Novel food information: Event EF2-114 Pineapple
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Health Canada has notified Del Monte Fresh Produce Company (Del Monte) that it has no objection to the food use of Event EF2-114 pineapple. The Department conducted a comprehensive assessment of this pineapple variety according to its Guidelines for the Safety Assessment of Novel Foods. These Guidelines are based upon internationally accepted principles for establishing the safety of foods with novel traits.
The following provides a summary of the original notification from Del Monte and the evaluation by Health Canada and contains no confidential business information.
Del monte has developed a genetically modified Ananas comosus (L.) Merr. var. comosus, family Bromeliaceae (pineapple) variety which exhibits pink-coloured fruit flesh through elevated expression of lycopene.
Event EF2-114 was developed through the introduction of several gene expression constructs, some for expression of enzymes from other plant species (PSY gene from tangerine for lycopene biosynthesis; SuRBHra (ALS) from tobacco for transformant selection based on herbicide tolerance), and others for endogenous gene expression silencing using RNAi (bLcy and eLcy for conversion of lycopene into other carotenoids; flACC3′ for ethylene biosynthesis).
The safety assessment performed by Food Directorate evaluators was conducted according to Health Canada's Guidelines for the Safety Assessment of Novel Foods. These Guidelines are based on harmonization efforts with other regulatory authorities and reflect international guidance documents in this area (e.g., Codex Alimentarius). The assessment considered: how this pineapple variety was developed; how the composition and nutritional quality of this variety compared to non-modified pineapple varieties; and the potential for this pineapple variety to be toxic or cause allergic reactions. Del Monte has provided data that demonstrate that Event EF2-114 is as safe and of the same nutritional quality as traditional pineapple varieties used as food in Canada.
The Food Directorate has a legislated responsibility for pre-market assessment of novel foods and novel food ingredients as detailed in the Food and Drug Regulations(Division B.28). Event EF2-114 is considered a novel food under the following part of the definition of novel foods:
"c) a food that is derived from a plant, animal or microorganism that has been genetically modified such that:
- the plant, animal or microorganism exhibits characteristics that were not previously observed in that plant, animal or microorganism."
2. Development of the Modified Plant
The petitioner has provided information describing the methods used to develop Event EF2-114 and the molecular biology data that characterize the genetic change, which results in pink-coloured fruit flesh through elevated expression of lycopene.
Event EF2-114 was developed through Agrobacterium-mediated transformation of pineapple variety MD2 with two different transformation vectors: pHCW.T-2 and pHCWflACC3′-2.
The pHCW.T-7 vector contains a construct (T-DNA) of the PSY gene, encoding a phytoene synthase (PSY) from tangerine (Citrus reticulata). This enzyme catalyzes the synthesis of phytoene, an intermediate in the pathway for lycopene biosynthesis. The pHCW.T-7 vector also contains two RNAi constructs based on the sequence of two endogenous genes, bLcy and eLcy. When transcribed, these RNAi constructs reduce expression of a lycopene beta-cyclase and a lycopene epsilon-cyclase, respectively, which are involved in the catabolism of lycopene. Expression of the PSY protein and the two RNAi constructs are intended to promote accumulation of lycopene in the edible portion of the fruit.
The pHCWflACC3′-2 vector contains another RNAi construct designed to reduce expression of the endogenous aminocyclopropane-1-carboxylic acid synthase (ACS), an enzyme involved in ethylene biosynthesis. The purpose of reducing expression of this enzyme was to promote uniformity of flowering and fruit production. However, molecular and phenotypic characterization of the Event EF2-114 pineapple revealed that no intact insertion of this construct was present, resulting in a lack of expression of this RNAi construct in Event EF2-114. Consequently, the uniformity phenotype was not achieved in Event EF2-114.
Both vectors also encode a selectable marker gene from the tobacco plant (Nicotiana tabacum), SuRBHra, which expresses an acetolactate synthase (ALS). The sole purpose of this gene was to select for successful transformants during the development process. This enzyme confers tolerance to sulfonylurea herbicides.
Lastly, the expression cassettes for the RNAi constructs for bLcy, eLcy, and flACC3′ each include a LS1 intron from potato (Solanum tuberosum). The purpose of this element is to provide stability to the inverted repeat structures of the RNAi constructs, required for effective engagement of the plant's RNAi machinery. No protein-expressing potato genes are present in Event EF2-114.
