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Biotechnology Consultation - Note to the File
Biotechnology Notification File BNF No. 000127
Date: March 23, 2012
Subject: Glyphosate-tolerant MON 88302 Canola
Keywords: Canola, Brassica napus L., cp4 epsps, 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) from Agrobacterium sp. strain CP4, herbicide tolerance, glyphosate, MON 88302, OECD Unique Identifier MON-883Ø2-9, Monsanto, Roundup Ready®
This document summarizes our evaluation of biotechnology notification file (BNF) No. 000127. In a submission dated March 23, 2011, Monsanto Company (Monsanto) submitted to the Food and Drug Administration (FDA) a safety and nutritional assessment of bioengineered herbicide tolerant canola, transformation event MON 88302-9 canola (hereafter referred to as MON 88302 canola). Monsanto provided additional information on June 14, 2011, and September 16, 2011. FDA evaluated the information in Monsanto's submissions to ensure that regulatory and safety issues regarding the food and animal feed derived from the new plant variety have been resolved prior to commercial distribution. In our evaluation of BNF No. 000127, 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 effect of the modification in MON 88302 canola is to confer tolerance to the herbicide glyphosate and, in particular, improved tolerance to glyphosate in male reproductive tissues, thus allowing the application of glyphosate during reproductive growth stages. To accomplish this objective, Monsanto introduced a gene, cp4 epsps, which encodes for 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) protein, into a conventional canola cultivar. The CP4 EPSPS protein confers tolerance to the herbicide glyphosate. The expression cassette utilizes a specific chimeric promoter and intron combination (P-FMV/TSf1) for the cp4 epsps gene, which results in increased expression in male reproductive tissues. This enhances resistance to glyphosate for these tissues, which in previous glyphosate-tolerant varieties had remained sensitive to glyphosate.
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 88302 canola, 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 88302 canola.
Genetic Modification and Characterization
Monsanto transformed the recipient commercial line Ebony to obtain MON 88302 canola.
Transformation Plasmid and Method
Monsanto described the transformation plasmid, PV-BNHT2672. This plasmid contained the cp4 epsps gene 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 FMV/Tsf1 chimeric promoter, the Tsf1 leader and intron sequence, the CTP2 targeting sequence, the cp4 epsps gene, and the e9 3’ non-translated region. MON 88302 canola was generated using Agrobacterium-mediated transformation of immature Ebony embryo tissue utilizing PV-BNHT2672. The kanamycin resistance gene aad2, present in the intact PV-BNHT2672 plasmid, is not expected to be present in the transformed cells because it was outside the T-DNA.
Characteristics, Inheritance, and Stability of the Introduced DNA
Monsanto characterized the insert in MON 88302 canola using restriction enzyme digestion of genomic DNA followed by Southern blot analysis. Monsanto concluded that results of this analysis demonstrate that a single intact copy of the gene cassette was integrated into the plant genome. Monsanto further noted that Southern blot analyses of the entire MON 88302 canola genome using probes designed to detect plasmid backbone sequences from PV-BNHT2672 confirmed that there were no plasmid vector backbone sequences present in MON 88302 canola. Therefore, the aad2 gene from PV-BNHT2672 was not inserted into the canola 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 canola genomic DNA by comparing the sequences at the insertion site to conventional canola. Monsanto assessed the stability of the DNA insert across four breeding generations through restriction enzyme digestion of genomic DNA followed by Southern blot analysis. Monsanto states that these data indicated that MON 88302 canola contained one copy of the cp4 epsps expression cassette, which is stably inherited across multiple generations. Monsanto also assessed heritability and stability of the introduced DNA in MON 88302 canola through analyses of segregation data as determined through phenotype. Monsanto states that Chi-square analysis demonstrated that there was no statistically significant difference between the observed and expected segregation patterns, supporting the conclusion that the cp4 epsps expression cassette contained in MON 88302 canola 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 88302 canola insert and flanking genomic DNA sequences to determine whether insertion of the introduced DNA may have created any ORF that may encode a potential 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 would not constitute a safety concern.
Identity and Function of Introduced Protein
MON 88302 canola was genetically engineered to express the CP4 EPSPS protein, which conveys resistance to the herbicide glyphosate. This protein has been previously evaluated by FDA in many other biotechnology consultations. 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 discussed analyses to confirm the identity of the CP4 EPSPS protein introduced into MON 88302 canola. Monsanto states that all the results from these analyses confirmed the identity of the CP4 EPSPS protein expressed in MON 88302 to be 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 88302 canola obtained from samples collected during the 2009 growing season from three field sites in the United States and three field sites in Canada. Protein levels were determined by enzyme-linked immunosorbent assay (ELISA) conducted on several canola tissue types. Monsanto reported that mean concentrations of CP4 EPSPS across canola tissue types ranged from 27 micrograms per gram (dry weight) in seed to 230 micrograms per gram dry weight in over-season leaf stage 3 (OSL3). Monsanto also notes that the total protein content of canola oil is very low (less than 0.2 parts per million) and therefore the levels of CP4 EPSPS in canola oil would also be less than 0.2 parts per million.
