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Biotechnology Consultation - Note to the File
Biotechnology Notification File BNF No. 000137
April 30, 2013
Event VCO-Ø1981-5 glyphosate-tolerant corn
Corn, maize, Zea mays L., Event VCO-Ø1981-5, 5-enolpyruvylshikimate-3-phosphate synthase, EPSPS ACE5, epsps grg23ace5, Arthrobacter globiformis, glyphosate tolerance, herbicide tolerance, OECD Unique Identifier VCO-Ø1981-5, Genective S.A., Bayer CropScience LP
This document summarizes our evaluation of Biotechnology Notification File (BNF) No. 000137.(1) In a submission dated March 5, 2012, Genective S.A. (Genective) submitted to the Food and Drug Administration (FDA) a safety and nutritional assessment of genetically engineered herbicide-tolerant corn(2), transformation event VCO-Ø1981-5 (corn event VCO-Ø1981-5).(3) Additional information was provided on August 27, 2012. FDA evaluated the information in Genective's submissions to ensure that regulatory and safety issues regarding human food and animal feed derived from the new plant variety have been resolved prior to commercial distribution.
In our evaluation of BNF No. 000137, we considered all information provided by the notifier as well as publicly available information and information in the agency’s files. Here we discuss the outcome of the consultation, but do not intend to restate the information provided in the final consultation in its entirety.
The intended technical effect of the modification in corn event VCO-Ø1981-5 is to confer tolerance to the herbicide glyphosate. To accomplish this objective, Genective introduced into the corn genome the epsps grg23ace5 gene, which encodes a modified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) protein that is 97.6% identical at the amino acid level to the EPSPS GRG23 protein from the soil bacterium Arthrobacter globiformis. The EPSPS ACE5 protein binds glyphosate with lower affinity when compared to the native corn EPSPS protein, but binds to the natural substrate, phosphoenolpyruvate, with similar affinity to corn EPSPS.
The purpose of this evaluation is to assess whether the developer has introduced a substance requiring premarket approval as a food additive or use of the new plant variety in food or animal feed raises other regulatory issues under the Federal Food, Drug and Cosmetic Act (FD&C Act). The Environmental Protection Agency (EPA) regulates the use of herbicides under the Federal Insecticide, Fungicide, and Rodenticide Act and the FD&C Act. Under EPA regulations, the herbicide, metabolic by-products, and residues in corn event VCO-Ø1981-5 that result from detoxification of applied herbicides by the expression products of corn event VCO-Ø1981-5 are considered pesticidal substances.
Genetic Modification and Characterization
Genective transformed the recipient corn line “Hi-II” (a publicly available corn line) to create corn event VCO-Ø1981-5. The Hi-II hybrid line was obtained from the crossing of the two separate lines Hi-IIA and Hi-IIB.
Introduced DNA and Transformation Method
Corn event VCO-Ø1981-5 was generated by the transfer of the T-DNA from plasmid pAG3541 into embryo tissue of corn line Hi-II via Agrobacterium-mediated transformation. Genective describes plasmid pAG3541, which contains the epsps grg23ace5 expression cassette. The expression cassette, located between the left and right T-DNA borders, contains a synthetic epsps grg23ace5 gene that is 97.6% identical at the amino acid level to the EPSPS GRG23 protein from the common soil bacterium A. globiformis. The epsps grg23ace5 expression cassette contains the following components:
- the Ubiquitin 4 promoter region from Saccharum officinarum L. (sugar cane), which includes the ScUbi4 promoter, the ScUbi4 5' untranslated region, and the ScUbi4 intron;
- the N-terminal chloroplast transit peptide coding sequence from acetohydroxyacid synthase gene from Zea mays (corn);
- the 3' untranslated region of the 35S gene from the cauliflower mosaic virus, which directs polyadenylation of the mRNA.
Characteristics, Inheritance, and Stability of the Introduced DNA
Genective characterized the introduced DNA using Southern blot hybridization and DNA sequencing of the end-products of polymerase chain reactions (PCR). Southern blot analyses showed that corn event VCO-Ø1981-5 contains a single copy of the T-DNA inserted at a single locus in the genome and lacks any detectable plasmid backbone sequences. Genective further analyzed the insertion site and flanking corn genomic sequences using a PCR-based approach followed by DNA sequencing and bioinformatics analysis of the obtained sequences. This analysis confirmed the integrity of the expression cassette, and also established that the expression cassette was inserted into the apparent 5' untranslated region of a putative gene coding for acanthoscurrin-homolog protein, which is described as an antimicrobial peptide. Genective states that while the insertion could potentially affect the expression of this gene, they did not observe any phenotypic changes in corn event VCO-Ø1981-5, nor were any differences in agronomic performance or disease resistance observed that would indicate a potential effect on the expression of an antimicrobial peptide.