The petitioner provided information to support the safety and long history of use of the donor organisms (A. comosus var. comosus, C. reticulata, and S. tuberosum) and the recipient organism (A. comosus var. comosus). None of these organisms pose a safety concern from a food perspective. For N. tabacum, the ALS enzyme is unrelated to the nicotine biosynthesis pathway and was assessed for similarity to known toxins and allergens.
3. Characterization of the Modified Plant
The number of T-DNA inserts in Event EF2-114 was characterized by using a combination of Southern blot analysis and a PCR-based assay, Thermal Asymmetric Interlaced PCR (TAIL-PCR). The results of the analyses demonstrated that there are multiple integration sites in Event EF2-114. A comparison of the data between the Southern blot and TAIL-PCR analyses confirmed the presence of at least one (1) intact-copy of the pHWC.7-2 T-DNA construct, a truncated copy of the pHWCflACC3′-2 T-DNA construct, and additional complete and/or partial copies of each T-DNA construct (pHCW.T-7 specific elements: four copies of PSY coding region; two complete and two partial copies of bLcy;elements present in both plasmid cassettes: ALS-ALS3' region that spans SuRBHra: four copies associated with the pHCW.T-7 cassette; two copies associated with pHCW-flACC3′; one additional partial copy) in the pineapple genome. The safety of potential open reading frames (ORFs) generated from these additional sequences were further analysed. No vector backbone sequences from were detected in Event EF2-114.
Given the large number of rearranged copies of the T-DNA inserts in Event EF2-114, the petitioner conducted a sequence level analysis of the Left and Right Border regions. The aim was to determine whether there was any correlation between these sequences and the number of integrations seen in the Southern blot data. This approach was also used to predict and characterize the potential risk that any potential new unintended coding sequences or ORFs could be allergens or toxins.
ORF finder software from NCBI was used to analyse the Left Border (LB) and Right Border (RB) TAIL-PCR sequences of the integration sites to search for possible novel proteins or peptides. The LB analysis discovered ORFs in the 38 to 63 amino acid range. ORFs discovered within the RB sequences ranged from 35 to 110 amino acids in length. None of these showed greater than 35% identity to any sequence contained in the NCBI databases. The predicted ORFs were further screened for similarity to known allergens and toxins. The first approach was to restrict the BLASTP results using the keyword delimiters "allergen" or "toxin". These searches did not yield any results.
The second approach involved searching the University of Nebraska's FAARP AllergenOnline databases using default parameters. Using the FASTA3.04 algorithm with an 80 residue scanning search found two ORFs of length greater than 80 amino acids, one 97 and one 110 amino acids in length. Other hits were below 80 amino acids. The two larger ORFs were analysed, and none matched with greater than 35% identity or over 80 amino acids to known allergens from the FAARP database. A search using an 8 amino acid scanning window likewise did not find matches above 35% identity to any sequence strings in this database.
Stability of the T-DNA inserts in the Event EF2-114 genome was demonstrated by assessing individual Event EF2-114 plants from four generations. From the initial transformed and regenerated plant (T0), three successive generations were grown through clonal propagation (T1, T2, T3) and tested, as was a tissue culture grown sample derived from the T1 generation (T1-TC). All generations were analyzed by means of Southern blot analysis. The results indicate that the T-DNA inserts (including the partial sequences) are intact and stable over all four generations of Event EF2-114.
Generational stability was also assessed at the phenotypic level by observing the fruit colour over five generations grown by successive clonal propagation (T1, T2, T3, T4, T5) as well as the tissue culture sample described above (T1-TC). Mature fruit from the untransformed MD2 control pineapple was golden in colour. Fruits from plants containing the transformation event had pink flesh as a result of accumulated lycopene. Cross sections of each fruit generation showed uniformity in colour across the different generations.
4. Product Information
Event EF2-114 differs from its traditional counterparts by the addition of several gene expression constructs, some for expression of enzymes from other plant species (PSY gene from tangerine for lycopene biosynthesis; SuRBHra (ALS) from tobacco for transformant selection based on herbicide tolerance), and others for endogenous gene expression silencing using RNAi (bLcy and eLcy for conversion of lycopene into other carotenoids; flACC3′ for ethylene biosynthesis).
