Biotechnology Consultation Note to the File BNF No. 000126
Return to inventory: Completed Consultations on Foods from Genetically Engineered Plant Varieties
Biotechnology Consultation - Note to the File
Biotechnology Notification File BNF No. 000126
Date: March 23, 2012
Subject: MON 87427, tissue-selective herbicide tolerant corn
Keywords: Corn; maize; Zea mays; cp4 epsps, 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) from Agrobacterium sp. strain CP4, herbicide tolerance, glyphosate, male sterility, tissue selective, hybrid seed production, MON 87427, OECD Unique Identifier MON 87427-7, Roundup Ready®, Monsanto
This document summarizes our evaluation of biotechnology notification file (BNF) No. 000126. In a submission dated December 15, 2010, Monsanto Company (Monsanto) submitted to the Food and Drug Administration (FDA) a safety and nutritional assessment of bioengineered herbicide tolerant corn, transformation event MON 87427-7 corn (hereafter referred to as MON 87427 corn). Monsanto provided additional information on April 21, 2011, and April 28, 2011. FDA evaluated the information in Monsanto's submissions to ensure that regulatory and safety issues regarding human food and animal feed from the new plant variety have been resolved prior to commercial distribution. In our evaluation of BNF 000126, we considered all information provided by Monsanto 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 MON 87427 corn is to confer tolerance to the herbicide glyphosate in a tissue-selective manner. To accomplish this objective, Monsanto introduced a gene, cp4 epsps, which encodes 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) protein into a conventional corn cultivar (LH198 × HiII). The CP4 EPSPS protein confers tolerance to the herbicide glyphosate. A specific promoter and intron combination (e35S-hsp70) is used in the expression cassette for the cp4 epsps gene that results in CP4 EPSPS protein production in vegetative and female reproductive tissue, providing tolerance to glyphosate within these tissues. Reduced production of CP4 EPSPS in specific male tissues results in these tissues being sensitive to the herbicide glyphosate and thus eliminates the need for detasseling during the production of hybrid seed corn.
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 with respect to the Federal Food, Drug and Cosmetic Act (FD&C Act). The Environmental Protection Agency (EPA) regulates herbicides under the FD&C Act and the Federal Insecticide, Fungicide, and Rodenticide Act. Under EPA’s regulations, the herbicide residues and metabolic by-products in MON 87427 corn, resulting from the detoxification of the applied herbicide by the expression product, are considered pesticide residues. In its submission to FDA, Monsanto indicated that it has submitted a regulatory package to EPA for the use of glyphosate on MON 87427 corn.
Genetic Modification and Characterization
Monsanto transformed the recipient line LH198 × HiII to obtain MON 87427 corn.
Transformation Plasmid and Method
Monsanto describes the transformation plasmid, PV-ZMAP1043. This plasmid contains the cp4 epsps expression cassette within the transfer DNA (T-DNA) of this plasmid, delineated by left and right border sequences. The expression cassette contained the following genetic elements: the e35S promoter, the hsp70 intron, the CTP2 targeting sequence, the cp4 epsps gene, and the nos 3’ non-translated region of nopaline synthase. MON 87427 corn was generated using Agrobacterium-mediated transformation of immature LH198 × HiII embryo tissue utilizing PV-ZMAP1043. The kanamycin resistance gene, aads, present in the intact PV-ZMAP1043 plasmid, would not be expected to be transferred to the transformed cells because it was outside the T-DNA.
