown in Canada.

The CFIA has concluded that the introduced genes and their corresponding novel traits do not confer to apple events GD743 and GS784 any characteristic that would result in unintended environmental effects following unconfined environmental release.

The CFIA has therefore concluded that the impact on biodiversity of apple events GD743 and GS784 is unlikely to be different from that of the apple varieties that are currently grown in Canada.

V. Criteria for the Livestock Feed Assessment

The AFD considered nutrient and anti-nutrient profiles; the safety of feed ingredients derived from apple events GD743 and GS784, including the presence of gene products, residues and metabolites in terms of 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; and whether feeds derived from apple events GD743 and GS784 meet the definitions and requirements of feeds as listed in Schedule IV of the Feeds Regulations.

1. Potential Impact of Apple Events GD743 and GS784 on Livestock Nutrition

Nutrient and Anti-Nutrient Composition

The nutritional equivalence of apple events GD743 and GS784 to those of their respective unmodified control apple varieties was determined using fruit harvested from US field trials in Washington and New York that were planted in 2004 and 2005, respectively. Mature fruit of these events and the unmodified control apple varieties were harvested in 2009 from these field trials for compositional analysis. Fruit samples were randomly collected and stored at 2ºC. Composite samples were prepared by combining one–quarter slices from 4 fruits from each tree, where they were cut, cored and placed in Ziploc bags. The composite samples were then shipped on ice to the lab where they were analysed for moisture, crude protein, carbohydrates, calories, crude fat, ash, dietary fibre, sugar, potassium, vitamin C and total phenolics. Composition data was analysed statistically using analysis of variance, and any statistically significant differences between apple events GD743 and GS784 and their respective unmodified control apple varieties were identified and assessed (P<0.05). The biological relevance of any statistically significant differences observed between apple events GD743 and GS784 and their respective unmodified control apple varieties was also assessed within the natural variation observed in conventional apple composition data found in the USDA's National Nutrient Database for Standard References 2009, and other scientific published literature values.

No statistically significant differences were observed in either field trial between apple events GD743 and GS784 and their respective unmodified control apple varieties for moisture, crude protein, calories, ash, carbohydrates, dietary fibre, sugars and potassium levels. Crude fat was not detected in apple events GD743 and GS784 or their respective unmodified control apple varieties. All mean values for apple events GD743 and GS784 and their respective unmodified control apple varieties were within the values reported in the USDA's National Nutrient Database for Standard references 2009, and/or other published scientific literature. For both apple events GD743 and GS784 and their respective unmodified apple controls, samples from Washington had significantly higher moisture contents and correspondingly lower protein, carbohydrate, sugars and fibre contents compared to samples from New York.

Samples of cut fruit from apple events GD743 and GS784 had significantly higher vitamin C and total phenolics values than those of their respective unmodified control apple varieties. However, vitamin C and total phenolics values in apple events GD743 and GS784 were within the range of values of published scientific literature for conventional apples. Mean phenolic values of samples of apple events GD743 (182 mg Gallic Acid Equivalent (GAE)/100 g) and GS784 (194 mg GAE/100 g) were significantly higher than those of samples of their respective unmodified control apple variety (75 mg GAE/100 g and 90 mg GAE/100 g, respectively), but these were within the natural variability of values reported for conventional apples in the USDA's National Nutrient Database for Standard References 2009, Okanagan Specialty Fruits Inc. proposed that the lower levels of phenolic compounds observed in the unmodified control apple variety samples were due to higher PPO enzyme levels in these varieties, which use phenolic compounds as substrates, when cut. They also proposed that the lower vitamin C levels in the unmodified control apple variety samples may have been the result of PPO enzymes degrading and converting vitamin C to a form which was not detected by the assay used.

Conclusion

It was concluded, based on the evidence provided by Okanagan Specialty Fruits Inc., that except for the reduced enzymatic browning when cut, the nutritional composition of apple events GD743 and GS784 is similar to that of their respective unmodified control apple varieties grown in the trials and/or to that of other conventional apples in published scientific literature. Feed ingredients derived from apple events GD743 and GS784, such as apple pomace, are considered to meet the present ingredient definition in the Feeds Regulations.

2. Potential Impact of Apple Events GD743 and GS784 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

Apple events GD743 and GS784 display reduced enzymatic browning due to RNAi-mediated suppression of ppo genes and are resistant to kanamycin due to production of the NPTII protein. The assessment of apple events GD743 and GS784 evaluated the impact of the following potential hazards relative to the safety of feed ingredients derived from this event:

  • RNAi-mediated suppression of ppo genes
  • The presence of novel protein NPTII

RNAi-Mediated Suppression of ppo Genes

Expression of the PPO suppression cassette does not result in production of novel proteins; therefore, there is no risk of new protein toxins or allergens being introduced into apple events GD743 and GS784. The livestock feed safety assessment of apple events GD743 and GS784 focused on the safety of the suppression of the expression of endogenous PPO enzyme levels and the small noncoding RNAs. Suppression of the expression of endogenous PPO enzyme levels in apple events GD743 and GS784 was not considered to present a risk to human or animal health or the environment since there was no evidence of association with a toxic mode of action and there was no evidence that reduction of PPO enzyme levels would impact endogenous apple toxin or allergen levels. Small noncoding RNAs present in apple events GD743 and GS784 were not considered to present a risk to human or animal health or the environment. Bioinformatic analyses demonstrated that the DNA sequences inserted into apple events GD743 and GS784 did not have significant similarity to protein-coding genomic regions of livestock or humans. A lack of significant similarity was observed between DNA sequences inserted into apple events GD743 and GS784 and protein-coding genomic regions in apple (excluding the ppo genes), suggesting that the DNA sequences were not expected to affect expression of other apple genes. Finally, the systemic absorption of exogenous small noncoding RNAs in crops by livestock or human bystanders is considered to be unlikely based on information in the scientific literature.