Regarding expression levels of the PSY and SuRBHra proteins, the petitioner explained that the extraction of proteins and nucleic acids from pineapple tissue is extremely difficult. This is mainly due to low concentrations in fresh fruit, high activity of proteases and nucleases, and high levels of interfering compounds such as polyphenols and polysaccharides (Song et al., 2006Footnote 1).
To produce sufficient quantities of both the PSY and SuRBHra proteins to perform further safety studies recommended by Codex guidance, the petitioner attempted to overexpress in an Escherichia coli expression system. Unfortunately, the PSY protein failed to express in this system, while the SuRBHra protein localized exclusively in inclusion bodies within the E. coli cells, requiring harsh conditions for purification that were not suitable for use in in vitro digestibility, heat stability, or acute toxicity assays (i.e., the purification conditions denatured SuRBHra significantly).
The petitioner elected instead to use real-time quantitative reverse transcription PCR (qRT-PCR) to measure the RNA transcript levels of PSY and SuRBHra, as well as the genes targeted by RNAi mediated silencing, bLcy, eLcy, and flACC3′. qRT-PCR requires nanogram quantities of template RNA and uses fluorescent dye reporter molecules to monitor the amplification products during each cycle of the PCR reaction. Unlike Northern blot analysis, qRT-PCR does not require post-handling of RNA, making it a more accurate method for comparing gene expression levels.
The petitioner reported that expression of the tangerine PSY gene was not detected in Event EF2-114 by qRT-PCR, meaning that the level of expression was below the limit of detection (LOD). It was suggested that some internal regulatory process may be preventing expression of exogenous PSY, however the precise mechanism was not investigated. Expression of endogenous phytoene synthase genes UT1 and UT2 was detected by qRT-PCR. The level of RNA expression of SuRBHra was found to be very low; however, it appeared to be sufficient to confer the herbicide tolerance and enable selection during the transformation process.
It also was demonstrated by qRT-PCR that expression level of both the bLcy and eLcy genes was successfully reduced in Event EF2-114 relative to the unmodified control (MD2). It was suggested that the expression of endogenous phytoene synthasegenes, together with reduced expression of the bLcy and eLcy genes, contributed to the observed pink fruit tissue phenotype in Event EF2-114.
Given the presence of only a partial copy of the RNAi construct designed to reduce expression of flACC3′ in the Event EF2-114 genome as well as the absence of the desired flowering uniformity phenotype, it was expected that flACC3′ transcript levels would have been comparable in Event EF2-114 and the unmodified control (MD2). The qRT-PCR analysis was, however, unable to reliably detect or quantify the flACC3′ transcript in either Event EF2-114 or the parental MD2 pineapple line.
5. Dietary Exposure
It is expected that Event EF2-114 (which will be marketed as PinkGlow™ pineapple) will be consumed similarly to the parental MD2 pineapple line currently sold in the Canadian marketplace. The petitioner does not anticipate a significant change in the consumption of pineapple with the introduction of Event EF2-114.
Nutrient comparisons were conducted on fruits harvested from a single region at Corporación de Desarrollo Agrícola Del Monte (CDADM), Buenos Aires, Costa Rica. Plants were grown and maintained under standard agricultural practices. Greenhouse grown, 15-20 week old seedlings were transplanted in a completely randomized block design with three replicates with unequal numbers, consisting of a total of 2,541 Event EF2-114 plants (606+1,477+458) and 145 MD2 plants (25+61+59). One year later, the experimental and control plants were forced to flower and produce fruit.
At ABC Research Laboratories, Gainesville, FL, 18 fruits each of Event EF2-114 and MD2 were randomly selected for compositional analyses. The remaining fruits were saved for further analyses. Compositional analyses for a total of 19 analytes were conducted to compare the nutrient levels in test and control fruits. The analysis included proximates (ash, carbohydrates, moisture, protein, and fat), dietary fiber, amino acids (leucine, isoleucine, methionine, and valine), carotenoids (lycopene, beta-carotene, alpha-carotene, and lutein), sugars (sucrose, fructose, and glucose), vitamin C, and potassium. All analyses were conducted using AOAC or other acceptable methods.
The data were analyzed using the InfoStat-Statistical Software. A student's t test was used to compare the nutrient levels of Event EF2-114 and MD2 pineapples at a significance level of P<0.05.