Characteristics, Inheritance, and Stability of the Introduced DNA
Monsanto characterized the insert in MON 87427 corn using restriction enzyme digestion of genomic DNA followed by Southern blot analysis. Monsanto concluded that results of this analysis demonstrate that a single intact gene cassette was integrated into the plant genome. Monsanto further notes that Southern blot analyses using probes designed to detect plasmid sequences in the entire sequence of PV-ZMAP1043 confirmed that no plasmid vector backbone sequences were present in MON 87427. Therefore, the aad2 gene from PV-ZMAP1043 was not inserted into the corn genome. Monsanto states that results obtained from the Southern blot analyses were complemented by DNA sequencing of the insert and flanking genomic sequences, which confirmed the complete sequence of the insert and demonstrated there were no rearrangements in the insert or at the insertion site. Monsanto further confirmed the junctions between the expression cassette and the corn genomic DNA by comparing the sequences at the insertion site to conventional corn. Monsanto assessed the stability of the DNA insert across five generations through restriction enzyme digestion of genomic DNA followed by Southern blot analysis. Monsanto states that these data demonstrated that MON 87427 corn contained one copy of the cp4 epsps expression cassette and is stably inherited across multiple generations. Monsanto also assessed heritability and stability of the introduced DNA in MON 87427 corn through analyses of segregation data as determined through tissue sensitivity to glyphosate. Monsanto states that Chi-square analysis demonstrated no statistically significant difference between the observed and expected segregation patterns. This supports the conclusion that the cp4 epsps expression cassette contained in MON 87427 corn is located at a single locus and is inherited according to Mendelian principles. Monsanto performed bioinformatic analyses to detect potential open reading frames (ORF) of the MON 87427 corn insert and flanking genomic DNA sequences to determine whether insertion of the introduced DNA may have created any ORF that may encode a toxin, allergen, or biologically active putative polypeptide. Based on the bioinformatic analyses, Monsanto concludes that even in the unlikely occurrence of translation of any of these ORFs, the putative polypeptides share no homology with known allergens or toxicants and therefore would not constitute a safety concern.
Identity and Function of Introduced Protein
MON 87427 corn was genetically engineered to express the CP4 EPSPS protein, which confers resistance to the herbicide glyphosate. This protein has been previously evaluated by FDA in many other biotechnology consultations.1 Monsanto notes that the gene encoding the CP4 EPSPS protein was derived from Agrobacterium sp. strain CP4, a well-described non-pathogenic, non-allergenic, and non-toxigenic organism. Monsanto discusses analyses2 to confirm the identity of the CP4 EPSPS protein introduced into MON 87427 corn. Monsanto reports that these analyses confirm that CP4 EPSPS from MON 87427 corn is equivalent to CP4 EPSPS expressed in Escherichia coli and CP4 EPSPS expressed in other bioengineered crops.
Protein Expression Level
Monsanto conducted field expression studies of the CP4 EPSPS protein in MON 87427 corn obtained from samples collected during the 2008 growing season from five field locations in the United States. Protein levels were determined by enzyme-linked immunosorbent assay (ELISA) conducted on several corn tissue types.3 Monsanto reports mean concentrations of CP4 EPSPS in corn tissues ranged from below the limit of detection in pollen to 680 micrograms per gram (dry weight) in over-season leaf stage 1 (OSL1). Monsanto reports the mean concentration of CP4 EPSPS in grain to be 4.2 micrograms per gram dry weight.
Potential for Toxicity of the Introduced Protein
Monsanto provides bioinformatics analyses for the CP4 EPSPS protein using standard methods, including FASTA algorithm comparison of CP4 EPSPS protein against known protein toxins that are in the TOX_2010 database compiled by Monsanto. Monsanto states that this analysis revealed no relevant sequence alignments between CP4 EPSPS and known protein toxins, as determined by the calculation of an E-score (expectation score) of less than or equal to 1 × 10−5 between MON 87427 corn CP4 EPSPS and protein toxin sequences in the TOX_2010 database. Monsanto notes that an E-score of less than or equal to 1 x 10−5 indicates significant homology. Monsanto concludes that no sequence similarity exists between CP4 EPSPS protein and any known toxin or other biologically active proteins that would be harmful to human or animal health. Monsanto also describes results from an acute toxicity study in mice conducted by Harrison et al. (1996). In this study, male and female CD-1 mice were gavaged with doses up to 572 milligrams of CP4 EPSPS protein per kilogram of body weight. Monsanto reports that no adverse events were observed at any dose level, as determined by survival, clinical observations, body weight gain, food consumption or gross pathology.
