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In a submission dated May 26, 2006, Monsanto Company (Monsanto) submitted to FDA a safety and nutritional assessment of the genetically engineered Roundup RReady2Yield™ (glyphosate-tolerant) soybean line MON 89788. Monsanto submitted additional information on January 17, 2007. Monsanto concluded that food and feed derived from MON 89788 are as safe and nutritious as food and feed derived from conventional soybean.
This current submission is in addition to a previously completed consultation for glyphosate-tolerant soybean event 40-3-2 in BNF 000001. Monsanto has previously completed consultations for other Roundup Ready® crops which are also tolerant to glyphosate. These other crops include bioengineered sugar beet (BNF 000056, BNF 000090), canola (BNF 000020, BNF 000077), corn (BNF 000035, BNF 000051, BNF 000071), wheat (BNF 000080), bentgrass (BNF 000079), alfalfa (BNF 000084) and cotton (BNF 000026, BNF 000098).
The intended effect of the genetic engineering is to confer tolerance to the herbicidal compound glyphosate [N-phosphonomethyl-glycine], which is the active ingredient in Roundup agricultural herbicides. In glyphosate-sensitive plants, glyphosate binds to the plant's endogenous 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and prevents the biosynthesis of aromatic amino acids that are necessary for plant growth. Glyphosate-tolerant soybean line MON 89788 contains one cp4 epsps gene from Agrobacterium sp. strain CP4, encoding the CP4 EPSPS enzyme, which has a much reduced affinity for glyphosate.
The soybean variety used as the recipient for the DNA insertion to create MON 89788 was A3244, a non-transgenic conventional variety developed by Asgrow Seed Company. Monsanto states that the A3244 is an elite maturity group III soybean variety, which was developed and selected based on its superior agronomic performance over other soybean lines.
Monsanto constructed the double-border, binary plasmid vector PV-GMGOX20 for transformation of soybean variety A3244. The plasmid contains a cp4 epsps gene expression cassette in the T-DNA. Agrobacterium tumefaciens-mediated transformation of the meristem tissue from soybean variety A3244 was carried out; this resulted in the integration of the T-DNA region of PV-GMGOX20 into the genome of A3244.
The T-DNA region of PV-GMGOX20 contains the following genetic elements:
| Genetic elements in the T-DNA | Description of the genetic elements |
|---|---|
| RB- Right border | Right border sequence derived from Agrobacterium tumefaciens |
| P-FMV/Tsf1 | Chimeric promoter that combines the enhancer sequences from the 35S promoter of the Figwort Mosaic virus and the promoter from the Tsf1 gene in Arabidopsis thaliana encoding elongation factor EF-1α. |
| L-Tsf1 | 5'-end untranslated leader (exon 1) from the Tsf1 gene of Arabidopsis thaliana encoding elongation factor EF-1α. |
| I-Tsf1 | Untranslated intron from the Tsf1 gene of Arabidopsis thaliana encoding elongation factor EF-1α. |
| TS-CTP2 | Sequence encoding the chloroplast transit peptide from the ShkG gene of Arabidopsis thaliana. |
| CS-cp4 epsps | Codon-optimized coding sequence of the aroA (epsps) gene from the Agrobacterium sp. strain CP4 encoding the CP4 EPSPS protein. |
| T-E9 | 3'-end untranslated sequence from the ribulose-1,5-bisphosphate carboxylase small subunit (RbcS2) E9 gene of pea (Pisum sativum). |
| LB- Left border | Left border sequence derived from Agrobacterium tumefaciens |
Four genetic elements exist outside the T-DNA borders. These are: origins of replication for maintenance of the PV-GMGOX20 plasmid in Agrobacterium (ori-V), and in E. coli (ori-pBR322); coding sequence for the repressor of primer (rop) protein which allows maintenance of plasmid copy number in E. coli; and the aadA gene which confers spectinomycin and streptomycin resistance, and is used for molecular cloning and selection in bacterial hosts.