Genective assessed the heritability and stability of the introduced DNA over four segregating generations through analyses of segregation data as determined through sensitivity to glyphosate. Mendelian segregation analysis using Chi-square distribution testing demonstrated no statistically significant difference between the observed and expected phenotypic segregation ratios. This supports the conclusion that the insertion of the T-DNA occurred at a single locus inherited according to Mendelian principles. Genective also assessed the stability of the corn event VCO-Ø1981-5 T-DNA insertion across four generations by Southern blot analysis. Genective states that these data demonstrate a stable insertion site inherited across multiple generations.
Genective performed bioinformatic analyses using genomic DNA sequence data to assess the potential for new open reading frames (ORFs) that may encode a biologically active putative polypeptide as a result of insertion of the introduced DNA. No homologies with known allergen sequences were identified using a short (8) amino acid search algorithm when these potential ORFs were analyzed for their putative allergenicity. Using the 80 amino acid sliding window (> 35% homology) approach, two areas of potential homology were identified, which are also present in the native corn genome indicating that they were not a result of the insertion present in corn event VCO-Ø1981-5. No significant homologies with known toxins or other harmful proteins were found, when ORFs were analyzed against publicly available databases using the BLASTP algorithm. Genective further noted that it was unlikely any of the ORFs identified would generate translatable mRNA, since no start codons were found.
Corn event VCO-Ø1981-5 was genetically engineered to express the EPSPS ACE5 protein, which confers resistance to the herbicide glyphosate. The EPSPS ACE5 protein was derived from the native A. globiformis glyphosate-tolerant EPSPS GRG23 protein using a directed evolution protein engineering process. This process altered the amino acid sequence to more closely match native corn EPSPS and impart greater temperature stability than native A. globiformis EPSPS GRG23 at 37°C. Genective states that the EPSPS ACE5 protein is 97.6% identical at the amino acid level to the EPSPS GRG23 protein, and that A. globiformis is a common Gram positive soil bacterium that is not known to produce mammalian toxins or allergens.
The EPSPS enzymatic reaction is very important in plant biology, as it is a non-branching step in the shikimate pathway, leading to the biosynthesis of a large number of aromatic plant metabolites, including essential aromatic amino acids (phenylalanine, tyrosine, and tryptophan), tetrahydrofolate, ubiquinone and vitamin K. EPSPS protein family members expressed in glyphosate-tolerant crops have been previously evaluated by FDA in many other biotechnology consultations.(4) The safety of the EPSPS ACE5 protein was previously described in an early food safety evaluation (NPC 12) submitted to FDA in 2009.
Protein Expression Level
Genective conducted field studies to assess expression of the EPSPS ACE5 protein in corn event VCO-Ø1981-5. Protein levels were determined by enzyme-linked immunosorbent assay (ELISA) conducted on the leaf, root, and whole plants at multiple developmental stages and in pollen and grain in samples collected from three locations in Iowa during the 2009 growing season. At each location, three replicated plots of corn event VCO-Ø1981-5 and the non-transgenic control were planted in a randomized complete block design. Genective reports that the EPSPS ACE5 protein was detected by ELISA in all tissues, except grain, and that expression was highest in rapidly growing vegetative tissue. The highest protein concentrations observed were approximately 25 nanograms per milligram dry weight in V4 and V8 vegetative-stage leaves. By maturity the EPSPS ACE5 protein was below the limit of detection in all tissues sampled.
Safety Assessment of the EPSPS ACE5 Protein
Potential for Toxicity of the Introduced Protein
Genective compared the EPSPS ACE5 amino acid sequence to the amino acid sequence of known toxins using standard bioinformatics methods, including BLASTP algorithm. Genective reports that the firm examined sequence matches with an Expectancy value of 1.0 or less and found no significant homology between EPSPS ACE5 and known toxins.
To obtain sufficient quantities of the EPSPS ACE5 protein for conducting safety assessment studies, Genective produced the EPSPS ACE5 protein using an Escherichia coli protein expression system. Genective confirmed the identity and biochemical equivalence of the E. coli-derived and plant-derived EPSPS ACE5 proteins using several analytical techniques.