As expected, lycopene (a carotenoid) accumulated in the fruit of Event EF2-114 and resulted in production of pink/red-coloured flesh. Lycopene concentration in Event EF2-114 fruit varied between 14.3 and 32.9 ppm (average 21.3 ppm). Lycopene concentration in Event EF2-114 was within the range of lycopene produced in other foods with a history of safe use. For example, lycopene concentrations are 26 ppm for tomato and 45 ppm for watermelon.
The mean value for beta-carotene (pro-vitamin A) in Event EF2-114 was 5.3 µ/100 g, which is significantly lower than the mean value reported in MD2 (16.8 µ/100 g). The effect is predictable since the conversion of lycopene to beta-carotene is decreased by suppressing lycopene bLcy. It should be noted that pineapple is not a major source of beta-carotene; beta-carotene content in sweet potato, carrot, cantaloupe is 9444, 8285, and 2020 µ/100 g, respectively.
Potassium concentration was significantly higher in Event EF2-114 (156 mg/100 g) than in MD2 (129 mg/100 g). It was also noted that individual variation in potassium content was generally high for both varieties; however, the range for Event EF2-114 was relatively higher than MD2. The variation seen in potassium content in both Event EF2-114 and MD2 can be due to environmental factors. Experience with other crops indicates that there is a positive correlation between lycopene content and potassium content in the fruit. A published study reports that concentration of potassium in fruits of high lycopene tomato ′Fla. 8153′ was significantly (P<0.01) correlated with the fruit carotenoids, indicating a possible role for potassium in increasing lycopene and reducing beta-carotene in the fruit.
Vitamin C is a major nutrient in pineapple. The results of the analysis showed that the mean value for the Event EF2-114 (40.3 mg/100 g) was relatively lower than MD2 (46.8 mg/100 g). However, the differences were small and the mean values were within the range of values reported for the control (MD2).
Levels for all other nutrients analyzed from Event EF2-114 were within the ranges normally seen in commercially produced pineapple varieties.
Potential nutrient intakes were calculated by the petitioner using Food consumption data collected in What We Eat in America (WWEIA), the dietary interview portion of the National Health and Nutrition Examination Survey (NHANES) from the combined survey years of 2003-2010 and nutrient composition data from the U.S. Department of Agriculture (USDA) and Del Monte.
For the potential nutrient uptake modeled by the petitioner and based on a scenario where Event EF2-114 is substituted for all pineapple consumed in Canada, effect on total lycopene intake would be very small. Despite differences observed in the concentration of beta-carotene, vitamin C, and potassium, nutrient composition is within the range of natural variation of MD2 and potential changes in dietary intake of these nutrients from pineapple would be insignificant within the context of the Canadian diet.
Based on the information provided on the composition of Event EF2-114 and control MD2, there are no safety concerns with the use of Event EF2-114 pineapple as a food in Canada from a nutritional perspective.
The petitioner provided evidence to support the safety of the two novel proteins, PSY and SuRBHra, and the three novel RNAi constructs to suppress the endogenous genes bLcy, eLcy, and flACC3′, expressed in Event EF2-114. The evidence included a history of safe food use, bioinformatics, expression levels, and dietary exposure.
The PSY protein is an endogenous protein in all plants. The PSY transgene is from tangerine, a common food in the diet. Therefore, the PSY protein has a history of safe food use, and would not be expected to pose a toxicological concern.
Bioinformatics analysis of the amino acid sequence for the PSY protein was conducted to compare identity with known toxins, using the NCBI Entrez protein database (March 28, 2013). Searches were also conducted on the flanking regions of the inserts, based on putative proteins from open reading frames (ORFs). There were no biologically relevant sequence similarities with known toxins.
The novel PSY protein was not detected in Event EF2-114, and PSY transcript levels were below the threshold level of detection (threshold cycle > 35 for PCR analysis). The novel PSY protein is present in Event EF2-114 at extremely low concentrations. Therefore, exposure to the novel PSY protein is expected to be negligible, from a toxicological perspective.
