Novel Food Information - Herbicide Tolerant Maize Event VCO-Ø1981-5

Health Canada has notified Genective SA that it has no objection to the food use of Herbicide tolerant maize event VCO-Ø1981-5. The Department conducted a comprehensive assessment of this maize 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.

Background

The following provides a summary of the notification from Genective SA and the evaluation by Heath Canada and contains no confidential business information.

1. Introduction

Genective SA has developed an herbicide tolerant maize line containing event VCO-Ø1981-5. This line was developed through Agrobacterium-mediated transformation of immature embryos Hi-II maize to introduce the epsps grg23ace5 gene, which encodes a single polypeptide designated EPSPS ACE5. The EPSPS ACE5 protein is a modified 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) derived from the native Arthrobacter globiformis EPSPS protein which exhibits an inherent tolerance to glyphosate.

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 herbicide tolerant maize event VCO-Ø1981-5 was developed; how the composition and nutritional quality of this maize line compared to conventional varieties; and the potential for this line to be toxic or cause allergic reactions. Genective SA has provided data that demonstrates that herbicide tolerant maize event VCO-Ø1981-5 is as safe and of the same nutritional quality as conventional maize varieties used as food in Canada.

The Food Directorate has a legislated responsibility for pre-market assessment of novel foods and novel food ingredients as detailed in the Food and Drug Regulations (Division 28). Herbicide tolerant maize event VCO-Ø1981-5 is considered a novel food under the following part of the definition of a novel food:

"c) a food that is derived from a plant, animal or microorganism that has been genetically modified such that

1. the plant, animal or microorganism exhibits characteristics that were not previously observed in that plant, animal or microorganism."

2. Development of the Modified Plant

Herbicide tolerant maize event VCO-Ø1981-5 was developed using Agrobacterium-mediated transformation of immature Hi-II maize embryos with the superbinary plasmid pAG3541 (containing the epsps grg23ace5 coding sequence and its required regulatory elements). Immature embryos were excised approximately eight days after pollination and incubated with an A. tumefaciens strain containing the plasmid pAG3541. After three days of co-cultivation on solid culture media, embryos were transferred to media containing the antibiotic TIMENTIN (to eliminate Agrobacterium bacteria from the system post-transformation). After approximately two weeks of culture, embryos were transferred to selection media containing glyphosate. Successfully transformed calli tolerant to glyphosate were identified and transferred to fresh selection media. Embryogenic callus was ultimately regenerated into whole plants and transferred to a greenhouse for further analysis. The regenerated plants (designated T0) were evaluated further for tolerance to glyphosate and through molecular techniques for the presence of the gene of interest (i.e., epsps grg23ace5).

3. Characterization of the Modified Plant

Hi-II T0 plants were crossed with the non-transgenic inbred B110 maize line (Committee for Agricultural Development, Iowa State University), and the seed was harvested and dried. These F1 (designated BC0B) seeds were then backcrossed with B110 to produce BC1B seeds for molecular analysis of the inserted transgenic DNA in the maize genome. The copy number and integrity of the DNA insert in BC1B plants, and the absence of any genetic element present on the backbone of the plasmid pAG3541, were confirmed using Southern blot analysis. The analysis confirmed the presence of a single copy of the inserted DNA at a single insertion site in the maize genome.

The maize flanking genomic sequences of the DNA insert were identified using a PCR-based approach that included the Genome Walker™ strategy, followed by a bioinformatics analysis of the obtained sequences. The insertion site was mapped using a BLAST search against the Maize Genetics and Genomics Database and it was determined that the insertion site is located on chromosome 1 of the maize genome. Further analysis comparing the sequence of the DNA insert to the pre-insertion Hi-II locus sequence indicated that short deletions exist at the extremities of the DNA insert (i.e., 22-base pair (bp) and 16-bp deletions at the 5' and 3' junctions, respectively). Nucleotide sequence alignment of the 5' and 3' flanking genomic sequences of the DNA insert with that of the pre-insertion locus also revealed that a 21-bp deletion to the Hi-II genome occurred as a result of the DNA insertion.