Potential for Toxicity of the Introduced Protein
Monsanto includes bioinformatics analysis including a comparison of the CP4 EPSPS protein to known protein toxins contained in the TOX_2010 database, a sequence 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 88302 canola 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 allergenicity of the CP4 EPSPS protein. Monsanto assessed the structural similarity of CP4 EPSPS to known allergens through standard bioinformatics methods using the AD_2010 database (FAARRP, 2010). This analysis revealed no significant sequence alignments 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 no contiguous stretches of 8 or greater amino acids are 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 intestinal fluid and in simulated gastric fluid. Monsanto states that these studies indicate that CP4 EPSPS is rapidly digested. Monsanto notes that the expression level of CP4 EPSPS in MON 88302 canola seed is very low, with a mean concentration of 27 micrograms per gram dry weight. Monsanto highlights that the low concentration of CP4 EPSPS in MON 88302 canola seed, 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 discusses the historical development of canola (Brassica napus L.) as an agricultural product and describes its current use in food and feed. For food applications, Monsanto notes that canola is principally grown for the oil that is extracted from its seed. Canola oil is a high quality oil used in many foods including frying and baking oils, salad oils, and margarines and shortenings. As a feed source, Monsanto states that the meal and solids left as by-products of the canola oil extraction process represent the primary forms of canola used in feed. Monsanto notes that the meal and solids are used as high protein animal feed. Canola meal can be used in poultry, pig, beef and dairy cattle feeds and is also used in aquaculture diets for farmed fish. Monsanto notes that canola forage is rarely used as animal feed.
Scope of Analysis
Monsanto reported data on seventy components in seed derived from MON 88302 canola, including key nutrients, anti-nutrients and toxicants, and compared it with corresponding levels of components in a non-transgenic variety with a similar genetic background (control). Monsanto also compared the composition of MON 88302 canola with seven commercial reference varieties (reference varieties) grown alongside MON 88302 canola and the control.
Study Design - Compositional Analyses
Monsanto states that seed was obtained from canola grown in four replicate plots, planted in a randomized complete block design, at each of five field sites in the U.S. (two sites) and Canada (three sites) during the 2009 growing season. Monsanto notes that MON 88302 canola plots were treated with glyphosate herbicide and all plants were treated with maintenance pesticides as necessary. Monsanto details that data from the reference varieties were used to establish a 99% tolerance interval for each measured component. Monsanto assessed the data using a mixed model analysis of variance where component data from MON 88302 canola and the control were combined from all five sites for statistical comparison (combined-site analysis). Data were also compared between sites individually, yielding five additional comparisons (individual-site analysis). Monsanto reported that statistically significant differences were identified at a 5% level of significance (P<0.05). Where statistically significant differences were detected between MON 88302 canola and the control, Monsanto assessed whether the difference was biologically meaningful with regard to food and feed safety. This assessment included a discussion of the magnitude of the observed differences, if any; the reproducibility of the differences at each field site using the individual-site analysis; comparisons between data from MON 88302 canola and a 99% tolerance interval calculated from data derived from analysis of the reference varieties, as well as comparisons to values in the published literature.
Results of analyses:
Monsanto analyzed seed for proximates (moisture, crude protein, crude fat, ash, carbohydrates by calculation), acid detergent fiber (ADF), neutral detergent fiber (NDF), total dietary fiber (TDF), fatty acids (C8-C24), 18 amino acids, vitamin E, and 9 minerals. Monsanto reports that for the combined-site analysis, statistically significant differences were detected for TDF and seven fatty acids (16:1 palmitoleic, 18:0 stearic, 18:1 oleic, 18:2 linoleic, 18:3 linolenic, 20:0 arachidic, and 22:0 behenic acids). Monsanto notes that individual site mean values for all nutrients with statistically significant differences were within the 99% tolerance interval that was calculated for the reference varieties and within the natural variability of these components in canola as reported in the literature. Monsanto also states that the magnitude of differences between MON 88302 canola and control for the components in the combined-site analysis ranged from 7.56% to 20.01%. Monsanto notes that these differences are small relative to the natural variability of these components as demonstrated by the established 99% tolerance interval. Monsanto highlights that all the compositional components identified as statistically significantly different from control were within the 99% tolerance interval. Monsanto thus concludes that the differences observed in some of the measured components are not biologically meaningful to food and feed safety.
Anti-Nutrients and Toxicants
Monsanto reports that MON 88302 canola was analyzed for anti-nutrients and naturally-occurring toxicants including phytic acid, sinapine, erucic acid, alkyl glucosinates, indolyl glucosinates, and total glucosinates. Monsanto notes that erucic acid was below the limit of detection (0.04% of total fatty acids) and was excluded from statistical analysis. Monsanto reports that a statistically significant difference was detected for mean alkyl glucosinates in the combined-sites analysis for MON 88302 canola compared to the control. Monsanto notes that alkyl glucosinates was found to be 27.58% lower in MON 88302 canola compared to the control, which Monsanto states is a relatively small difference compared to natural variability as determined by the established 99% tolerance interval. Monsanto also notes that the mean level of alkyl glucosinates observed in MON 88302 canola is within the range of natural variability as reported in the literature. Monsanto concludes that this difference is not biologically meaningful to food and feed safety.
Summary of Compositional Analyses
Monsanto states that the compositional analysis supports the conclusion that MON 88302 canola is compositionally equivalent to conventional canola varieties. Monsanto concludes that the small differences in the levels of the components described above are not considered to be biologically meaningful for food and feed safety and nutrition.
FDA evaluated Monsanto’s submission to determine whether MON 88302 canola 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. Monsanto has concluded that its glyphosate-tolerant canola variety, MON 88302, and the foods and feeds derived from it are as safe as conventional canola varieties and, with the exception of the herbicide tolerance trait, are not materially different in composition or any other relevant parameter from other canola 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 88302 canola to be complete.
Richard E. Bonnette
BNF 1, 20, 26, 35, 51, 56, 71, 77, 79, 80, 84, 90, 97, 98, 104, 109, and 126 (http://www.fda.gov/bioconinventory)
These analyses included N-terminal sequence analysis, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, western blot analyses, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), glycosylation analysis, and CP4 EPSPS enzyme activity.
Forage, seed, over-season leaf (OSL1-OSL4) and two root stages (Root-1 and Root-2) were analyzed.
FDA 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.