Monsanto conducted Southern blot analysis of the genomic DNA in order to characterize the introduced T-DNA in MON 89788. From the analyses, Monsanto concludes that event MON 89788 contains a single copy of the T-DNA integrated at a single locus of the genome. The integrated T-DNA contains the cp4 epsps expression cassette with all the components of the cassette intact. Monsanto also concludes that event MON 89788 does not contain backbone sequences (ori-V, ori-pBR322, rop, and aadA coding sequences) from the transformation vector PV-GMGOX20.
To assess whether the inserted DNA in MON 89788 was stably integrated into the genome, Monsanto performed Southern blot analyses across four generations, designated R4, R5, R6, and R7. The four generations of MON 89788 grain utilized to assess generational stability were also examined for the absence of backbone sequences. Monsanto states that these analyses demonstrate that the inserted DNA containing the full-length cp4 epsps expression cassette was stably integrated at a single locus in all progeny, and that the generations examined do not contain backbone sequence from the transformation vector PV-GMGOX20.
Monsanto used DNA sequence analysis to confirm that the arrangement of the genetic elements in the inserted DNA is identical to that found in plasmid PV-GMGOX20.
To examine the inheritance of the glyphosate tolerance trait, Monsanto conducted crosses using conventional breeding techniques and generated phenotypic segregation data across several generations. Chi-square analysis of the data indicates that the introduced trait (glyphosate tolerance) was stably inherited, and segregated through multiple generations following the expected Mendelian inheritance pattern.
EPSPS enzyme is involved in the biosynthesis of aromatic amino acids that are necessary for growth and development of the plant. Monsanto states that the CP4 EPSPS protein from Agrobacterium sp. strain CP4 is structurally similar and functionally identical to native plant EPSPS enzyme. In plants, the chloroplast is the primary site for aromatic amino acid biosynthesis and is where the EPSPS enzyme is located. The native EPSPS has a high affinity for glyphosate and is inhibited by it. In contrast, the CP4 EPSPS has a much-reduced affinity for glyphosate. Thus, CP4 EPSPS helps the plant to synthesize the aromatic amino acids and grow in the presence of glyphosate. The CP4 EPSPS expressed in soybean MON 89788 is a 47.6 kDa protein consisting of a single polypeptide of 455 amino acids. CP4 EPSPS protein was also expressed in E. coli to obtain sufficient amount of pure protein for use in various studies. Monsanto used the following methods to characterize MON 89788-produced CP4 EPSPS protein and to demonstrate its equivalence with E. coli-derived CP4 EPSPS protein: sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Brilliant Blue G-Colloidal staining; immunoblotting and densitometric analysis of the blot; matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry; N-terminal sequence analysis; CP4 EPSPS enzyme assay; and glycosylation analysis. Monsanto concludes from the studies that MON 89788-derived CP4 EPSPS and E. coli-derived CP4 EPSPS proteins are equivalent in their biochemical and physiological characteristics.
Monsanto reports that levels of CP4 EPSPS protein in MON 89788 over-season leaf (OSL) collected at different growth stages, grain, root, and forage were determined using a validated enzyme-linked immunosorbent assay (ELISA). Tissue samples were collected from MON 89788 produced in replicated field trials across five U.S. field locations during 2005. Monsanto reports that mean levels of the CP4 EPSPS protein for OSLl, OSL2, 0SL3, OSL4, grain, root and forage of MON 89788 were 54, 60, 58, 75, 140, 22, and 59 μg/g, respectively, on a fresh weight (FW) basis. These levels translate to 300, 340, 330, 290, 150, 74 and 220 μg/g, respectively, on a dry weight (DW) basis. Results indicate the CP4 EPSPS protein represents only a small portion of total protein in the MON 89788 grain (approximately 0.037% of total protein).