Novel NPTII protein

The NPTII protein was previously expressed in an E. coli production system and characterized in published safety studies by Fuchs et al. (1993a, 1993b), as described above (see Section III, part 3: Resistance to Kanamycin). Equivalency was demonstrated between the NPTII protein produced in apple events GD743 and GS784 and the E. coli-produced NPTII protein previously characterized by Fuchs et al. (1993a, 1993b) on the basis of identical amino acid sequence and recognition by anti-NPTII antiserum.

The potential allergenicity and toxicity of the NPTII protein to livestock were evaluated. With respect to its potential allergenicity, no single experimental method yields decisive evidence, thus a weight of evidence approach was taken, taking into account information obtained with various test methods. The source of the nptII gene, E. coli, is not commonly associated with allergenicity. The NPTII protein amino acid sequence lacks relevant similarities to known allergens. Finally, unlike many allergens, the E. coli-produced NPTII protein was shown experimentally to be rapidly degraded in simulated gastric fluid and simulated intestinal fluid. The weight of evidence thus indicates that the NPTII protein is unlikely to be allergenic.

In terms of its potential toxicity to livestock, the information indicates that the NPTII protein is unlikely to be toxic to livestock. The NPTII protein lacks a mode of action to suggest that it is intrinsically toxic to livestock and a bioinformatics evaluation of the NPTII protein amino acid sequence confirmed the lack of relevant similarities between the NPTII protein and known toxins. In addition, no adverse effects were observed in studies by Fuchs et al. (1993a, 1993b) when E. coli-produced NPTII protein was ingested by mice at doses of approximately 5000 mg/kg bwt.

The livestock exposure to the NPTII protein is expected to be negligible as the NPTII protein is expressed at undetectable levels in fruit of apple events GD743 and GS784 and in studies by Fuchs et al. (1993a, 1993b), E. coli-produced NPTII protein was rapidly degraded under conditions which simulate the mammalian digestive tract.

Conclusion

It was concluded, based on the evidence provided by Okanagan Specialty Fruits Inc., that the novel reduced enzymatic browning and resistance to kanamycin traits will not confer to apple events GD743 and GS784 any characteristic that would raise concerns regarding the safety of apple events GD743 and GS784. Feed ingredients derived from apple events GD743 and GS784 are considered to meet present ingredient definitions for apple.

VI. New Information Requirements

If at any time, Okanagan Specialty Fruits Inc. 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 apple events GD743 and GS784 or lines derived from them, Okanagan Specialty Fruits Inc. 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 apple events GD743 and GS784 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 apple events GD743 and GS784.

VII. Regulatory Decision

Based on the review of the data and information submitted by Okanagan Specialty Fruits Inc. 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 apple events GD743 and GS784 does not present altered environmental risk when compared to apple varieties that are currently grown in Canada.

Based on the review of the data and information submitted by Okanagan Specialty Fruits Inc. and input from other relevant scientific sources, the Animal Feed Division of the Animal Health Directorate, CFIA, has concluded that the novel reduced enzymatic browning trait will not confer to apple events GD743 and GS784 any characteristic that would raise concerns regarding the safety or nutrition of apple events GD743 and GS784. Livestock feeds derived from apple events GD743 and GS784 are currently listed in Schedule IV of the Feeds Regulations. Apple events GD743 and GS784 have been found to be as safe as and as nutritious as currently and historically grown apple varieties. Apple events GD743 and GS784 and its products are considered to meet present ingredient definitions.

Unconfined release into the environment and use as livestock feed of apple events GD743 and GS784 are therefore authorized by the Plant Biosafety Office of the Plant Health and Biosecurity Directorate and the Animal Feed Division of the Animal Health Directorate as of March 20, 2015. Any apple lines derived from apple events GD743 and GS784 may also be released into the environment and used as livestock feed, provided that:

  1. no inter-specific crosses are performed;
  2. the intended uses are similar;
  3. it is known, based on characterization, that these plants do not display any additional novel traits and are substantially equivalent to apple varieties that are currently grown and permitted to be used as livestock feed in Canada, in terms of their potential environmental impact and livestock feed safety; and
  4. the novel genes are expressed at levels similar to those of the authorized lines.

Apple events GD743 and GS784 are subject to the same phytosanitary import requirements as unmodified apple varieties. Apple events GD743 and GS784 are required to meet the requirements of other jurisdictions, including but not limited to, the Food & 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 apple events GD743 and GS784.

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