Genective also describes the result of an acute toxicity study in mice. Male and female mice were administered by oral gavage a negative control or single dose of EPSPS ACE5 at 2000 milligrams per kilogram bodyweight. Genective reports that no treatment-related effects were observed in this study. On the basis of the results of the homology search and the toxicity study, Genective concluded that the EPSPS ACE5 protein is safe for consumption and would pose little or no risk to human or animal health.
Potential for Allergenicity of the Introduced Protein
To assess the potential for allergenicity of the EPSPS ACE5 protein, Genective considered the similarity of the EPSPS ACE5 amino acid sequence to known allergens, digestibility in simulated gastric fluid, and heat stability. Genective provides results of bioinformatics analyses, including FASTA algorithm and 8-mer comparison, between EPSPS ACE5 protein and allergens in the public allergen database, AllergenOnline (www.allergenonline.com); release 11, 2011. Genective reports that no sequence alignment met or exceeded the threshold of 35% identity over 80 amino acids and that there were no contiguous stretches of 8 or greater amino acids shared between the EPSPS ACE5 protein and proteins in the allergen database.
Genective describes in vitro assessment of the stability of EPSPS ACE5 in simulated gastric fluid. Genective states that the results of this assessment indicate that the EPSPS ACE5 protein is rapidly digested. Genective also states that EPSPS ACE5 lacks the potential for glycosylation. Finally, Genective reports that while EPSPS ACE5 has been optimized for heat stability under environmental conditions relative to the native A. globiformis EPSPS GRG23 protein, it remains heat-labile under industrial conditions and its digestibility remains comparable to other EPSPS proteins.
Food & Feed Use
Genective states that corn event VCO-Ø1981-5 will be grown for the same uses as currently commercialized corn varieties. Corn (Zea mays L.) originated in Mexico and was grown as a food crop as early as 2700 B.C. Today, corn is grown worldwide for food, feed, and industrial uses. Corn grain is used in food primarily in the form of processed products, such as high fructose corn syrup, cereals, oil, meal, flour, starch, and grits. Corn oil is high in polyunsaturated fatty acids and is used mainly as a salad and cooking oil and in margarine production. Corn is also used for fuel ethanol and beverage alcohol production.
Corn is also used extensively for animal feed and feed products. Corn grain is fed to many animal species including cattle, poultry, and swine, either as intact or processed grain or as dry or wet milling byproducts. Corn silage is a common feed for ruminants. Corn is a good source of energy, polyunsaturated fatty acids, some B vitamins, vitamin E, and several essential amino acids, although corn-based animal diets are typically supplemented (as needed) with vitamins, minerals, and limiting amino acids.
Scope of Analysis
Genective states that seeds from corn event VCO-Ø1981-5 and a negative segregant were crossed with the inbred line B116 to produce near-isogenic hybrids: BC0BS2 x B116 positive VCO-Ø1981-5 corn hybrid (VCO-Ø1981-5 hybrid) and the corresponding non-transgenic control hybrid (control hybrid). Genective reports data on 71 components, including key nutrients, anti-nutrients, and secondary metabolites in grain and forage derived from VCO-Ø1981-5 corn and control hybrids.(5) Genective also compares the composition of VCO-Ø1981-5 hybrid with three commercial hybrid corn varieties (reference varieties). No herbicide sprays were used post-emergence.
Study Design - Compositional Analyses
Genective states that grain and forage were obtained from corn grown at each of five field locations in North America during the 2009 growing season. Each field site utilized a randomized block design with three blocks per site of VCO-Ø1981-5 hybrid, the control hybrid, and the three reference varieties. Grain samples (150 grams of grain from the center of each of two mature (R6) ears) and forage samples (one whole plant at the R4 stage) were collected from each of three blocks. Genective analyzed the data using analysis of variance methods comparing compositional data combined across all field sites for VCO-Ø1981-5 hybrid and control hybrid grain and forage. Statistical differences in composition were considered significant at the P ≤ 0.05 level. Genective also cited composition information on corn from the published scientific literature in its discussion as a comparison. Genective considered component mean values that fell within the ranges of the reference varieties and literature values to be within the normal variation for commercial corn.
Results of Analyses
Compositional Analysis of Corn Forage
Genective reports the results of compositional analysis for nine components in corn forage (crude protein, fat, ash, crude fiber, carbohydrates (by calculation), acid detergent fiber (ADF), neutral detergent fiber (NDF), calcium, and phosphorus). Genective reports that no statistically significant differences were observed in the mean values for any of the measured components from forage obtained from VCO-Ø1981-5 hybrid and control hybrid.