Further characterization on the insertion site was performed to determine if putative genes were present in close proximity to the DNA insert. The corresponding region in the Hi-II genome was analyzed using the FGENESH algorithm. The results indicate that the transgenic DNA was inserted into the maize genome 14-bp upstream of the translation start site of a putative transcription unit. The unit includes a transcription start site, a unique coding region and a polyadenylation site.

A BLAST search identified the coding region as a homolog of acanthoscurrin-2. Acanthoscurrin homologs have been found in other crops such as rice and sorghum. The acanthoscurrin homolog sequence identified in herbicide tolerant maize event VCO-Ø1981-5 shares 54% homology with maize acanthoscurrin-2, 62% and 71% homology with rice and sorghum homologs, respectively.

From the FGENESH analysis, it was suggested that the transgenic DNA insertion probably occurred at the putative promoter or 5' untranslated region (UTR) level. A promoter search was performed using  TSSP / Prediction of Plant Promoters (RegSite Plant DB, Softberry Inc.). A putative promoter region was identified upstream of the acanthoscurrin homolog coding sequence and two polyadenylation signals downstream of the coding sequence, the transgenic DNA inserted between the promoter and the translation start site of the acanthoscurrin homolog coding sequence. It is unlikely that this transcriptional unit is functional, considering both the length of the DNA insert (i.e., 3.7 Kb) and the presence of a terminator region (within the DNA insert) in a functional orientation to the 5' UTR to which it is located. There is no anticipated impact on the transcription of the native maize acanthoscurrin-2 gene due to the DNA insert.

Lastly, an analysis of potential open reading frames (ORFs) at the transgenic DNA insertion site was conducted to investigate whether any ORFs were created as a result of the insertion. As a conservative approach, an ORF was defined as any DNA sequence between two stop codons (i.e., TGA, TAG, or TAA). By this definition, 12 ORFs (including one comprising the existing acanthoscurrin homolog) were identified as potential polypeptides that could be produced from the DNA insert and the flanking genomic sequences in herbicide tolerant maize event VCO-Ø1981-5. A homology search for each ORF against a database of known allergens (i.e., AllergenOnline Database, version 11 from February 2011) was performed using AllergenOnline (accessed May 23, 2011). Two different search types were performed: a homology search using an 80-amino acid (AA) sliding window and an 8-AA sliding window exact match search. The 80-AA sliding window search indicated one ORF as having significant homologies with a new allergen, Amb a4, a recently identified as an allergen in ragweed. Another ORF showed similarity to a collagen protein identified as an allergen however according to AllergenOnline data this was an extremely rare event (i.e., a single person) with an allergic response to an injected vaccine.

The ORF showing homology to the ragweed allergen Amb a4 is located in the acanthoscurrin homolog coding region of the DNA insert 3' flanking genomic sequence, with no overlap to the inserted transgenic DNA. This indicates that non-transgenic maize already contains an ORF showing homology to the ragweed allergen Amb a4. To date there has been no reported allergenic issue with maize related to Amb a4.

A homology search against known toxins was conducted for each ORF using the BLASTP algorithm on the  National Center for Biotechnology Information website using the PAM30 matrix (which is more suitable for short sequences). All resulting sequence matches were manually inspected for homology with known toxic or anti-nutritional proteins as indicated in the United States (US) Environmental Protection Agency (EPA) Code of Federal Regulations document 40 CFR725.421. None of the inspected ORFs exhibited any significant homology to known toxins or other harmful proteins.

Generational stability of the transgenic DNA insert was determined across multiple generations of herbicide tolerant maize event VCO-Ø1981-5. Genomic DNA from four generations was analyzed by Southern blot analysis and the presence of the DNA insert was confirmed in each generation. Inheritance of the epsps grg23ace5 gene in progeny plants was examined using segregation analysis. Glyphosate spray treatment was used to identify plants transformed with the epsps grg23ace5 which confers tolerance to glyphosate. Observed segregation patterns were compared to the expected patterns and these data were compared using chi-squared (c2) distribution analysis. A c2 value of ³0.05 was treated as the cut-off for statistical support of a 1:1 segregation in each generation. This value was exceeded for each of the analyzed generations, thus the results are consistent with the results of the molecular characterization. These results also confirm the stable inheritance of a single copy of the epsps grg23ace5 gene into the progeny of herbicide tolerant maize event VCO-Ø1981-5 in accordance with Mendelian inheritance principles.