Monsanto has provided estimates of potential exposure to MON 89788-derived CP4 EPSPS protein in soy-derived foods consumed in the U.S. using the Dietary Exposure Evaluation Model (DEEM-FCID version 2.03) and food consumption data from the USDA Continuing Survey of Food Intakes by Individuals (CSFII). In the estimation, all soybean food items consumed were assumed to be derived from MON 89788 soybeans. Monsanto states that since soybean oil contains negligible amount of protein, the analysis estimated exposure from three main sources of exposure, that is, the grain, flour and milk. Monsanto reports that according to the analysis, the 95th percentile for the acute dietary intake estimate (ADIE) of CP4 EPSPS from consumption of MON 89788 is 9.9 μg/kg body weight for the adult U.S. population and 391 μg/kg body weight for non-nursing infants. Monsanto concludes that there is no meaningful risk to human health from dietary exposure to CP4 EPSPS from consumption of MON 89788 soybeans.
Monsanto searched TOXIN5, a sequence database of known protein toxins compiled by Monsanto, for amino acid sequence homology to the CP4 EPSPS protein. Monsanto reports that no biologically relevant sequence similarities were observed between the CP4 EPSPS protein and known protein toxins.
Monsanto describes an acute toxicity study by Harrison et al. (1996) conducted in mice. Male and female CD-1 mice were dosed by gavage with up to 572 mg/kg body weight of CP4 EPSPS protein produced in E. coli. 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.
Monsanto provides information about Agrobacterium sp. strain CP4, the donor of the cp4 epsps gene. Agrobacterium species are non-pathogenic and non-toxigenic. The safety of the Agrobacterium sp. strain CP4 has been previously demonstrated during Monsanto's consultations with FDA on other glyphosate-tolerant crops.
Monsanto describes its assessment of the allergenic potential of the CP4 EPSPS protein in soybean event MON 89788. Monsanto states that there are no reports of allergy to Agrobacterium sp. strain CP4, which is the source of the CP4 EPSPS protein.
Monsanto searched AD5, a sequence database of known allergen, gliadin, glutenin proteins compiled by Monsanto, for amino acid sequence homology to the CP4 EPSPS. Monsanto concludes that CP4 EPSPS in soybean event MON 89788 does not share structurally relevant or immunologically significant identity with any known allergens, gliadins or glutenins.
Monsanto discusses studies performed by Harrison et al. (1996) to assess the in vitro stability of E. coli-expressed CP4 EPSPS in simulated gastric fluids (SGF) and in simulated intestinal fluids (SIF). Western blot analysis of the SGF- and SIF-incubated CP4 EPSPS protein showed that its half-life was less than 15 seconds in SGF and less than 10 min in SIF. In subsequent studies, the stability/degradation of the CP4 EPSPS protein was assessed by SDS-PAGE followed by colloidal blue gel staining, western blot analysis, and activity assay. Monsanto reports that SDS-PAGE colloidal blue staining indicated that 98% of the CP4 EPSPS protein was digested within 15 seconds. Western blot analysis indicated that greater than 95% of the CP4 EPSPS protein was digested within 15 seconds; and the enzymatic activity assay indicated that the CP4 EPSPS activity was reduced to less than 10% of the initial activity within 15 seconds of exposure to SGF.
Monsanto describes historical and current uses of soybean in food and animal feed, and states that MON 89788 is intended to be utilized in the same manner and for the same uses as conventional soybeans. Soybeans are used for a wide variety of food and feed purposes and are the main source of plant protein consumed by humans and animals. Soybeans are also the leading source of vegetable oil of all crops produced in the world. Soybean meal is the major supplemental protein source in U.S. livestock and poultry rations. On average, dry soybeans contain roughly 40% protein and 20% oil.
Monsanto reports that the levels of various analytes measured in MON 89788 were compared to those measured in A3244, the conventional soybean variety that was used for transformation; hence, the genetic background of MON 89788 is similar to that of A3244. Additional conventional soybean varieties, currently in the marketplace, were also included in the analyses to establish the range of natural variability for each analyte.