Potential for Allergenicity of the Introduced Protein
Monsanto provides an analysis of the potential for allergenicity of the CP4 EPSPS protein. Monsanto assessed the structural similarity of CP4 EPSPS to known allergens through standard bioinformatic methods using the AD_2010 database (FAARRP, 2010). This analysis revealed no significant sequence homology as determined by the E-score. Monsanto also reports that no sequence alignment met or exceeded the threshold of 35% identity over 80 amino acids and there were no contiguous stretches of 8 or greater amino acids shared between the CP4 EPSPS protein and the proteins in the allergen database. Monsanto describes a series of in vitro assessments of the stability of E. coli-produced CP4 EPSPS in simulated gastric fluid and simulated intestinal fluid. Monsanto states that these studies indicate that CP4 EPSPS is rapidly digested. Monsanto also notes that the expression level of CP4 EPSPS in MON 87427 corn grain is very low, with a mean concentration of 4.2 micrograms per gram dry weight, calculated to be 0.004% of total protein. Monsanto highlights that the low concentration of CP4 EPSPS in MON 87427 grain, the lack of homology to known allergens, and the digestibility studies support the conclusion that CP4 EPSPS is unlikely to be allergenic.
Food & Feed Use
Monsanto reviews the historical development of corn (Zea mays L.) as an agricultural product and describes the current uses of corn in food and feed. For food applications, Monsanto notes that corn grain is usually either wet-milled or dry-milled prior to its use in food. Wet-milling corn grain will produce starches and corn syrup based sweeteners, which are then used ubiquitously in the food supply. Dry-milled corn grain produces corn grits and corn meal, both of which are consumed as foods or as ingredients of foods. Monsanto notes that corn is commonly used in animal feed. Monsanto states that in 2009 43% of the corn produced in the U.S. was used for animal feed. Whole corn grain is usually ground or flaked and mixed with a high-protein feed compound and supplemented with vitamins and minerals before use as feed. Corn milling also produces by-products that often are used in animal feeds, including corn gluten and corn gluten meal. The whole corn plant is also used as an animal feed as corn silage, which is produced through a process of harvesting, chopping and storing the material in silos.
Scope of Analysis
Monsanto reports data on seventy-eight components in forage and grain derived from MON 87427 corn, including key nutrients, anti-nutrients and secondary metabolites, and compared it with corresponding levels of components in a non-transgenic control variety with a similar genetic background (LH198 × LH287; control). Monsanto also compared the composition of MON 87427 corn with four commercial reference varieties (reference varieties) grown alongside MON 87427 corn and control.
Study Design - Compositional Analyses
Monsanto states that forage and grain were obtained from corn grown in three replicate plots, planted in a randomized complete block design, at each of three field sites in corn growing regions of the U.S. during the 2008 growing season. Monsanto notes that MON 87427 corn plants were treated with glyphosate herbicide. Monsanto states that data from the reference varieties were used to calculate a 99% tolerance interval for each component. Monsanto assessed the data using a mixed model analysis of variance where compositional data from MON 87427 corn and the control were combined from all three sites for statistical comparison (combined-site analysis). Data were also compared between sites individually; yielding three additional comparisons (individual-site analysis). Monsanto reports that statistically significant differences were identified at a 5% level of significance (p<0.05). Where statistically significant differences were detected between MON 87427 corn and the control, Monsanto assessed whether the difference was biologically meaningful4 with regard to food and feed safety. This assessment included a discussion of the magnitude of the difference observed, the reproducibility of the difference at each field site, comparisons between data from MON 87427 corn and a 99% tolerance interval calculated from data derived from analysis of the reference varieties, as well as values in the published literature, including the International Life Sciences Institute - Crop Composition Database (ILSI-CCD).5
Results of analyses
Compositional Analysis of Corn Forage
Monsanto reports the results of compositional analysis for nine components in corn forage (moisture, crude protein, crude fat, ash, carbohydrates by calculation, acid detergent fiber (ADF), and neutral detergent fiber (NDF), calcium and phosphorus). Monsanto states that no statistically significant differences were observed in the combined-site analysis of components from MON 87427 corn when compared to the control. Monsanto concludes that this analysis supports the conclusion that forage from MON 87427 corn is compositionally equivalent to conventional corn forage.