4. Product Information

Herbicide tolerant maize event VCO-Ø1981-5 differs from its conventional counterpart by the addition of the epsps grg23ace5 coding sequence and its required regulatory elements. The insertion of the epsps grg23ace5 gene results in the expression of the EPSPS ACE5 protein: a modified 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) derived from the native Arthrobacter globiformis EPSPS protein. The native A. globiformis EPSPS (designated as GRG23) naturally exhibits tolerance to glyphosate. The GRG23 protein was modified (i.e., a total of 10 amino acids changed) using directed evolution protein engineering to obtain an EPSPS with an activity temperature range similar to the native maize EPSPS (i.e., 37 °C). These amino acid changes are not related to the herbicide tolerance trait of the EPSPS ACE5 protein. The EPSPS ACE5 protein is 97.6% identical and 98.5% homologous at the amino acid level to the original EPSPS GRG23 enzyme.

Expression levels of the EPSPS ACE5 protein in various tissues (i.e., leaf, root, whole plant, pollen, and grain) of herbicide tolerant maize event VCO-Ø1981-5 were determined using an enzyme-linked immunosorbent assay (ELISA). Plant material was produced at three field locations and harvested over the entire growing season of 2009. Each field trial included both the transgenic test hybrid BC02 × B116 (containing the event VCO-Ø1981-5) and the non-transgenic isogenic hybrid BC02 × B116 (control). Plants were collected from each replicate at five growth phases throughout the growing season (i.e., V4, V8, R1, R4, and R6). Expression levels were in the tens of nanograms (ng) per milligram (mg) of dry weight tissue (dwt) (leaf, 7.47-24.86 ng/mg dwt; root, 0.00-14.97 ng/mg dwt; whole plant, 5.42-21.50 ng/mg dwt) during the early growing season (i.e., V4-V8), when the maize plant undergoes rapid growth. However, the expression levels were very low and even below the limit of detection (LOD) (leaf, 1.40-8.70 ng/mg dwt; root, 0.00-1.86 ng/mg dwt; whole plant, 0.00-6.93 ng/mg dwt; pollen, 0.00-9.60 ng/mg dwt; grain, 0.00 ng/mg dwt) in latter reproductive stages (i.e., R1-R6), when the plant`s energy is going towards development of the grain and pollen. No detectable levels of EPSPS ACE5 protein were detected in any tissues from the control replicates.

5. Dietary Exposure

It is expected that herbicide tolerant maize event VCO-Ø1981-5 will be used in applications similar to conventional maize varieties. The petitioner does not anticipate a significant change in the food use of maize with the introduction of herbicide tolerant maize event VCO-Ø1981-5.

6. Nutrition

The petitioner provided data from a compositional study to assess key nutritional components in forage and grain samples from herbicide tolerant maize event VCO-Ø1981-5 and control hybrids. The study and results pertaining to the grain samples are described as follows.

The petitioner conducted a field trial during the 2009 growing season at five sites located in the US Corn Belt (i.e., Boone County, IN; Valley County, NE; Kossuth County, IA; Webster County, IA; and Wright County, IA). The following maize lines were grown in a randomized block design with three replicates per site: herbicide tolerant maize event VCO-Ø1981-5, a control hybrid (a near isogenic, non-modified parent variety), and 3 commercial maize varieties that were used as reference. As indicated by the petitioner, the hybrids and all agronomic practices were representative of those used for commercial maize production in each study location. No herbicides were used post-emergence (conventional or glyphosate). For grain samples, two ears were harvested at physiological maturity (i.e., R6) and 150 grams if grain were collected and analyzed for 71 components using standard methods. The grain compositional analysis includes all proximates, amino acids, fatty acids, micronutrients, and naturally occurring anti-nutritional factors mentioned in the Organization for Economic Co-operation and Development (OECD) Consensus Document on Compositional Consideration for New Varieties of Maize (Zea mays). All compositional analyses were conducted at EPL Bio-Analytical Services (EPL-BAS, Niantic, IL) or Eurofins Scientific Inc. (Des Moines, IA). An analysis of variance (ANOVA) was done with the statistical software JMP 8 (SAS Institute, Cary, NC) to compare measured analyte mean levels between herbicide tolerant maize event VCO-Ø1981-5 and the control hybrid. Measured data ranges were compared with published results from the International Life Sciences Institute (ILSI) crop composition database.