The SuRBHra protein, also known as acetolactate synthase (ALS), is a well-characterized enzyme in plants. The petitioner demonstrated that two modified ALS enzymes approved in soybean and corn by Health Canada (GM-HRA, ZM-HRA) had a high similarity in amino acid sequence with the SuRBHra protein. The two mutations in the SuRBHra protein were the same as the approved ALS enzymes. The evidence suggests that the novel SuRBHra protein has a history of safe food use.
Bioinformatics analysis of the amino acid sequence for the SuRBHra protein was conducted as described for the PSY protein. There were no biologically relevant sequence similarities with known toxins.
Expression of SuRBHra transcripts was detectable but low; however, SuRBHra protein was not detected in Event EF2-114. The petitioner estimated a dietary exposure to SuRBHra protein based on the Limit of Detection (LOD = 12.5 ng) and assuming a per capita pineapple fruit consumption of 2 g/day. The estimated dietary exposure is equal to or less than 0.0001875 mg SuRBHra protein/day. This amount is negligible from a toxicological perspective.
The novel RNAi (bLcy, eLcy, and flACC3′) partial coding sequences are derived from pineapple. A bioinformatics analysis of the putative novel siRNAs generated did not find any relevant matches with the human transcript or genome databases. This indicates off-target effects are unlikely, and unintended gene suppression is not expected to occur.
Based on the high intake level for pineapple reported in Japan, the petitioner provided a dietary estimate of novel siRNA exposure that would result from Event EF2-114 pineapple consumption. The petitioner measured the total siRNA in Event EF2-114, then calculated the amount that would represent a single unique siRNA. Exposure was estimated to result in four copies of novel siRNA per liver cell for the general population. This value is well below the reported threshold of 100 copies of siRNA or greater per cell estimated to affect suppression of a targeted transcript. The level of expressed novel siRNA in Event EF2-114 is considered sufficiently low to prevent unintended suppression of the genome in human cells.
Health Canada calculated the dietary exposure to total siRNA content per body basis for the adult human body. The estimated exposures determined by the petitioner and by the PTAS are conservative and are well below the threshold of concern for novel siRNA (100 copies/cell). On this basis, the presence of the novel siRNA in Event EF2-114 are not considered a safety concern.
The petitioner and Health Canada determined that the higher level of lycopene present in Event EF2-114 pineapple, compared to conventional pineapple, is not a safety concern, because it is within the range found in other foods with a history of safe use. Lycopene concentration in Event EF2-114 is a mean of 21.3 ppm, compared to 26 ppm in tomato and 45 ppm in watermelon.
The novel PSY and SuRBHra proteins were assessed for potential allergenicity by considering bioinformatics, possible protein glycosylation, and potential dietary exposure.
Bioinformatics analysis of the amino acid sequences for the PSY and SuRBHra proteins was conducted to compare overall identity with known or putative allergens. As well, 80-mer and 8-mer sliding windows for matches were conducted to identify significant homology and the presence of allergenic epitopes, respectively. The flanking regions of the inserts were also searched, based on putative proteins and ORFs. Searches were performed using AllergenOnline (Version 15.0), on January 12, 2015, as well as the NCBI Entrez protein database (January 20, 2015). The results did not detect any matches with known or putative allergens.
Protein glycosylation is a feature of some allergens. Analysis of potential glycosylation sites in PSY and SuRBHra proteins demonstrated neither protein is highly glycosylated. This finding supports a lack of allergenicity.
The novel PSY and SuRBHra proteins were not detected in Event EF2-114 (LOD = 12.5 ng). This result indicates exposure to these novel proteins is expected to be negligible, and is unlikely to stimulate an allergic response.
Based on the information provided, evaluators did not identify any safety concerns regarding the food use of Event EF2-114 pineapple from a toxicological or allergenic perspective.
Health Canada's review of the information presented in support of the food use of Event EF2-114 pineapple does not raise concerns related to food safety. Health Canada is of the opinion that food derived from this pineapple variety is as safe and nutritious as food derived from current commercial pineapple varieties.
This Novel Food Information document has been prepared to summarize the opinion regarding the subject product provided by the Food Directorate, Health Products and Food Branch, Health Canada. This opinion is based upon the comprehensive review of information submitted by the petitioner according to the Guidelines for the Safety Assessment of Novel Foods.
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For further information, please contact:
Novel Foods Section
Health Products and Food Branch
Health Canada, PL2204A1
251 Frederick Banting Driveway
Ottawa, Ontario K1A 0K9
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