Compositional Analysis of Corn Grain
Genective reports results of compositional analyses for 60 components in corn grain. Genective analyzed grain for proximates (moisture, crude protein, fat, ash, crude fiber, starch, carbohydrates (by calculation)), ADF, NDF, total dietary fiber, fatty acids, amino acids, B vitamins, α-tocopherol, total tocopherols, β-carotene, minerals, secondary metabolites (ferulic acid, p-coumaric acid, and inositol), and anti-nutrients (raffinose, phytic acid, and trypsin inhibitor). Genective reports statistically significant differences in the following VCO-Ø1981-5 hybrid components when compared to the control hybrid: three fatty acids (linoleic acid (C18:2), palmitic acid (C16:0), and eicosenoic acid (C20:1)), three amino acids (arginine, phenylalanine, and tyrosine), β-carotene, niacin, total tocopherols, three minerals (calcium, copper, and potassium), and ferulic acid.
Genective notes that where a statistical difference was detected in a component between VCO-Ø1981-5 hybrid and control hybrid, the ranges still overlapped; in addition, mean values for vitamins, minerals, amino acids, palmitic acid, eicosenoic acid, secondary metabolites,(6) and anti-nutrients from VCO-Ø1981-5 hybrid were within the range established for the reference varieties and within published literature ranges for corn. For linoleic acid, Genective reports that mean values for both VCO-01981-5 and control hybrids were slightly above both range of values for the reference varieties and the values provided in ILSI (2006).(7) Genective notes that these differences are small, and that VCO-Ø1981-5 hybrid was closer than control hybrid to the maximum value observed for reference varieties and values in the crop composition database. Genective thus concludes that these differences are unlikely to have biological significance.
Summary of Compositional Analyses
Genective concludes that forage and grain from VCO-Ø1981-5 hybrid are nutritionally comparable to forage and grain from conventional corn hybrids. Genective bases its conclusion on compositional analysis of VCO-Ø1981-5 hybrid, control hybrid, and reference varieties, as well as comparison with publicly available composition data for conventional corn varieties. Genective concludes that the differences in the observed levels of components in grain between VCO- Ø1981-5 hybrid and other corn hybrids are not meaningful for food and feed safety or nutrition.
FDA evaluated Genective’s submission to determine whether corn event VCO- Ø1981-5 raises any safety or regulatory issues with respect to the intended modification or with respect to the food and feed itself. Based on the information provided by the company and other information available to the agency, FDA did not identify any safety or regulatory issues under the FD&C Act that would require further evaluation at this time.
Genective concludes that, with the exception of the intended modification (tolerance to glyphosate), corn event VCO-Ø1981-5 and foods and feeds derived from it are not materially different in composition, safety or any other relevant parameter from other corn varieties now grown, marketed, and consumed in the U.S. At this time, based on Genective’s data and information, the agency considers Genective’s consultation on corn event VCO-Ø1981-5 to be complete.
(1) The protein expressed in corn event VCO-Ø1981-5 (the EPSPS ACE5 protein) was the subject of New Protein Consultation No.12 (NPC 12) from Athenix Corp., now an affiliate of Bayer CropScience LP (Bayer). FDA responded to Athenix Corp. on October 15, 2010, with a letter stating that we had no questions about Athenix’s conclusion in NPC 12 that the potential inadvertent presence in the food supply of low levels of the EPSPS ACE5 protein would not raise food safety concerns. The letter is available at http://www.fda.gov/npcinventory.
(2) This document uses the word “corn” instead of “maize,” in accordance with common practice in the United States.
(3) Corn event VCO-Ø1981-5 was developed in collaboration with Bayer.
(4) See BNF 1, 20, 26, 35, 51, 56, 71, 77, 79, 80, 84, 90, 97, 98, 104, 106, 109, 122, 126, and 127. FDA had no questions about the safety of EPSPS proteins in any of these consultations. (http://www.fda.gov/bioconinventory)
(5) Components for analysis were selected based on the OECD Consensus Document on Compositional Considerations for New Varieties of Maize (Zea mays): Key Food and Feed Nutrients, Anti-Nutrients and Secondary Plant Metabolites (2002).
(6) For ferulic acid, Genective reports that control corn but not corn event VCO-Ø1981-5 fell outside the range of the reference varieties (but was within range of values provided in the ILSI Crop Composition Database, Version 3.0, www.cropcomposition.org.).
(7) FDA notes that the linolenic acid values for VCO-Ø1981-5 hybrid and control hybrid are within the range of more recent ILSI values than those reported by Genective (ILSI Crop Composition Database, Version 4.2, www.cropcomposition.org. [accessed May 2, 2012]).