Of the 71 components analyzed, 57 analyte comparisons were suitable for statistical analysis. No statistical differences were found between herbicide tolerant maize event VCO-Ø1981-5 and the control hybrid for 44 of 57 analytes. The most important differences between herbicide tolerant maize event VCO-Ø1981-5 and the control hybrid were observed for phenylalanine (10.6% lower than the control hybrid), beta carotene (20.7% higher than the control hybrid), vitamin B3 (21.4% higher than the control hybrid), total tocopherols (46% higher than the control hybrid), calcium (13.3% lower than the control hybrid), copper (23.1% higher than the control hybrid), and secondary metabolite ferulic acid (14.5% lower than the control hybrid). Small, but statistically significant differences were also noted for palmitic acid, linoleic acid, eicosenoic acid, tyrosine, arginine, and potassium. However, these differences were deemed too small (<10%) to be biologically relevant. With the exception of linoleic acid levels, all data ranges and mean standard deviations of herbicide tolerant maize event VCO-Ø1981-5 and the control hybrid overlapped and the values were not distinctly different from the normal range of variability.

For linoleic acid, the mean values of both herbicide tolerant maize event VCO-Ø1981-5 and the control hybrid were slightly above the data range of reference hybrids, with the control hybrid being outside the published range from the ILSI composition database. The petitioner concluded that the absolute level of difference was small and not biologically relevant. The high levels of linoleic acid were attributed to the specific genotype of the hybrid and therefore not deemed related to the transgenic event.

The petitioner concluded that herbicide tolerant maize event VCO-Ø1981-5 is comparable to the non-transgenic near-isogenic hybrid, reference hybrids, and the published data ranges, regarding the nutritional composition. In addition, no consistent patterns emerged to suggest that biologically significant changes in the composition of the grain had occurred as an unintended consequence of the genetic modification resulting in herbicide tolerant maize event VCO-Ø1981-5. In general Food Directorate evaluators found that the quantity and quality of the information provided was satisfactory. The compositional analysis for the grain samples did not raise any nutritional concerns.

7. Chemistry/Toxicology

Expression of the EPSPS ACE5 protein in herbicide tolerant maize event VCO-Ø1981-5 is low and thus the protein could not be purified in sufficient quantity for use in subsequent safety testing and protein characterization. Therefore, the EPSPS ACE5 protein was produced using an Escherichia coli expression system and compared to the EPSPS ACE5 protein extracted from the leaves of herbicide tolerant maize event VCO-Ø1981-5 plants. Equivalence between the plant-derived and microbial-derived EPSPS ACE5 protein was demonstrated by physical methods including apparent molecular weight (i.e., SDS-PAGE), amino acid sequence homology at the gross level (i.e., MALDI-TOF mass spectrometry) and fine level (i.e., N-terminal amino acid sequencing), immunological similarity (i.e., western blot analysis), and glycosylation assay. Based on the information provided, Food Directorate evaluators concluded that the plant- and microbial-derived EPSPS ACE5 proteins are substantially equivalent.

The potential toxicity of microbial-derived EPSPS ACE5 protein was assessed in a 14-day acute oral toxicity study, where mice (5 animals per sex) were administered microbial-derived EPSPS ACE5 protein at a dose of 2,000 mg/kg body weight (bw). Animals were observed for 14 days after administration, then euthanized and necropsied. The parameters that were recorded included mortality, signs of clinical toxicity, bw, as well as external and internal examination conducted at termination. The test substance was well tolerated and did not induce any treatment-related or adverse effects.

An in vitro digestibility assay for microbial-derived EPSPS ACE5 protein was evaluated in simulated gastric fluid (SGF) at 37 °C containing the mammalian digestive enzyme pepsin. SDS-PAGE and western blot analyses were performed to detect microbial-derived EPSPS ACE5 protein and potential stable protein fragments after 0-60 minutes of digestion. Both assays showed that microbial-derived EPSPS ACE5 protein was rapidly digested in SGF, within 30 seconds of incubation in the presence of pepsin at pH 1.2. This is supportive that the intact protein is unlikely to enter the bloodstream and cause systemic toxicity.