Monsanto states that grain and forage tissues were collected from MON 89788 and A3244 soybeans grown in three replicated plots at each of five field sites across the U.S. during 2005. In addition, 12 conventional soybean varieties were also included as references where three varieties were grown at each of two sites and two varieties were grown at each of three sites, for a total of 12 reference varieties. Statistical analyses of the compositional data were conducted using a mixed model analysis of variance with data from each of five sites, and a combination of all five field sites. Each individual analyte for MON 89788 was compared to that of A3244. Statistical significance was declared at 5% level (p≤0.05).
Monsanto measured the following groups of components:
A list of specific analytes within each group is shown in Table 2.
| Proximates | Amino Acids | Fatty Acids | Anti-Nutrients | Isoflavones | Vitamins |
|---|---|---|---|---|---|
| ash fat moisture protein carbohydrate acid detergent fiber (ADF) neutral detergent fiber (NDF) |
arginine histidine isoleucine leucine lysine methionine phenylalanine threonine valine tryptophan cystine glycine alanine aspartic acid glutamic acid proline serine tyrosine |
palmitic (16:0) stearic (18:0) oleic (18:1) linoleic (18:2) linolenic (18:3) arachidic (20:0) eicosenoic (20:1) behenic (22:0) |
phytic acid trypsin inhibitor lectin raffinose stachyose |
daidzein genistein glycitein |
vitamin E |
For the combined-site analyses for soybean grain, the only statistical differences between MON 89788 and the control (A3244) were for the isoflavones daidzein and glycitein, and Vitamin E. A3244 grain had higher levels of daidzein and glycitein than MON 89788. MON 89788 had higher levels of Vitamin E than A3244. Monsanto reported that the differences were generally small (1.6 - 11 %) and the mean levels for all analytes measured for both MON 89788 and A3244 fell within the 99% tolerance intervals calculated by Monsanto for the conventional soybean reference varieties, and within the range of literature data. Monsanto also found statistically significant differences in some nutrient levels at specific locations, but except for the nutrients already cited, these differences were not consistent across all locations.
Monsanto determined the levels of the following components of forage from soybean event MON 89788 and the control (A3244):
Proximates - Ash, fat, protein, carbohydrates (by difference) and moisture.
Fiber - Neutral detergent fiber (NDF) and acid detergent fiber (ADF).
Of all the analytes measured in forage, only the moisture level was significantly different in MON 89788 when compared with its control, A3244. All values measured for the analytes fell within the 99% tolerance interval calculated by Monsanto for the conventional soybean reference varieties, and with the exception of moisture, all fell within published literature ranges. Moisture levels were only slightly below the literature values, and it is commonly know that moisture levels are a function of harvest time.
To assess whether the transformation process may have increased the overall allergenicity of MON 89788 soybean compared to conventional soybean, Monsanto conducted IgE-binding enzyme linked immunosorbent assays (ELISA) with extracts from MON 89788, A3244, and 24 commercial soybean varieties. The commercial varieties were included to establish the normal variations of responses expected to soybeans. Sera used in these assays were obtained from 16 individuals with IgE-mediated allergy to soy as evidenced by clinical history of anaphylactic reactions to soy or positive response in a double-blind placebo controlled food challenge, and six non-allergic individuals. From these experiments, Monsanto concluded that the allergic potential of MON 89788 soybean is no greater than soybean varieties currently on the market.
Monsanto concludes that MON 89788 is compositionally equivalent to conventional soybeans.
Monsanto has concluded that their glyphosate-tolerant soybean, event MON 89788, and the feeds and foods derived from them are not materially different in safety, composition, or any other relevant parameter from soybeans now grown, marketed, and consumed. At this time, based on Monsanto's data and information, the agency considers Monsanto's consultation on glyphosate-tolerant soybean line MON 89788 to be complete.
Supratim Choudhuri, Ph.D.
Mary D. Ditto, Ph.D.