Compositional Analysis of Corn Grain
Monsanto reports results of compositional analysis for 64 nutrient components in corn grain. Monsanto analyzed grain for proximates (moisture, crude protein, crude fat, ash, carbohydrates by calculation), ADF, NDF, total dietary fiber (TDF), fatty acids (C16-C22), 18 amino acids, 7 vitamins, and 8 minerals. Monsanto reports that, in the combined-site analysis, statistically significant differences in palmitic (16:0), stearic (18:0), oleic (18:1), linoleic (18:2) acid, arachidic (20:0) acid, and total fat levels in MON 87427 corn were detected when compared with the control. Monsanto notes that individual site mean values for all nutrients with statistically significant differences were within the 99% tolerance interval established from the commercial varieties grown concurrently. Monsanto also states that the relative magnitude of the differences for each of these components was small, ranging from 1.96% to 5.09% for MON 87427 corn compared to the control. Monsanto further notes that the observed differences are less than differences typically found in corn hybrids developed through traditional breeding. Monsanto states that all the compositional components identified as statistically different from control were within the natural variability for these components in corn (ILSI-CCD). Monsanto thus concludes that these differences are not biologically meaningful to food and feed safety and nutrition.
Anti-Nutrients and Secondary Metabolites
Monsanto reports that MON 87427 corn and control samples were analyzed for anti-nutrients (phytic acid and raffinose) and secondary metabolites (furfural, ferulic acid and p-coumaric acid). In the combined-site analyses, no statistically significant differences were detected for any of the secondary metabolites. A statistically significant decrease in phytic acid was observed in MON 87427 corn compared to control. Monsanto notes that the magnitude of the decrease was relatively small (5.92 %) and that no significant differences in phytic acid were observed for the individual site analysis. The mean phytic acid values for MON 87427 corn and control were within the range of natural variability reported in the literature (ILSI-CCD). Monsanto thus concludes that this observed difference is not biologically meaningful to food and feed safety and nutrition.
Summary of Compositional Analyses
Monsanto states that the compositional analysis supports the conclusion that MON 87427 corn is compositionally equivalent to conventional corn varieties. Monsanto concludes that the small differences in the levels of the components described above are not considered to be biologically meaningful to food and feed safety and nutrition.
FDA evaluated Monsanto’s submission to determine whether MON 87427 corn 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 or current FDA regulations that would require further evaluation at this time. Monsanto has concluded that its glyphosate-tolerant corn variety, MON 87427 corn, and the foods and feeds derived from it are as safe as conventional corn varieties and, with the exception of the herbicide tolerance trait, are not materially different in composition or any other relevant parameter from other corn varieties now grown, marketed, and consumed in the U.S. At this time, based on Monsanto’s data and information, the agency considers Monsanto’s consultation on MON 87427 corn to be complete.
Richard E. Bonnette
1BNF 1, 20, 26, 35, 51, 56, 71, 77, 79, 80, 84, 90, 97, 98, 104, and 109 (http://www.fda.gov/bioconinventory)
2These analyses included matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, western blot analyses and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), glycosylation analysis, and CP4 EPSPS enzyme activity.
3Forage, seed, over-season leaf (OSL1-OSL4), pollen, silk, over-season root (OSR1-OSR4), senescent root, over-season whole plant were analyzed.
4FDA considers “biologically meaningful” to connote that there is a notable impact on dietary requirements, or an impact that otherwise would affect human or animal health.
5Monsanto used version 3.0 (accessed on August 12, 2010) of the ILSI-CCD in its analysis. The database is maintained by ILSI and can be accessed at http://www.cropcomposition.org/.