A heat stability study with microbial-derived EPSPS ACE5 protein was conducted at temperatures ranging from 4 °C to 95 °C for 30 or 60-minute incubations. SDS-PAGE and western blot analyses were performed to detect the presence of microbial-derived EPSPS ACE5 protein. The results of the study indicate that microbial-derived EPSPS ACE5 protein becomes denatured after heat treatment at temperatures ≥60 °C. Therefore, active protein in unlikely to be consumed from products that are normally processed by heat treatment (e.g., cooking).

A bioinformatics search was conducted using the NCBI Protein dataset by BLASTP bioinformatics analysis to compare amino acid sequence from EPSPS ACE5 protein for similarity to known toxins. From the searches conducted, no relevant matches were made between the EPSPS ACE5 protein and known toxins.

The level of EPSPS ACE5 in grain is below the level of detection (i.e., 1.79 ng/mg dwt). By comparison, no adverse effects were observed at 2,000 mg/kg bw in a 14-day acute oral toxicity study with mice. The amount of grain that must be consumed by children and adults to match the limit protein dose was calculated. A 15-kg child would need to consume 16,759 kg maize/day and a 70-kg adult would need to consume 78,212 kg maize/day to reach the equivalent amount of EPSPS ACE5 protein in the highest dose administered to mice where no adverse effects were observed. The potential for the EPSPS ACE5 protein to cause a toxic reaction is considered negligible and thus it is concluded that this protein does not pose a toxicological concern in herbicide tolerant maize event VCO-Ø1981-5.

8. Allergenicity

As mentioned above, the microbial-derived EPSPS ACE5 protein is rapidly digested when exposed to SGF and therefore this protein would not be expected to incite an allergenic reaction. Additionally, as the protein is denatured at high temperatures (e.g., during food processing, cooking, etc.) it is less likely to elicit an allergic reaction.

The petitioner provided an assessment on the similarity of the EPSPS ACE5 protein to known or putative allergens using the AllergenOnline database. A bioinformatics analysis for potential allergenicity was performed for sequence similarity at the whole protein level, an 80-amino acid (AA) sliding window, and the short 8-AA contiguous level. The results of the searches found no significant similarity between the EPSPS ACE5 protein and any known or putative allergens. It was thus concluded that the EPSPS ACE5 protein expressed in herbicide tolerant maize event VCO-Ø1981-5 does not present any allergenicity concerns.

Conclusion

Health Canada's review of the information presented in support of the food use of herbicide tolerant maize event VCO-Ø1981-5 does not raise concerns related to food safety. Health Canada is of the opinion that food derived from herbicide tolerant maize event VCO-Ø1981-5 is as safe and nutritious as food from current commercial maize varieties.

Health Canada's opinion deals only with the food use of herbicide tolerant maize event VCO-Ø1981-5. Issues related to its use as animal feed have been addressed separately through existing regulatory processes in the Canadian Food Inspection Agency (CFIA). The CFIA evaluated information provided on the environmental, animal, and human health safety of herbicide tolerant maize event VCO-Ø1981-5 with the intended use in animal feed. From their assessment, the CFIA concluded that there are no concerns from an environmental and feed safety perspective. This perspective is applicable to the food and feed products derived from herbicide tolerant maize event VCO-Ø1981-5 destined for commercial sale.

It is the continuing responsibility of the food manufacturer or importer to ensure that their products are in compliance with all applicable statutory and regulatory requirements. Any new information obtained in relation to these products which have potential health and safety implications should be forwarded to Health Canada for our consideration in order to ensure the continued safety and integrity of all foods available in the Canadian marketplace. The sale of a food which poses a hazard to the health of consumers would contravene the provisions of the Food and Drugs Act.

This Novel Food Information document has been prepared to summarize the opinion regarding the subject product provided by the Food Directorate, Health Products and Food Branch, Health Canada. This opinion is based upon the comprehensive review of information submitted by the petitioner according to the Guidelines for the Safety Assessment of Novel Foods.

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For further information, please contact:

Novel Foods Section
Food Directorate
Health Products and Food Branch
Health Canada, PL2204A1
251 Frederick Banting Driveway
Ottawa, Ontario K1A 0K9
novelfoods-alimentsnouveaux@hc-sc.gc.ca